Inhibitory propeptides

Field of the invention

The present invention relates to compounds, peptides or peptidomimetics that inhibit, reduce or prevent protease activity and the use of these compounds, peptides or peptidomimetics to treat or prevent a condition. In particular the condition may be periodontal disease. The protease activity may be activity of a gingipain. The compounds, peptides or peptidomimetics of the invention may also be used in assays for the identification of protease inhibitors.

Background of the invention

Periodontal diseases are bacteria associated inflammatory diseases of the supporting tissues of the teeth and are a major public health problem. Nearly all of the human population is affected by periodontal diseases to some degree. A US Dental Health survey in 1989 reported that 85% of the studied population has periodontal diseases. The major form of periodontal disease is gingivitis which is associated with the non- specific accumulation of dental plaque at the gingival margin. The more destructive form of periodontal disease (periodontitis) is associated with a subgingivial infection by specific Gram-negative bacteria. The major bacterial pathogens implicated in this disease are known as the "red complex", which is composed of Tannerella forsythia, Porphyromonas gingivatis and Treponema denticola. P. gingivalis is the main aetiological agent in chronic periodontitis.

The main virulence factors of P. gingivalis are its extracellular cysteine proteases, known collectively as the gingipains. Most common are RgpA and RgpB (the Arg- gingipains) and Kgp (the Lys-gingipain). The Arg-gingipains cleave proteins at the carboxyl side of Arg residues and the Lys-gingipains cleave at the carboxyl side of Lys residues.

These cell surface cysteine proteases are thought to be important for the degradation of proteins to provide peptides for growth as well as other intrinsic and extrinsic functions for survival and virulence. Several of these functions for survival and virulence may be bacterial adhesion to host tissue, hemagglutination, and the processing of bacterial cell- surface and secretory proteins. The catalytic domains of RgpA and Kgp can bind as a complex on the cell surface with a series of non-covalently bound sequence-related hemagglutinin/adhesin domains while RgpB has been shown to exist as not part of the protease adhesin complex and may consist of the catalytic domain only.

Like other cysteine proteases, the gingipains are synthesized as inactive forms with a propeptide region at the N-terminus that is removed to yield the mature, active form. The gingipains are highly conserved and the amino acid sequences of both the mature enzyme and propeptides reveal that they are only distantly related to other cysteine proteases.

There exists a need for a better or alternative inhibitor of bacterial enzymes involved in the pathogenesis of various diseases, particularly periodontal disease.

Reference to any prior art in the specification is not, and should not be taken as. an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

Summary of the invention

The field of the invention relates to a compound, peptide or peptidomimetic for inhibiting, reducing or preventing the activity of a bacterial enzyme, the compound, peptide or peptidomimetic comprising an amino acid sequence of a gingipain propeptide or fragment thereof. In one embodiment the enzyme may be an extracellular protease. Preferably, the extracellular protease is a cysteine protease, more preferably a gingipain. The protease maybe RgpA, RgpB or Kgp that is derived from a strain of Porphyromonas gingivalis.

In certain embodiments the compound, peptide or peptidomimetic is a peptide or peptidomimetic that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 10 (shown in Figure 1 ).

In other embodiments the peptide or peptidomimetic comprises paralogous and orthologous sequences to those sequences shown in SEQ ID NO: 1 to 10.

In other embodiments the peptide or peptidomimetic comprises conservative subsitutions in the above amino acid sequences. These substitutions are described further below. A peptide of the invention may be isolated, purified, enriched, synthetic or recombinant. According to the present invention there is also provided a compound, peptide or peptidomimetic comprising an amino acid sequence of a Kgp propeptide or fragment thereof, wherein the compound, peptide or peptidomimetic does not have the capacity to form a dimer. The dimer may be a homodimer or a heterodimer.

According to the present invention there is also provided a compound, peptide or peptidomimetic comprising an amino acid sequence of a Kgp propeptide or fragment thereof, wherein the compound, peptide or peptidomimetic is monomeric.

In one embodiment, the compound, peptide or peptidomimetic comprises an amino acid sequence of a Kgp propeptide or fragment thereof, wherein the compound, peptide or peptidomimetic does not have the capacity to form a dimer via a disulphide bond. Typically, the compound, peptide or peptidomimetic does not contain a cysteine residue.

According to the present invention there is also provided a compound, peptide or peptidomimetic comprising an amino acid sequence having about 70% identity to a Kgp propeptide or fragment thereof, wherein the compound, peptide or peptidomimetic does not have the capacity to form a dimer by a disulphide bond with another peptide.

In certain embodiments of the invention there is provided a compound, peptide or peptidomimetic that is a peptide or peptidomimetic that comprises, consists essentially of or consists of an amino acid sequence of a Kgp propeptide or fragment thereof, wherein the amino acid sequence does not contain a cysteine residue at a position equivalent to position 16 in SEQ ID NO: 1 (numbering wherein Gin at the N-terminus is position 1 and Arg at the C-terminus is position 209). Typically, the amino acid sequence has identity to a Kgp propeptide or fragment thereof that occurs naturally with a cysteine a position equivalent to position 16 in SEQ ID NO: 1. Typically, the compound, peptide or peptidomimetic is a peptide or peptidomimetic that comprises an amino acid sequence that does not contain a lysine residue at any one or more positions equivalent to 4, 41, 69, 100, 110, 129, 168 or 204 in SEQ ID NO: 1.

In certain embodiments the compound, peptide or peptidomimetic is a peptide or peptidomimetic that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 4, 5, or 10 (shown in Figure 1) wherein the amino acid residue at the position equivalent to position 16 in SEQ ID NO: 1 is not a cysteine. Typically, the amino acid residue that is not a cysteine is any other naturally occurring or artificial amino acid wherein the compound, peptide or peptidomimetic retains the ability to inhibit, reduce or prevent the activity of a bacterial enzyme, preferably gingipain, even more preferably Kgp. Typically, the amino acid residue that is not a cysteine is any one of the other 19 naturally occurring amino acids. Preferably, the amino acid that is not a cysteine is selected from the group consisting of serine, alanine, glycine or threonine. Even more preferably, the amino acid that is not a cysteine is an alanine. Typically, the compound, peptide or peptidomimetic is a peptide or peptidomimetic that comprises an amino acid sequence that does not contain a lysine residue at any one or more positions equivalent to 4, 41 , 69, 100, 110, 129, 168 or 204 in SEQ ID NO: 1.

In particular embodiments the peptide or peptidomimetic consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 wherein the amino acid residue at position 16 is not a cysteine.

In particular embodiments the peptide or peptidomimetic consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 4 wherein the amino acid residue at position 16 is not a cysteine.

In particular embodiments the peptide or peptidomimetic consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 5 wherein the amino acid residue at position 16 is not a cysteine.

In particular embodiments the peptide or peptidomimetic consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 wherein the amino acid residue at position 25 and 57 is not a cysteine.

In any embodiment of the invention described herein, unless otherwise expressly stated, the amino acid residue that is not a cysteine is any other naturally occurring or artificial amino acid (including modified-cysteine that does not form disulphide bonds) wherein the compound, peptide or peptidomimetic retains the ability to inhibit, reduce or prevent the activity of a bacterial enzyme, preferably gingipain, even more preferably Kgp. Determining whether the compound, peptide or peptidomimetic retains the ability to inhibit, reduce or prevent the activity of a bacterial enzyme can be readily determined empirically, for example by any method described herein. In one embodiment, the amino acid residue that is not a cysteine is selected from the group consisting of serine, alanine, glycine or threonine. Preferably, the amino acid that is not a cysteine is an alanine. In any embodiment or aspect of the invention described herein, the peptide that does not have the capacity to form a dimer with another peptide by a disulphide bond, may be modified to be more resistant to proteolysis. To improve resistance to proteolysis, any one or more of the non-structurally conserved, non-interacting, surface exposed lysines and arginines can be substituted, by any natural or non-natural amino acid, preferably by glutamines or asparagines, thus maintaining polarity and alpha-helical propensity. For peptides derived from or with similarity to Kgp propeptide or fragment, substitution or modification can be at any one or more lysines namely 4, 110, 100, 129, 69, 168, and 41 or equivalent to account for minor variations and deletions between strains. For peptides derived from or with similarity to RgpB propeptide or fragment thereof, substitution can be at any one or more of Arg 102, Lys 97, Lys 121, Arg 66, Arg 158, and Lys 39 or equivalent to account for minor variations and deletions between strains. For peptides derived from or with similarity to RgpA propeptide or fragment thereof, substitution or modification can be at any one or more of Arg 103, Lys 98, Lys 122, Arg 67, Arg 159, and Lys 40 or equivalent to account for minor variations and deletions between strains. The peptides may include an amino acid sequence that does not contain a lysine residue at any one or more positions equivalent to 4, 41, 69, 100, 110, 129, 168 or 204 in SEQ ID NO: 1. The peptides may include an amino acid sequence that does not contain a lysine or arginine residue at any one or more positions equivalent to Lys 39, Arg 66, Lys 97, Arg 102, Lys 121 and Arg 159 in SEQ ID NO: 2. The peptides may include an amino acid sequence that does not contain a lysine or arginine residue at any one or more positions equivalent to Lys 39, Arg 66, Lys 97, Arg 102, Lys 121 and Arg 159 in SEQ ID NO: 3.

A peptide or peptidomimetic of the invention includes an isolated, purified or recombinant amino acid sequence of a propeptide or fragment thereof as it would occur naturally when part of the cognate gingipain. In other embodiments, the peptide or peptidomimetic of the invention may include a synthetic amino acid sequence of a propeptide or fragment thereof, optionally with post-translational modifications.

In particular embodiments the peptide or peptidomimetic consists of or consists essentially of an amino acid sequence selected from the group consisting of any one of SEQ ID NOS: 1 to 10 inclusive.

In particular embodiments the peptide or peptidomimetic consists of or consists essentially of an amino acid sequence selected from the group consisting of any one of SEQ ID NOS: 1, 4, 5 or 10 (shown in Figure 1) wherein the amino acid residue at the position equivalent to position 16 in SEQ ID NO: 1 is not a cysteine.

In other embodiments, a peptide or peptidomimetic of the invention comprises an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 28. Preferably, the group consisting of SEQ ID Nos: 1 to 10, even more preferably the group consists of SEQ ID Nos: 1 to 3. Preferably, the amino acid sequence does not contain any cysteine residues or at least not a cysteine residue at the position equivalent to position 16 in SEQ ID NO: 1.

In other embodiments, a peptide or peptidomimetic of the invention comprises an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1, 4, 5 and 10, wherein the amino acid sequence does not contain any cysteine residues or at least not a cysteine residue at the position equivalent to position 16 in SEQ ID NO: 1.

In other embodiments, a peptide or peptidomimetic of the invention consists of or consisting essentially of an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 28. In these embodiments, a compound, peptide or peptidomimetic that includes SEQ ID NOs: 1 to 28 as well as additional amino acid residues would "consist essentially of SEQ ID NOs: 1 to 28 as long as it exhibits activity for inhibiting, reducing or preventing the activity of a bacterial enzyme, as may be determined in accordance with the assays described below. Similarly, a compound, peptide or peptidomimetic "consists essentially of one of SEQ ID NO: 1 to 28 where it is shorter than the corresponding SEQ ID as long as it exhibits activity for inhibiting, reducing or preventing the activity of a bacterial enzyme, as may be determined in accordance with the assays described below. These embodiments thus do not include a full-length gingipain sequence. Preferably, a compound, peptide or peptidomimetic of the invention consists of or consisting essentially of an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 10. Even more preferably the group consists of SEQ ID Nos: 1 to 3. Preferably, the amino acid sequence does not contain any cysteine residues or at least not a cysteine residue at the position equivalent to position 16 in SEQ ID NO: 1.

The invention extends to any peptide or peptidomimetic as described herein wherein the amino acid residue at the position equivalent to position 16 in SEQ ID NO: 1 is not a cysteine.

The invention also provides a cysteine-free propeptide comprising, consisting essentially of or consisting of an amino acid sequence of a Kgp propeptide or fragment thereof. In one embodiment, a peptide or peptidomimetic of the invention comprises, consists essentially of or consists of an amino acid sequence that is at least 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1, 4, 5 and 10, wherein the amino acid sequence does not contain any cysteine residues or at least not a cysteine residue at the position equivalent to position 16 in SEQ ID NO: 1. Preferably, the cysteine-free propeptide includes an amino acid sequence that does not contain a lysine residue at any one or more positions equivalent to 4, 41, 69, 100, 110, 129, 168 or 204 in SEQ ID NO: 1.

A peptide or peptidomimetic of the invention comprises, consists essentially of or consists of the inhibitory loop of the naturally occurring propeptide or a functionally similar sequence. In one embodiment the sequence is Lys113- Glu128 of RgpB or a functionally similar sequence having a sequence identity of at least about 60, 70, 80 or 90%.

A peptide or peptidomimetic of the invention comprises, consists essentially of or consists of a gingipain propeptide amino acid sequence of 1) a peptide corresponding to an amino acid sequence equivalent to Lys113- Glu128 of RgpB, or 2) a peptide corresponding to an amino acid sequence of a Kgp propeptide that is equivalent to Lys113- Glu128 of RgpB. In one embodiment, the gingipain propeptide amino acid sequence is the sequence shown in any one of the Figures as the inhibitory loop or a functionally similar sequence. Preferably, the inhibitory loop is as shown in Figure 25A.

The invention also includes a peptide or peptidomimetic comprising, consisting essentially of or consisting of an amino acid sequence corresponding to the inhibitory lysine of a Kgp propeptide and the 5 amino acids N -terminal and C -terminal to that lysine. Preferably the lysine is, or equivalent to, the lysine at position 110 in Kgp. More preferably, the amino acid sequence is as shown in SEQ ID NO: 29 or a functionally similar sequence having a sequence identity of at least about 60, 70, 80, 90, 91 , 92, 93, 94,95, 96, 97, 98 or 99% to SEQ ID NO: 29.

The invention also includes a peptide or peptidomimetic comprising, consisting essentially of or consisting of an amino acid sequence corresponding to the inhibitory arginine of an Rgp propeptide and the 5 amino acids N -terminal and C -terminal to that arginine. Preferably the arginine is, or equivalent to, the arginine at position 102 in RgpB or 103 in RgpA. More preferably, the amino acid sequence is as shown in SEQ ID NO: 30 or 31 or a functionally similar sequence having a sequence identity of at least about 60, 70, 80, 90, 91 , 92, 93, 94,95, 96, 97, 98 or 99% to SEQ ID NO: 30 or 31.

A 'compound' of the invention is a compound identified as an inhibitor by an assay described herein. A compound may be a protein (such as an antibody or fragment thereof or an antibody mimetic), peptide, nucleic acid (including RNA, DNA, antisense oligonucleotide, peptide nucleic add), carbohydrate, organic compound, small molecule, natural product, library extract or from a bodily fluid.

In some embodiments, a compound, peptide or peptidomimetic of the invention has an amino acid length of between about 10 to about 300. In other embodiments, the length is between about 20 and 205 or about 50 and about 210. In other embodiments, the length is about 100 to about 200 amino acids.

Porphyromonas gingivalis is an example of gram negative bacteria that has evolved to grow under protein-rich, anaerobic conditions. The genomes of several other bacteria and archaea that exist in either protein-rich, anaerobic or more extreme conditions have recently been sequenced; some of these species have yet to be grown in vitro. These genomic studies have provided evidence for proteins that have sequence similarities with the gingipains and significant sequence similarities with the propeptides of the gingipains. Except in the case of Desulfatibacillum alkenivorans AK-01 the significant sequence similarities with the gingipain propeptide are found in the N-terminal regions as expected for propeptides.

These bacteria include: Candidates Cloacamonas acidaminovorans, a syntrophic bacterium that is present in many anaerobic digesters; Candidatus Kuenenia stuttgartiensis. an ammonium oxidising bacteria; Chloroherpeton thalassium, a non- filamentous, flexing and gliding green sulfur bacterium that is an obligate phototroph; Desulfatibactltum alkenivorans AK-01 , a mesophilic sulfate-reducer isolated from estuarine sediment that utilizes C13 to C18 alkanes, -alkenes (C15 and C16) and 1- alkanols (C15 and C16) as growth substrates; Desulfococcus oieovorans (strain DSM 6200/Hxd3) an alkane-degrading sulfate-reducing bacterium isolated from the saline water phase of an oil-water separator from a northern German oil field (Hxd3 is a delta- proteobacterium that is able to grow anaerobically on C12 to C20 alkanes) and Photobacterium profundum, which is classified as a piezophile, because it lives under high pressure, having been isolated at a depth of 2500 m.

Two species from the superkingdom archea have also revealed sequences that show significant similarity with the gingipain propeptide. Methanosaeta thermophHa is an anaerobic thermophilic obigately aceticlastic methanogen isolated from flooded rice paddies and sewage digesters. Aciduliprofundum boonei is a cultivated obligate thermoacidophilic euryarchaeote from deep-sea hydrothermal vents.

Propeptides from these bacteria that show similarity with gingipain propeptides of SEQ ID NO: 1 to 10 are within the scope of the invention. Examples of such peptides are, but not limited to, those which have sequences of SEQ ID NO: 11 to 28 (shown in Figure 2).

In certain embodiments there is provided a composition for inhibiting a bacterial enzyme comprising a compound, peptide or peptidomimetic of the invention and a pharmaceutically acceptable carrier. The composition can further include a divalent cation.

A composition of the invention may include propeptides with different amino acid sequences such that the composition inhibits more than one type of bacterial enzyme. For example, a composition of the invention may include two more propeptides that each exhibit selectivity for a specific gingipain, e.g. RgpA or RgpB and Kgp. in one embodiment, a composition of the invention includes propeptides having the sequence of any one or more of SEQ ID NO: 1 to 28, preferably SEQ ID NO: 1 to 10. For example, some of the propeptides in the composition may have an amino acid sequence with identity to a propeptide derived from a Kgp, while the remainder of the propeptides in the composition may have an amino acid sequence with identity to a propeptide derived from a Rgp. The level of sequence identity has already been referred to herein. A composition of the invention includes a gingipain propeptide or fragment thereof that has been purified or enriched from a biological tissue or fluid. The gingipain propeptide or fragment thereof that has been purified or enriched from a biological tissue or fluid is modified such that it cannot form a homodimer or heterodimer.

In one embodiment, there is provided a method for treating or preventing one or more of the conditions described herein comprising administering to a subject an effective amount of compound, peptide, peptidomimetic or composition of the invention. In one embodiment, the compound, peptide, peptidomimetic or composition is administered directly to the gums of the subject.

In another embodiment a method of the invention further comprises administering an agent selected from the group consisting of anti-inflammatory agents, antibiotics and antibiofilm agents. The antibiotic may be selected from the group consisting of amoxicillin, doxycycline and metronidazole. Anti-inflammatory agents include Nonsteroidal Anti-inflammatory Drugs (NSAIDs). Examples of NSAIDs include compounds than inhibit a cyclooxygenase. Specific examples of NSAIDs include aspirin, ibuprofen and naproxen.

In another embodiment there is provided a method for treating or alleviating a symptom of periodontal disease in a subject, the method comprising administering to the subject a compound, peptide, peptidomimetic or composition of the invention. In another embodiment the method further includes administering a protein for inducing an immune response to bacteria involved in periodontal disease initiation or progression. In one embodiment, the bacteria are P. gingivalis.

In another embodiment there is provided a method of preventing, treating or alleviating a symptom of chronic periodontitis in a subject, the method comprising administering to the subject a compound, peptide, peptidomimetic or composition of the invention. The chronic periodontits is typically associated with P. gingivalis. In another embodiment the method further includes administering a protein for inducing an immune response to bacteria involved in periodontal disease initiation or progression. In one embodiment, the bacteria are P. gingivalis,

In another embodiment the invention provides a use of an effective amount of a compound, peptide, peptidomimetic or composition of the invention in the preparation of a medicament for the treatment or prevention of periodontal disease and/or the other conditions identified herein as suitable for treatment.

The present invention also provides a pharmaceutical composition for the treatment or prevention of periodontal disease (and/or the other conditions identified above as suitable for treatment) comprising an effective amount of a compound, peptide or peptidomimetic of the invention and a pharmaceutically acceptable carrier. The composition may further include an agent selected from the group consisting of antiinflammatory agents, antibiotics and antibiofilm agents. The antibiotic may be selected from the group consisting of amoxicillin, doxycycline and metronidazole.

In another embodiment the invention provides a composition for the treatment or prevention of periodontal disease (and/or the other conditions identified above as suitable for treatment) comprising as an active ingredient a compound, peptide or peptidomimetic of the invention. The composition can further include a divalent cation.

In another embodiment the invention provides a pharmaceutical composition comprising an effective amount of a compound, peptide or peptidomimetic of the invention as a main ingredient. The composition may be used for example for the treatment or prevention of periodontal disease and/or the other conditions identified herein as suitable for treatment. Preferably, the composition further comprises a divalent cation.

In another embodiment the invention provides a compound, peptide or peptidomimetic of the invention for use in the treatment or prevention of periodontal disease and/or the other conditions identified herein as suitable for treatment.

In another embodiment the invention provides a composition comprising a compound, peptide or peptidomimetic of the invention for use in the treatment or prevention of periodontal disease. Preferably, the composition further comprises a divalent cation. The divalent cation is preferably selected from the group consisting of Zn ²⁺ , Ca ²⁺ . Cu ²⁺ , Ni ² *, Co ² *, Fe ² *, Sn ² *, and Mn ² *. In addition, the divalent cation may be in association with fluoride such as SnF* and CuF*. It is currently preferred, however, that the divalent cation is Ca ² * or Zn ² *.

It is further preferred that the ratio of the divalent cation to the peptide is in the range of 1.0:2.0 to 1.0: 10.0, preferably in the range of 1.0:4.0. The present invention also provides an assay for identifying an inhibitor of a cysteine protease comprising the steps of:

- contacting a cysteine protease with a candidate compound in the presence of a compound, peptide or peptidomimetic of the invention, - determining whether the candidate compound competes with the compound, peptide or peptidomimetic of the invention;

wherein competition indicates that the candidate compound is an inhibitor of a cysteine protease.

The present invention also provides an assay for identifying an inhibitor of a cysteine protease comprising the steps of:

- contacting a cysteine protease with a compound, peptide or peptidomimetic of the invention in the presence or absence of a candidate compound,

- determining the level of the compound, peptide or peptidomimetic bound to the protease, wherein a reduction in the level of the compound, peptide or peptidomimetic in the presence of the candidate compound compared to the absence of the candidate compound thereby identifies the candidate compound as an inhibitor of a cysteine protease.

The present invention also provides an assay for identifying an inhibitor of a cysteine protease comprising the steps of:

- contacting a cysteine protease with a candidate compound in the presence or absence of a compound, peptide or peptidomimetic of the invention,

- determining the level of the candidate compound bound to the protease, wherein a reduction in the level of the candidate compound in the presence of the compound, peptide or peptidomimetic compared to the absence of the compound, peptide or peptidomimetic thereby identifies the candidate compound as an inhibitor of a cysteine protease.

The present invention also provides an assay for identifying an inhibitor of a cysteine protease comprising the steps of: - providing a compound, peptide or peptidomimetic of the invention, in the presence or absence of a candidate compound, in conditions that allow binding of the compound, peptide or peptidomimetic of the invention to a cysteine protease,

- determining the level of the compound, peptide or peptidomimetic bound to the protease,

wherein a reduction in the level of the compound, peptide or peptidomimetic in the presence of the candidate compound compared to the absence of the candidate compound thereby identifies the candidate compound as an inhibitor of a cysteine protease. The present invention also provides an assay for identifying an inhibitor of a cysteine protease comprising the steps of:

- providing a candidate compound, in the presence or absence of a compound, peptide or peptidomimetic of the invention, in conditions that allow binding of the candidate compound to a cysteine protease,

- determining the level of the candidate compound bound to the protease,

wherein a reduction in the level of the candidate compound in the presence of the compound, peptide or peptidomimetic compared to the absence of the compound, peptide or peptidomimetic thereby identifies the candidate compound as an inhibitor of a cysteine protease.

Preferably, the candidate compound identified as an inhibitor of a cysteine protease is assayed one or more times in accordance with the steps described herein with a further cysteine protease and the same or a further compound, peptide or peptidomimetic of the invention to determine whether the candidate compound inhibits one or more cysteine proteases.

Preferably the candidate compound is an antibody or fragment thereof, or an antibody mimetic such as an anticalin. The candidate compound may be part of a library in which case the assay is performed in high-throughput.

Preferably, the cysteine protease is a gingipain, more preferably Kgp, RgpA or RgpB. Even more preferably the gingpain is Kgp.

A compound, peptide or peptidomimetic of the invention useful in an assay of the invention has already been defined herein. Preferably, the compound, peptide or peptidomimetic of the invention comprises an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 98, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 28. Preferably, the group consisting of SEQ ID Nos: 1 to 10, even more preferably the group consists of SEQ ID Nos: 1 to 3. In other embodiments, a compound, peptide or peptidomimetic of the invention consists of or consisting essentially of an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 28. Preferably, the compound, peptide or peptidomimetic of the invention does not have the capacity to form a dimer. Preferably, the compound, peptide or peptidomimetic does not have the capacity to form a dimer via a disulphide bond. Typically, the compound, peptide or peptidomimetic of the invention does not contain a cysteine residue. The compound, peptide or peptidomimetic may include, consist essentially of or consist of the amino acid sequence is as shown in SEQ ID NO: 29 or a functionally similar sequence.

In one embodiment, the invention provides a compound, peptide or peptidomimetic of the invention for use in an assay of the invention. In one embodiment, the invention provides a compound, peptide or peptidomimetic of the invention when used in an assay of the invention. In one embodiment, the invention provides a compound, peptide or peptidomimetic labelled for use in an assay of the invention.

The invention also provides a recombinant or synthetic protein consisting of or consisting essentially of an amino acid sequence of the catalytic domain of Kgp. In other words, the protein consisting of an amino acid sequence of the catalytic domain of Kgp is not linked or does not interact with an adhesin domain.

In one embodiment, the invention provides a use of the recombinant or synthetic protein consisting of or consisting essentially of an amino acid sequence of the catalytic domain of Kgp or Rgp in an assay of the invention to identify an inhibitor of a cysteine protease, preferably Kgp or Rgp. In one embodiment, the invention provides the recombinant or synthetic protein consisting of or consisting essentially of an amino acid sequence of the catalytic domain of Kgp or Rgp when used in an assay of the invention to identify an inhibitor of a cysteine protease, preferably Kgp or Rgp.

The present invention also provides use of a compound identified by an assay described herein to inhibit a cysteine protease. Preferably, the cysteine protease is Kgp or Rgp. Even more preferably the cysteine protease is Kgp. In one embodiment the invention also provides use of a compound identified as an inhibitor by an assay described herein to treat or prevent periodontal disease.

The invention also provides a method of treating or preventing periodontal disease and/or the other conditions identified herein as suitable for treatment or prevention, including administering a peptide or peptidomimetic of the invention and/or a compound identified by an assay as described herein as an inhibitor of a cysteine protease.

As it is the physical nature of the peptides rather than the specific sequence of the peptide which results in their protease inhibitory activity so called conservative substitutions may be made in the peptide sequence with no substantial loss of activity. It is intended that such conservative substitutions which do not result in a substantial loss of activity are encompassed in the present invention. Any conservative substitutions can be made provided that the peptide does not have the capacity to form a dimer by a disulphide bond with another peptide. Whilst the concept of conservative substitution referred to above is well understood by the person skilled in the art, for the sake of clarity conservative substitutions are those set out below.

Gly, Ala, Val, He, Leu, Met;

Asp, Glu; Asn, Gin;

Ala, Ser, Thr

Lys, Arg, His;

Phe, Tyr, Trp, His; and

Pro, Na-alkalamino acids. As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Brief description of the drawings

Figure 1: Amino acid sequences of gingipain propeptides from various strains of Porphryomonas gingivalis. Figure 2: Amino acid sequences of propeptides from bacteria other than Porphryomonas gingivalis.

Figure 3: ion exchange chromatography of the desalted, acetone-precipitated proteins from the P. gingivalis KgpcatAABMl mutant ECR368 culture supernatant, using a Q- Sepharose column attached to an AKTA-Basic FPLC system. The column was eluted with 10 mM Sodium acetate pH 5.3 and then a linear gradient of 0-1 M NaCI in 10 mM sodium acetate was applied. The eluant was monitored at an absorbance of 280 nm. The collected fractions were measured for Lys- and Arg-specific proteolytic activity. The fractions containing Lys-activity were pooled and collected for further purification. Figure 4: Gel filtration purification of the concentrated samples of ECR368 culture supernatant after desalting using a Superdex G75 column attached to an AKTA-Basic FPLC system. The column was eluted with TC50 buffer, pH 8.0, 1.0 mL min. The eluant was monitored at an absorbance of 280 and 215 nm. Fractions A8-A9 contains active KgpcatAABMl Figure 5: SDS-PAGE of Kgpca _t AABMl enriched fraction from P. gingivalis ECR368. Lanes contain; lane 1: See-Blue® Pre-Stained standard, where sizes in kDa are indicated, lane 2: culture supernatant, lane 3: culture supernatant after acetone precipitation, lane 4: culture supernatant after acetone precipitation and ultracentrifugation, lane 5: KgpcatAABMl enriched fraction after gel filtration purification. The gel was Coomassie® stained.

Figure 6: (A) Gel filtration chromatography of the rKgp propeptide using Superdex G75 column equilibrated with 50 mM NH4HCO3 attached to an AKTA-Basic FPLC system. The eluant was monitored at an absorbance of 280 and 215 nm. (B) MALDI-TOF MS analysis of the rKgp propeptide with the His-tag still attached showed a singly-charged (m/z 25,446.9 [M*Hf ). a dual-charged (m/z 12728.8 [M*2H) ² *) and a triply charged (m/z 8486.5 [M*3H] ³ *) signal, each corresponding to the target molecular mass (25,285 Da).

Figure 7: KgpcatAABMl enriched fraction proteolytic activity (Units/mg) with 20.0 and 40.0 mg/L rKgp propeptide (rKgpPro) at 1 mM cysteine in the assay with the chromogenic GPKNa substrate. The final concentration of Kgpca _t AABMl enriched fraction per well is 1.16 mg/L. All samples were significantly different (p < 0.05) from the control. Figure 8: Proteolytic assay using chromogenic substrate GPKNa confirming that the rate of substrate hydrolysis was linear throughout the assay. Kgp _ca( AABM1 enriched fraction proteolytic activity (Units mg) with 0 mg L and 40.0 mg L rKgp propeptide (rKgpProK at 1 mM cysteine in the assay with the chromogenic

GPKNa substrate. The final concentration of Kgp _Mt AABM1 enriched fraction per well was 1.16 mg/L. The rate of substrate hydrolysis was linear throughout the assay.

Figure 9: RP-HPLC profile of the chromogenic assay (GPKNa) post-incubation mixtures applied to an analytical RP-HPLC column (C18) and eluted using a linear gradient of 0- 100% buffer B in 30 min at a flow rate of 1.0 mL/min. The eluant was detected at 214 nm. (A) Incubation mixture of the KgpcatAABMI enriched fraction without propeptide (B) Incubation mixture of the Kgp«, _t AAB 1 enriched fraction and rKgp propeptide.

Figure 10: Analysis of Lys-specific chromogenic assay (GPK-NA) products by SDS- PAGE. The following assay contents were electrophoresed: KgpcatAABMI enriched fraction with rKgp propeptide (rKgpPro) (lane 2). Lane 1 shows the molecular weight (MW) markers (See-Blue® Pre-Stained standard, lane 1), labelled in kDa. The gel was Coomassie® stained.

Figure 11. A secondary plot for the estimation of inhibition constant (Ki') of Kgpcat&ABM1 enriched fraction by rKgp propeptide. The V _max observed values were plotted against the inhibitor concentration. The Ki' for Kgp propeptide was calculated to be 2.01 μΜ.

Figure 12: Kgp proteolytic activity measured using fluorescent BSA substrate (DQ™ BSA) with 1, 5, and 10 mg L rKgp propeptide (rKgpPro). The final concentration of Kgp per well is 1.16 mg L. The fluorescence value for the negative control (TLCK 1 mM- treated proteases) was subtracted from each value. The error bars were calculated as a standard deviation of 3-6 replicates. All samples were significantly different (p < 0.05) from the control.

Figure 13: Analysis of fluorescent BSA assay products by SDS-PAGE. The gels were Coomassie® stained. (A) The following assay contents were electrophoresed: Kgpca _t AABMI enriched fraction (from control wells, lanes 2-3), KgpcatAABMI enriched fraction with rKgp propeptide (rKgpPro) (lanes 4-5). Lane 1 shows the molecular weight (MW) markers (See-Blue® Pre-Stained standard, lane 1), labelled in kDa. (B) Samples from the assay were electrophoresed as follows; KgpcatAABMI enriched fraction (Kgp) (from control wells, lanes 2-3), Kgp _cat AABM1 enriched fraction with rKgp propeptide (rKgpPro) (lanes 4-5), Kgp«,tAABM1 enriched fraction with TLCK (lanes 6-7). MW indicates molecular markers (See-Blue® Pre-Stained standard, lane 1), where sizes in kDa are indicated.

Figure 14: Time course of RgpB proteolytic activity using the DQ-BSA fluorescent substrate. Fluorescence was measured over 11 hours at 37 °C with a reading taken every hour.

Figure 15: RgpB proteolytic activity measured using fluorescent BSA substrate (DQ™ BSA) with 0.1, 1, 5, 10 mg/L rRgp propeptide (rRgp Pro). The final concentration of RgpB per well is 1.16 mg/L. The fluorescence value for the negative control (TLCK 1 mM -treated proteases) was subtracted from each value. The error bars were calculated as a standard deviation of 3-6 replicates. All samples were significantly different (p < 0.05) from the control except the values for 0.1 mg/L and different from other values except between the values for 5 and 10 mg L.

Figure 16: A secondary plot for the estimation of inhibition constant (Κί') of RgpB enriched fraction by RgpB propeptide. The V _max observed values were plotted against the inhibitor concentration. The Κ for RgpB propeptide was calculated to be 11.8 nM.

Figure 17: Relative growth of P. gingivals in a protein-based minimal medium in the presence of rRgpB propeptide (R-pp) and/or Kgp propeptide (K-pp).

Figure 18. Cluster W 2.0.8 multiple sequence alignment of the Kgp, RgpB and RgpA propeptides. The distributions of the lysines (K) and arginines (R) are shown in pink and red, shaded in grayscale, respectively.

Figure 19. Purification of Kgp propeptide. (A) Non-reducing SDS-PAGE of recombinant Kgp propeptide expressed in E coli. Lane 1: See-Blue ^® Pre-stained standard, where sizes in kDa are indicated; Lane 2: Flow through the Ni Column; Lane 3: Wash from Ni column; Lane 4: Thrombin cleaved product; Lane 5: Total thrombin-free Kgp propeptide extract prior to size exclusion chromatography. The gel was stained with Coomassse ^® Brilliant Blue (G250). (B) Size exclusion chromatogram (Superdex G75) of Kgp propeptide revealing dimerisation. (C) SDS-PAGE of the purified Kgp-propeptide monomer and dimer forms incubated with and without 5 mM DTT but without boiling revealing the disruption of the dimer with 5 m DTT. Figure 20. Proteolytic activity time course profiles of rKgp and RgpB using chromogenic and fluorescent substrates. (A) Time course of rKgp measured as change in absorbance (405 nm) without an inhibitor (·) and with 40 mg/L Kgp propeptide (Kgp- PP) with (□) and without the hexahistidine tag (Δ) at 1 mM cysteine in the assay with the Lys-specific chromogenic substrate (GPKNA). The final concentration of rKgp per well is 1.16 mg L. (B) Time course of RgpB using the fluorescent natural substrate DQ- BSA without RgpB-PP (■), with 10 mg/L RgpB-PP ( ) and 1 mM TLCK (□).

Figure 21. Inhibition of rKgp and RgpB by their propeptides. Assays were performed using the chromogenic substrates GPKNA (□) and BapNA (O). The % proteolytic activity was also determined using the fluorescent substrate DQ-BSA with rKgp (■) and RgpB (·).

Figure 22. Characterisation of propeptide inhibition. Secondary plot of the reciprocal of max against inhibitor concentration♦ RgpB and■ rKgp. The K values were obtained from the x-intercept. Figure 23. Processing of rKgp in vivo. (A) Reduced SDS-PAGE analysis of incompletely processed precursors observed in Day 1 and 3 culture supematants of P. gingivalis ECR368 that releases rKgp. (B) SDS-PAGE analysis of the -60 KDa precursor form of rKgp with and without DTT revealing a single band under reducing conditions and two bands under non-reducing conditions, highlighting the disulphide bridge (CYS— CYS) that forms between the N -terminal half of the propeptide and the mature protease as summarized in (C) Processing steps: The N-terminal half of the propeptide is represented by the white rectangle, the C-terminal half by the black rectangle and the mature Kgp by the hatched rectangle.

Figure 24. Current models of RgpB and Kgp. (A) Models of Kgp and RgpB based on the coordinates from PDB (lcvr.pdb), highlighting the cysteine residues. (B) Model of RgpB highlighting the His-Cys Co distances in the two caspase sub-domains.

Figure 25. Interaction of gingipain catalytic domains with their propeptides. (A) Model of RgpB highlighting the N -term in us, catalytic Cys and His residues, and residues that differ between strains in red. The residues that form a surface-exposed conserved patch are predicted to interact with the propeptide. (B) Schematic representation of the inhibition of Kgp by its propeptide. Kgp was modelled using Orchestrar from within Sy by 1-8.1 [55] and based on the X-ray crystal structure of RgpB lcvr.pdb [56]. The propeptide is based on the A chain of the X-ray crystal structure of RgpB interacting with its propeptide 4ief.pdb [49].

Figure 26. Amino acid sequences inhibitory loops from Kgp (SEQ ID NO: 29), RgpB (SEQ ID NO: 30) and RgpA (SEQ ID NO: 31).

Figure 27. Kgp protease activity in 10 mM cysteine measured using fluorescent BSA substrate(DQ™ -BSA) and chromogenic substrate GPKNa with 0, 100 mg/L Kgp-PP dimer and 100 mg/L 5 mM DTT-stabilized Kgp-PP monomer. The monomer was purified in the presence of DTT and assayed immediately after size exclusion chromatography in the presence of 5m M DTT.

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All of the patents and publications referred to herein are incorporated by reference in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

Compounds, peptide or peptidomimetics that exhibit protease inhibitory activity have the potential to be developed in the area of oral care, functional foods, and pharmaceuticals. The present invention includes peptides that are characterized by the ability to inhibit extracellular protease activity. These peptides may be produced synthetically or expressed recombinantly. These peptides have several advantages including, but not limited to, that they are non-toxic, biocompatible and are derived from the cognate zymogen.

The present inventors have identified a problem with compounds, peptides or peptidomimetics that contain amino acid sequences from gingipain propeptides. Specifically, propeptide sequences that contain cysteine residues have been determined to form dimers via disulphide bonds which reduce the ability to inhibit, reduce or prevent a bacterial enzyme activity. As used herein, a "propeptide" is a sequence of amino acids N-terminal to the catalytic domain which when cleaved from the gingipain, such that it is no longer linked to the gingipain, results in the marked increase in catalytic activity of the gingipain.

The invention also includes functional fragments of the amino acid sequences of SEQ ID NO: 1 to 28. A functional fragment is an amino acid sequence that is shorter than the amino acid sequences corresponding to SEQ ID NO: 1 to 28 but still retains the function of the corresponding amino acid sequences to SEQ ID NO: 1 to 28. A functional fragment can be easily determined by shortening the amino acid sequence, for example using an exopeptidase, or by sythesizing amino acid sequences of shorter length, and then testing for any protease inhibitory activity.

Also within the scope of the invention are variants of the amino acid sequences of SEQ ID NO: 1 to 3 corresponding to orthologous or paralogous sequences. Examples of such sequences include those shown in SEQ ID NOS 4 to 28.

It will be understood by a person skilled in the art that one or more amino acid deletions to the amino acid sequence defined by any one of SEQ ID Nos: 1 to 28 may be made without losing the capacity of the compound, peptide or peptidomimetic to inhibit, reduce or prevent protease activity. Experiments, including those described herein, can be performed to determined whether a compound, peptide or peptidomimetic that has an amino acid sequence that differs to any one of SEQ ID Nos: 1 to 28 by one or more amino acid deletions can still inhibit, reduce or prevent protease activity.

As used herein "an amino acid residue at the position equivalent to position 16 in SEQ ID NO: 1" can be determined by any means known to a person skilled in the art. For example, an alignment of one or more sequences with an amino acid sequence of SEQ ID NO: 1 would allow a person skilled in the art to determine the amino acid at the position equivalent to position 16 in SEQ ID NO: 1. A person skilled in the art can compare the three dimensional structure of a peptide or peptidomimetic with the three dimensional structure of a peptide having the amino acid sequence of SEQ ID NO: 1 and determine the amino acid residue that is at an equivalent position to position 16 in SEQ ID NO: 1.

The peptides of the invention that cannot form a dimer, either a heterodimer or homodimer, due to a change in an amino acid or modification of an amino acid side chain as described further herein. In these circumstances, the peptides are not naturally occurring as they do not have the same amino acid sequence as a naturally occurring propeptide or are chemically modified to have a different chemical composition.

A compound, peptide or peptidomimetic of the invention does not have the capacity to form a dimer via a disulphide bond by removal of all cysteine residues or via chemical modification of the existing cysteine residues such that a disulphide bond cannot form with another peptide or peptidomimetic. Chemical modification may be by iodoacetylation. Other irreversible cysteine modifications that would be suitable include (1) oxidation to sulfinic acid (Cys-S0 ₂ H), a stable intermediate that rapidly oxidises to sulfonic acid (Cys-S0 ₃ H) - strong oxidizing agents such as halogens, hydrogen peroxide, and nitric acid can generate sulfonic acids from thiols, and (2) alkylation (methylation, acetylation) using thiol alkylating reagents N-Ethylmaleimide (NEM) or iodoacetamide (IAM).

In one embodiment, there is provided an isolated, recombinant, synthetic or purified compound, peptide or peptidomimetic that comprises, consists essentially of or consists of an amino acid sequence of having at least 80% identity with any one of SEQ ID NO: 1 , 4, 5 or 10, wherein the amino acid sequence contains a modified cysteine residue at a position equivalent to position 16 in SEQ ID NO: 1 that is modified such that it cannot form a disulphide bond. A compound, peptide or peptidomimetic of the invention may be prepared recombinantly or synthesized to comprise, consist essentially of, or consist of an amino acid sequence that is identical to a naturally occurring Kgp propeptide wherein one or more cysteine residues have been changed to an another amino acid, such that the compound, peptide or peptidomimetic cannot form a dimer. A compound, peptide or peptidomimetic of the invention may comprise, consist essentially of, or consist of an amino acid sequence that has 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a naturally occurring Kgp propeptide wherein one or more cysteine residues have been changed to an another amino acid, such that the compound, peptide or peptidom imetic cannot form a dimer.

The compound, peptide, peptidomimetic or composition of the invention may be administered directly to the gums of the subject in need of treatment or prevention of periodontal disease. Topical administration of the composition of the invention is preferred, however it will be appreciated by a person skilled in the art that a compound, peptide, peptidomimetic or composition may also be administered parenteral^, e. g, by injection intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously.

In one embodiment the compound, peptide or peptidomimetic may be a part of a composition applicable to the mouth such as dentifrice including toothpastes, toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums, dental pastes, gingival massage creams, gargle tablets, dairy products and other foodstuffs.

Alternatively, the compound, peptide, peptidomimetic of the invention may be formulated as a composition for oral administration (including sublingual and buccal), pulmonary administration (intranasal and inhalation), transdermal administration, and rectal administration.

Inhibition of an enzyme may be competitive or non-competitive. Without wishing to be bound by any theory or mode of action, it is believed that propeptides are noncompetitive inhibitors that do not compete with substrate for binding to the catalytic site of a target enzyme. It is believed that propeptides binds to the enzyme at a site other than the catalytic site.

A composition of the invention may include a peptide or peptidomimetic of the invention and a compound identified as an inhibitor of the catalytic site of a bacterial enzyme, such as a cysteine protease. Preferably, the cysteine protease is a gingipain, such as a Kgp or Rgp. In one embodiment, the composition includes a peptide or peptidomimetic of the invention and a compound identified as a competitive inhibitor of the same enzyme which the peptide or peptidomimetic of the invention inhibits.

Although the invention finds application in humans, the invention is also useful for veterinary purposes. The invention is useful for domestic or farm animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals.

A subject in need of treatment may be one which exhibits subclinical or clinical symptoms of periodontal disease. The subject may exhibit subclinical or clinical symptoms of chronic periodontitis. Subclinical or clinical manifestations of periodontal disease include acute or chronic inflammation of the gingiva. The hallmarks of acute inflammation may be present including an increased movement of plasma and leukocytes from the blood into the injured tissues. Clinical signs of acute infection of the gingiva may also be present including rubor (redness), calor (increased heat), tumor (swelling), dolor (pain), and functio laesa (loss of function). Chronic inflammation may be characterised by leukocyte cell (monocytes, macrophages, lymphocytes, plasma cells) infiltration. Tissue and bone loss may be observed. A subject in need of treatment may also be characterised by having an increased level of P. gingivalis bacteria present at a periodontal site, above a normal range observed in individuals without periodontal disease.

The route of administration may depend on a number of factors including the nature of the compound, peptide, peptidomimetic or composition to be administered and the severity of the subject's condition. It is understood that the frequency of administration of a compound, peptide, peptidomimetic or composition of the invention and the amount of compound, peptide, peptidomimetic or composition of the invention administered may be varied from subject to subject depending on, amongst other things, the stage of periodontal disease initiation or progression in the subject. The frequency of administration may be determined by a clinician.

It is also contemplated that any disease, condition or syndrome that is a consequence of or associated with protease activity of a gingipain or related protease, may be prevented or treated by a compound, peptide, peptidomimetic or composition of the invention. In addition, a symptom of a disease, condition or syndrome that is a consequence of or associated with protease activity of a gingipain or related protease, may be reduced in severity or incidence by a compound, peptide, peptidomimetic or composition of the invention. Further more, other diseases, conditions or syndromes that are a consequence of or associated with periodontal disease may also be treated or the risk of developing these diseases, conditions or syndromes may be reduced. For example, periodontal disease may increase the risk of an individual developing cardiovascular disease. This increase risk of developing cardiovascular disease may be reduced by treating periodontal disease by administering a compound, peptide, peptidomimetic or composition of the invention to an individual with periodontal disease.

A representative assay to identify an inhibitor of a cysteine protease is a "competitive binding assay" or "competition binding assay". Competitive binding assays are serological assays in which unknowns (e.g. candidate compounds) are detected and quantitated by their ability to inhibit the binding of a labelled known compound to its specific target. The labelled known compound used herein may be a compound, peptide or peptidomimetic of the invention which when employed in such immunoassays may be labelled or unlabeled. A labelled compound, peptide or peptidomimetic may be employed in a wide variety of assays, employing a wide variety of labels. Detection of the formation of a compound-target complex between a compound, peptide or peptidomimetic of the invention and a cysteine protease can be facilitated by attaching a detectable substance to the compound, peptide or peptidomimetic. Suitable detection means include the use of labels such as radionucleotides, enzymes, coenzymes, fluorescers, chemi luminesces chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Such labelled reagents may be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and the like. See, for example, U.S. Patent Nos. 3,766,162; 3,791 ,932; 3,817,837; and 4,233,402.

Competition assays are known in the art. Competitive assays are widely used for different purposes such as agonist/antagonist interactions with a receptor or for concentration analysis for a drug of interest. In one example, an affinity-purified capture antibody pre-coated onto a microplate is used, to which a limited concentration of enzyme-linked analyte along with the non-labeled sample anaiyte are added simultaneously. Both analytes will then compete for the limited number of binding sites on the primary antibody. Substrate is added and hydrolyzed by the enzyme, thereby producing a color product that can be measured (exactly like an ELISA). The amount of labeled analyte bound is inversely proportional to the amount of unlabeled analyte presenting the sample (signal decreases as analyte concentration increases).

The candidate compound can be any compound which one wishes to test including, but not limited to, proteins (such as antibodies or fragments thereof or antibody mimetics), peptides, nucleic acids (including RNA, DNA, antisense oligonucleotide, peptide nucleic acids), carbohydrates, organic compounds, small molecules, natural products, library extracts, bodily fluids. The candidate compound may be part of a library, for example a collection of compounds containing variations or modifications.

Non-limiting examples of antibody mimetics or alternate immunoglobulin molecules include those described by Dimitrov, 2009, MAbs 1 26-28; whilst examples of non- immunoglobulin protein scaffolds are described in Skerra, 2007 Current Opinions in Biotechnology, 18295-304.

Anticalins are proteins that are not structurally related to antibodies but are a class of antibody mimetics. Anticalins are derived from human lipocalins which are a family of binding proteins. Anticalins are about eight times smaller than antibodies with a size of about 180 amino acids and a mass of about 20 kDa.

Anticalins have better tissue penetration than antibodies and are stable at temperatures up to 70 °C. Unlike antibodies, they can be produced in bacterial cells like E. colt in large amounts.

The assay methods of the invention include high- throughput screening applications. For example, a high-throughput screening assay may be used which comprises any of the assays according to the invention wherein aliquots of cysteine proteases are exposed to a plurality of candidate compounds within different wells of a multi-well plate. Further, a high-throughput screening assay according to the disclosure involves aliquots of cysteine protease which are exposed to a plurality of candidate compounds in a miniaturized assay system of any kind.

The method of the disclosure may be "miniaturized" in an assay system through any acceptable method of miniaturization, including but not limited to multi-well plates, such as 24, 48, 96 or 384-wells per plate, microchips or slides. The assay may be reduced in size to be conducted on a micro-chip support, advantageously involving smaller amounts of reagent and other materials. Any miniaturization of the process which is conducive to high-throughput screening is within the scope of the invention.

Prior to the present invention, there was no assay available which reliably identified an inhibitor of a cysteine protease. The work of the present inventors led to the production of isolated gingipain propeptides that retain the ability to inhibit gingipain activity. These propeptides can be used in assays to direct the identification of compounds that not only bind to a cysteine protease but also inhibit cysteine protease activity. These identified inhibitors of cysteine protease activity can then be used in a clinical setting to treat diseases that are involved with the activity of a cysteine protease. Alternatively, the identified inhibitor may be subject to optimisation such that its affinity and/or inhibitory activity for a cysteine protease is increased.

The assay of the invention also allows the identification of inhibitors that have inhibitory activity towards a specific type of cysteine protease. A candidate compound may be assayed repeatedly in the presence of different cysteine proteases to determine whether the candidate compound inhibits only one type of cysteine protease or have inhibitory activity towards more than one type of cysteine protease. For example, either a Kgp or a Kgp-like gingipain, or alternatively, inhibit a Rgp or Rgp-like gingipain.

The concentration of labeled compound, peptide or peptidomimetic of the invention bound to the cysteine protease is inversely proportional to the ability of the candidate compound to compete in the binding assay. Conversely, if the candidate compound is labelled then the ability of a compound, peptide or peptidomimetic of the invention to compete in the binding assay indicates that the candidate compound binds to a similar region of the cysteine protease as a gingipain propeptide.

A variety of other reagents may also be included in the screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc. that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between about 0 and about 40 °C, preferably, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 ^e C. Incubation periods are selected from about 0.05 to about 10 hours. Preferably the incubation period allows the molecular interactions occurring within the assay to reach equilibrium.

The present invention also provides methods for making a peptide of the invention. In one preferred embodiment, the method comprises the following steps:

(1) expressing a nucleic acid which encodes for a peptide according to any one of SEQ ID No: 1 to 28, in an appropriate prokaryotic or eukaryotic expression system; and

(2) isolating or purifying the expressed peptide.

Preferably, the method further includes the preceding step of generating a nucleic acid which encodes for a peptide of the invention (for example any one of SEQ ID No: 1 to 28), the nucleic acid being modified so as to inactivate a known or predicted cleavage site in the peptide. Lysine and arginine residues are the expected autocleavage sites during the processing of the mature gingipains, hence amino acid sequences and nucleic sequences that encode them which have these or other cleavage sites replaced by amino acids which inhibit or reduce cleavage, for example glutamines and asparagines, are within the scope of the invention.

Expression systems are well known in the molecular biology art as are methods for isolation and purification of expressed proteins.

The nucleic acid molecule encoding a peptide of the invention may, for example, be inserted into a suitable expression vector for production of the peptide by insertion of the expression vector into a prokaryotic or eukaryotic host cell. Successful expression of the recombinant peptide requires that the expression vector contains the necessary regulatory elements for transcription and translation which are compatible with, and recognised by the particular host cell system used for expression. A variety of host cell systems may be utilized to express the recombinant protein, which include, but are not limited to bacteria transformed with a bacteriophage vector, plasmid vector, or cosmid DNA; yeast containing yeast vectors; fungi containing fungal vectors; insect cell lines infected with virus (e.g. baculovirus); and mammalian cell lines transfected with plasmid or viral expression vectors, or infected with recombinant virus (e.g. vaccinia virus, adenovirus, adeno-associated virus, retrovirus, etc).

Using methods known in the art of molecular biology, various promoters and enhancers can be incorporated into the expression vector, to increase the expression of the recombinant peptide, provided that the increased expression of the amino acid sequences is compatible with (for example, non-toxic to) the particular host cell system used.

The selection of the promoter will depend on the expression system used. Promoters vary in strength, i.e. ability to facilitate transcription. Generally, it is desirable to use a strong promoter in order to obtain a high level of transcription of the coding nucleotide sequence and expression into recombinant protein. For example, bacterial, phage, or plasm id promoters known in the art from which a high level of transcription have been observed in a host cell system including £ coli include the lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters, lacUVS, ompF, bla, Ipp, and the like, may be used to provide transcription of the inserted nucleotide sequence encoding amino acid sequences.

Other control elements for efficient transcription or translation include enhancers, and regulatory signals. Enhancer sequences are DNA elements that appear to increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect to a nearby coding nucleotide sequence. Thus, depending on the host cell expression vector system used, an enhancer may be placed either upstream or downstream from the inserted coding sequences to increase transcriptional efficiency. Other regulatory sites, such as transcription or translation initiation signals, can be used to regulate the expression of the coding sequence.

In another embodiment, the expression plasmids may optionally contain tags allowing for convenient isolation and/or purification of the expressed proteins. The use of expression plasmids and the methods for isolating and purifying the tagged protein products are well known in the art.

Peptides of the invention can be produced by a variety of known techniques. For example such peptides or fragments thereof can be synthesized (eg chemically or recornbinantly), isolated, purified and tested for their ability to form complexes with mature gingipains using methods described herein or methods known in the art. Alternatively, peptides or fragments thereof may be recornbinantly produced using various expression systems (eg E coli, Chinese Hamster Ovary cells, COS cells baculovirus) as is well known in the art. A peptide of the invention may also be produced by digestion of naturally occurring or recornbinantly produced gingipain propeptide or gingipain precursors using for example a protease (eg trypsin, chymotrypsin). Computer analysis can be used to identify proteolytic cleavage sites. Alternatively peptides may be produced from naturally occurring or recombinant^ produced gingipain propeptide or gingipain precursors using such standard techniques in the art as by chemical cleavage (eg cyanogen bromide, hydroxylamine, formic acid).

A compound, peptide or peptidomimetic of the invention may comprise as many amino acids as are necessary to bind to the target protease, thereby inhibiting partially or completely protease activity. In one embodiment, the target protease in a gingipain and partial or complete inhibition of gingipain activity can be demonstrated in assays involving P. gingivalis whole cells or harvested outer membrane complex or purified gingipains.

A compound, peptide or peptidomimetic having a sequence of SEQ ID NO: 1 to 28, or compound, peptide or peptidomimetic does not have the capacity to form a dimer, may also have point mutations or other modifications introduced (including insertion, deletion and substitution) to improve a biochemical property, for example to enhance the activity or circulatory or storage half-life. In addition, as discussed further herein point mutations may be introduced into one or more proteolytic cleavage sites to prevent or inhibit proteolytic degradation of the compound, peptide or peptidomimetic in vivo. All variants discussed herein are within the scope of the invention provided such variants maintain the ability to inhibit, reduce or prevent the activity of a bacterial enzyme.

Accordingly, nucleic acids of the invention include, in addition to those encoding SEQ ID NO: 1 to 28 and encoding peptides that does not have the capacity to form a dimer, nucleic acids which differ in nucleotide sequence by allelic variations (naturally- occurring base changes in the species population which may or may not result in an amino acid change). The invention also includes nucleic acid sequence caused by point mutations or by induced modifications (e.g., insertion, deletion, and substitution) to enhance the activity, half-life or production of the gingipain propeptides encoded are also useful for the present invention. Computer programs that are used to determine DNA sequence homology are known in the art.

A 'peptidomimetic' is a synthetic chemical compound that has substantially the same structure and/or functional characteristics of a peptide of the invention, the latter being described further herein. Typically, a peptidomimetic has the same or similar structure as a peptide of the invention, for example the same or similar sequence of SEQ ID NO: 1 to 28 or fragment thereof, or a peptide as described herein that does not have the capacity to form a dimer. A peptidomimetic generally contains at least one residue that is not naturally synthesised. Non-natural components of peptidomimetic compounds may be according to one or more of: a) residue linkage groups other than the natural amide bond ('peptide bond') linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e , to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.

Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literatures, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymot.267:220-234.

The compounds, peptides or peptidomimetics of the invention can be administered in the form of a pharmaceutical composition. These compositions may be manufactured under GMP conditions or in some embodiments by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The ingredients may facilitate processing peptides or peptidomimetics into preparations which can be used pharmaceutically.

Administration for treatment can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular.

Pharmaceutical compositions for parenteral administration are generally sterile and substantially isotonic. Physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer may be used. The solution may also contain suspending, stabilizing and or dispersing agents. The peptides or peptidomimetics may be provided in powder form to be dissolved in solvent such as sterile pyrogen-free water, before use. "Percent (%) amino acid sequence identity" or " percent (%) identical" with respect to a peptide or polypeptide sequence, i.e. a peptide of the invention defined herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, i.e. a peptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples described below) needed to achieve maximal alignment over the full-length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity = X Y100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.

In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1 97) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22.4673- 4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non- limiting examples of a software program useful for analysis of ClustalW alignments is GENEDOC™ or JalView (http://www.jalview.org ). GENEDOC™ allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

An oral composition of this invention which contains the above-mentioned pharmaceutical composition can be prepared and used in various forms applicable to the mouth such as dentifrice including toothpastes, toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums, dental pastes, gingival massage creams, gargle tablets, dairy products and other foodstuffs. An oral composition according to this invention may further include additional well known ingredients depending on the type and form of a particular oral composition.

Optionally, the composition may further include one or more antibiotics that are toxic to or inhibit the growth of Gram negative anaerobic bacteria. Potentially any bacteriostatic or bactericidal antibiotic may be used in a composition of the invention. Preferably, suitable antibiotics include amoxicillin, doxycycline or metronidazole.

In certain preferred forms of the invention the oral composition may be substantially liquid in character, such as a mouthwash or rinse. In such a preparation the vehicle is typically a water-alcohol mixture desirably including a humectant as described below. Generally, the weight ratio of water to alcohol is in the range of from about 1 :1 to about 20:1 The total amount of water-alcohol mixture in this type of preparation is typically in the range of from about 70 to about 99.9% by weight of the preparation. The alcohol is typically ethanol or isopropanol. Ethanol is preferred.

The pH of such liquid and other preparations of the invention is generally in the range of from about 5 to about 9 and typically from about 5.0 to 70. The pH can be controlled with acid (e.g. citric acid or benzoic acid) or base (e.g. sodium hydroxide) or buffered (as with sodium citrate, benzoate, carbonate, or bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc).

In other desirable forms of this invention, the composition may be substantially solid or pasty in character, such as toothpowder, a dental tablet or a toothpaste (dental cream) or gel dentifrice. The vehicle of such solid or pasty oral preparations generally contains dentally acceptable polishing material.

In toothpaste, the liquid vehicle may comprise water and humectant typically in an amount ranging from about 10% to about 80% by weight of the preparation. Glycerine, propylene glycol, sorbitol and polypropylene glycol exemplify suitable humectants/carriers. Also advantageous are liquid mixtures of water, glycerine and sorbitol. In clear gels where the refractive index is an important consideration, about 2.5 - 30% w/w of water, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitol are preferably employed.

Toothpaste, creams and gels typically contain a natural or synthetic thickener or gelling agent in proportions of about 0.1 to about 10, preferably about 0.5 to about 5% w/w. A suitable thickener is synthetic hectorite, a synthetic colloidal magnesium alkali metal silicate complex clay available for example as Laponite (e.g. CP, SP 2002, D) marketed by Laporte Industries Limited. Laponite D is, approximately by weight 58.00% S1O2, 25.40% MgO, 3.05% Na ₂ 0, 0.98% Li ₂ 0, and some water and trace metals. Its true specific gravity is 2.53 and it has an apparent bulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose (e.g. available as Natrosol), sodium carboxymethyl cellulose, and colloidal silica such as finely ground Syloid (e.g. 244). Solubilizing agents may also be included such as humectant polyols such propylene glycol, dipropylene glycol and hexylene glycol, cellosolves such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing at least about 12 carbons in a straight chain such as olive oil, castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, the oral preparations will usually be sold or otherwise distributed in suitable labelled packages. Thus, a bottle of mouth rinse will have a label describing it, in substance, as a mouth rinse or mouthwash and having directions for its use; and a toothpaste, cream or gel will usually be in a collapsible tube, typically aluminium, lined lead or plastic, or other squeeze, pump or pressurized dispenser for metering out the contents, having a label describing it, in substance, as a toothpaste, gel or dental cream .

Organic surface-active agents may be used in the compositions of the present invention to achieve increased therapeutic or prophylactic action, assist in achieving thorough and complete dispersion of the active agent throughout the oral cavity, and render the instant compositions more cosmetically acceptable. The organic surface-active material is preferably anionic, non-ionic or ampholvtic in nature and preferably does not interact with the active agent. It is preferred to employ as the surface-active agent a detersive material which imparts to the composition detersive and foaming properties. Suitable examples of anionic surfactants are water-soluble salts of higher fatty acid monoglyceride monosulfates, such as the sodium salt of the monosulfated monoglyceride of hydrogenated coconut oil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate, alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higher alkylsulfo-acetates. higher fatty acid esters of 1,2-dihydroxy propane sulfonate, and the substantially saturated higher aliphatic acyl amides of lower aliphatic amino carboxylic acid compounds, such as those having 12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and the like. Examples of the last mentioned amides are N-lauroyl sarcosine, and the sodium, potassium, and ethanolamine salts of N-lauroy!, N-myristoyl, or N-palmitoyl sarcosine which should be substantially free from soap or similar higher fatty acid material. The use of these sarconite compounds in the oral compositions of the present invention is particularly advantageous since these materials exhibit a prolonged marked effect in the inhibition of acid formation in the oral cavity due to carbohydrates breakdown in addition to exerting some reduction in the solubility of tooth enamel in acid solutions. Examples of water-soluble non-ionic surfactants suitable for use are condensation products of ethylene oxide with various reactive hydrogen- containing compounds reactive therewith having long hydrophobic chains (e.g. aliphatic chains of about 12 to 20 carbon atoms), which condensation products ("ethoxamers") contain hydrophilic polyoxyethylene moieties, such as condensation products of poly (ethylene oxide) with fatty acids, fatty alcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate) and polypropyleneoxide (e.g. Pluronic materials).

The surface active agent is typically present in amount of about 0.1-5% by weight. Various other materials may be incorporated in the oral preparations of this invention such as whitening agents, preservatives, silicones, chlorophyll compounds and/or ammoniated material such as urea, diammonium phosphate, and mixtures thereof. These adjuvants, where present, are incorporated in the preparations in amounts which do not substantially adversely affect the properties and characteristics desired.

Any suitable flavouring or sweetening material may also be employed. Examples of suitable flavouring constituents are flavouring oils, e.g. oil of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon, and orange, and methyl salicylate. Suitable sweetening agents include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP (aspartyl phenyl alanine, methyl ester), saccharine, and the like. Suitably, flavour and sweetening agents may each or together comprise from about 0.1 % to 5% more of the preparation.

The compound, peptide or peptidomimetic of composition of the invention can also be incorporated in lozenges, or in chewing gum or other products, e.g. by stirring into a warm gum base or coating the outer surface of a gum base, illustrative of which are jelutong, rubber latex, vinylite resins, etc., desirably with conventional plasticizers or softeners, sugar or other sweeteners or such as glucose, sorbitol and the like.

The invention provides a method for treating or alleviating the symptoms of periodontal disease in a subject, the method comprising administering to the subject a compound, peptide, peptidomimetic or composition of the invention and a protein for inducing an immune response to P. gingivalis. The protein for inducing an immune response to P. gingiva!is includes those proteins described in PCT/AU2009/001112 (WO/2010/022463) which is herein incorporated by reference. In a further aspect, the present invention provides a kit of parts including (a) a compound, peptide, peptidomimetic or composition and (b) a pharmaceutically acceptable carrier. Desirably, the kit further includes instructions for their use for the treatment or prevention of periodontal disease in a patent in need of such treatment.

Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylceilulose, hydropropyl methylceilulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitoi such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitoi anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example benzoates, such as ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

To describe the invention in more detail, the following examples are described to illustrate some aspects and embodiments of the invention.

Example 1 - Preparation of Kgp Propeptide and inhibition of Kgp activity

Bacterial Strains and Growth Conditions

Glycerol or freeze-dried cultures of Porphyromonas gingivalis W50 and the KgpcatAABMI mutant ECR368 were grown anaerobically at 37°C on Horse Blood Agar (HBA; Oxoid). P. gingivalis was maintained by passage and only passage 3-7 were used to inoculate 20 mL and 200 mL Brain Heart Infusion broth (37 g L), supplemented with hem in (5 mg/L) and cysteine (0.5 g L) and erythromycin supplementation (10 ^ig/mL) for ECR368 (BHI). Growth was determined by measurement of culture optical density (OD) at a wavelength of 650 nm. Gram stains of the cultures were carried out to check for any contamination. The P. gingivalis cells were harvested during exponential growth phase by centrifugation (8000 g, 20 min, 4°C) and washed once with TC150 buffer (50 mM Tris-HCI, 150 mM NaCI, 5 mM CaCfe, pH 8.0) containing 0.5 g/L cysteine. The washed cells were resuspended in 2 mL of TC150 buffer (with 0.5 g/L cysteine), and kept at 4 °C to be used immediately in the proteolytic assays.

P. gingivalis W50 was grown in a minimal medium for at least 6 passages and stored in -80*C for subsequent growth experiments. The minimal medium was prepared as follows: basal buffer (10 mM NaH ₂ P0 ₄ , 10 mM KCI, and 10 mM MgCI ₂ ) was supplemented with haemoglobin (50 nM) and BSA (3% A-7906; Sigma-Aldrich Co.), pH 7.4, and filter sterilized (0.1 Mm membrane filter Filtropur BT50, Sarstedt). The cells (10* in 200 μΙ_) were inoculated into each well of the 96-well microtitre plate (Greiner Bio-One 96-Well Cell Culture Plates) with 100 mg/L of Kgp propeptide (Kgp-PP), RgpB propeptide (RgpB-PP) or Kgp-PP plus RgpB-PP. The plate was incubated overnight at 37 ^e C in the anaerobic chamber, sealed with a plateseal microtitre plate sealer (Perkin Elmer Life Sciences, Rowville, VIC, Australia). The absorbance was monitored at 620 nm for 50 h at 37°C, using a microplate reader (Multiskan Ascent microplate reader - Thermo Electron Corporation). The P. gingivafis W50 isogenic triple mutant lacking RgpA, RgpB, and Kgp was used as a negative control of growth in the minimal medium. The growth in presence of propeptide was compared against the growth of P. gingivalis in the minimal medium.

Purification of Lys-gingipain (Kgp)

For harvesting and purification of the mature KgpcatAABMI , 4 mL of the Kgpc«AABM1 mutant ECR368 starter culture was used to inoculate 200 mL BHI broth that was then incubated over three days at 37°C. The P. gingivalis cells were first removed by centrifugation at 8,000 g for 30 min at 4°C after which the supernatant was collected and ultracentrifuged at 100,000 gfor 1 h at -10°C to remove vesicles. The pellets were discarded and the supernatant was collected and stored on an ice salt mixture. Chilled acetone was slowly added to the chilled supernatant in a 3:2 ratio v/v and the proteins precipitated by centrifugation (8,000 gfor 30 min, -10°C). The supernatant was carefully discarded and the precipitate washed in TC50 buffer (50 mM Tris-HCI, 50 mM NaCI, 5 mM CaCb, pH 7.4). After centrifugation (8,000 g for 30 min, - 0°C), the precipitate was resuspended in TC50 buffer and filtered through a 0.22 μΜ filter. This extract was applied to a desalting column (Sephadex G25, XK26/40) attached to an AKTA-Basic FPLC system, and eluted with TC50 buffer at a flow rate of 5 mL/min, The eluate was monitored at 280 and 254 nm. The void volume was collected and concentrated to <10 mL by ultrafiltration using 10,000 W cut-off membranes (Vivaspins). The concentrated sample was applied to an anion exchange column (Q-sepharose), to separate the fractions with Lys-activity from those with Arg-activity (Figure 3). The pooled concentrated fractions with Lys-activity were then applied to a cation exchange column S-sepharose. The eluted fractions with Lys-activity were then size-fractionated using gel filtration column (Superdex G75, XK16/100) to separate Kgp proteases from the other proteins. The column was eluted with TC50 buffer at a flow rate of 1 mL/min. The eluate was monitored at 280, 254 and 215 nm, collected and stored at -70°C. Expression and Purification of Recombinant Kgp-propeptide

The genomic DNA encoding the propeptide of Kgp (amino acids 20-228) was amplified by polymerase chain reaction (PCR) using the genomic DNA of Kgp as a template. Primers 5' ACG CAG CAT ATG CAA AGC GCC AAG ATT AAG CTT GAT 3' and 5' ACG CAG CTC GAG TCA TCT ATT GAA GAG CTG TTT ATA AGC 3' were used for PCR These primers contained the Nde1 and Xhol restriction sites. An additional stop codon site was designed at the antisense position. The size of the DNA was checked by SDS-PAGE and the PCR product was cloned into PGEM-T easy vector (Promega) using TA cloning kit (Invitrogen). The PCR insert was removed after cleavage with enzymes Nde1 and Xhol, purified by gel extraction then inserted into the PET-28b expression vector (Novagen). The insert was sequenced to verify correct amplification and ligation.

For expression in Escherichia coii BL-21 (DE3) (Novagen), the PET-28b vector was transformed into the BL-21 (DE3) cells. Expression was induced by addition of 1 mM Isopropyl (3-D-1 -thiogalactopyranoside (IPTG). After 4 h of induced expression, the cells were harvested by centrifugation at 8,000 g for 20 min. The cells, containing the recombinant propeptide in inclusion bodies, were suspended in lysis buffer (50 mM Na ₂ HP0 . 300 mM NaCI, 10 mM imidazole, pH 8.0) and disrupted by sonication (15 min) and stirring (30 min, 4°C). The lysate was centrifuged and the resulting supernatant purified using Ni affinity chromatography to obtain purified recombinant propeptide.

A 50% Ni-NTA (Qiagen) slurry (4 mL) was added to the supernatant, which was then stirred for 15 min at 4°C. The mixture was loaded on an open column with a bed volume of 20 mL and the flow through was removed. The resin was washed twice with 10 mL purification buffer (50 mM potassium phosphate at pH 8.0, 150 mM NaCI, 20 mM imidazole). Then purification buffer (2 mL) containing 25 NIH units of thrombin (Sigma) was added to the slurry and allowed to incubate for 2 h at room temperature to enable thrombin to cleave the propeptide from its His-tag and release it from the nickel affinity resin. The released propeptides with the thrombin protease were collected with 15 mL purification buffer. The solution was loaded onto another column containing 1 mL of Benzamidine Sepharose resin (Pharmacia) and allowed to react for 15 min at room temperature, to enable the thrombin protease to bind to the Benzamidine Sepharose resin. The flow through fraction was collected. The Benzamidine Sepharose resin was washed twice with 2.5 ml_ of wash buffer (5 mM potassium phosphate at pH 7.0, 50 mM NaCI), and the washes were collected. The flow through fraction was combined with the two wash fractions, resulting in a 20 mL solution that was lyophilised. The redissolved extract was applied to a gel filtration column (Superdex G75, XK16 100) attached to an AKTA-Basic FPLC system and eluted with 50 mM NH4HCO3 at a flow rate of 1 mL min. The eluate was monitored at 280 and 215 nm. The eluate was collected, lyophilised and stored at -70°C.

Spectrophotometry Determination of Protein Concentration

The molar extinction coefficient (ε) (M ^'1 cm ^'1 ) at 280 nm and the molecular weights of the proteins were determined using the "ProtParam" program on the ExPASy server (Gasteiger et at., 2005). The ε of KgpcatAABMI was 105,340 M ^'1 cm ^'1 while the ε of the rKgp propeptide was 11 ,920 M ^"1 cm ^"1 . The concentrations of the Kgp _ca tAABM1 enriched fraction and the rKgp propeptide were determined using spectrophotometric means (Grimsley and Pace, 2003). The absorbance of each sample was measured by scanning the absorbance from 200 nm to 300 nm using the Varian Cary 50 Dual Beam spectrophotometer (Australia). The absorbance at 280 nm, which is absorbed by Trp, Tyr, and Cys residues, was used to calculate the protein concentration using Beer- Lamberts Law (A ₂ 80nm= ebC). The KgpcatAABMI enriched fractions were subsequently diluted for the protease inhibition assays.

MALDI TOF/TOFMS

Peptide samples were co-crystallized (1:1 vol/vol) on a MTP 384 target ground steel plate with saturated 2,5-dihydroxybenzoic acid (DHB) matrix in standard buffer (50% acetonitrile, 0.1% TFA). The samples were analysed on an Ultraflex MALDI TOF TOF Mass Spectrometer (Bruker, Bremen, Germany). Analysis was performed using Bruker Daltonics flexAnalysis 2.4 and Bruker Daltonics BioTools 3.0 software with fragmentation spectra matched to a casein database installed on a local MASCOT server.

Electrospray MS

Fractions collected from the RP-HPLC were analysed using an Esquire-LC MS/MS system (Bruker Daltonics) operating in the electrospray mass spectrometry mode. Sample injection was conducted at 340≠Jh, with nitrogen flow of 5 L/min and drying gas temperature of 300°C. Protease Inhibition Assays

Lys-specific proteolytic activity was determined using synthetic chromogenic substrate N-(/>Tosyl)-Gly-Pro-Lys 4-nitroanilide acetate salt (GPK-NA) (Sigma Aldrich). The Lys- specific reaction buffer contained 2 mM GPK-NA dissolved in 30% v/v isopropanol, 0.93 mM cysteine, 400 mM Tris-HCI pH 8.0, and 100 mM NaCI. Protease assays were conducted in sterile 96-well microtitre plates (Corning Incorporated, NY) with all fractions and controls assayed in triplicate. The rKgp propeptides were added to the wells in a final concentration of 20.0 mg/L (0.85 μΜ) and 40.0 mg L (1.71 μΜ) with 10 μΙ_ of 10 mM cysteine pH 8.0 and a final concentration of 1.16 mg L (0.02 μΜ) KgpcatAABMI enriched fraction, topped-up to a volume of 100 μΙ_ with TC150 buffer (50 mM Tris-HCI, 150 mM NaCI, 5 mM CaC , pH 8.0). Samples were incubated at 37°C for 15 min before the addition of 100 pL of chromogenic substrate (2 mM) (total volume 200 μΙ_). Protease activity was determined by measuring the absorbance at 405 nm with 10 s intervals for "20 min at 37°C, pH 8.0 using a PerkinElmer 1420 Multilabel Counter VICTOR3™ KgpcatAABMI enriched fraction proteolytic activity was determined as Units/mg.

Bacterial protease inhibitory activity was also determined using DQ™ Green bovine serum albumin (BSA) (Molecular Probes, USA) (Grenier et at., 2001 ; Yoshioka et at., 2003). The protein is labelled with a strong self-quenched amine dye which when cleaved emits maximally at 535 nm following excitation at 485 nm. The assay mixture contained Kgpca _t AABMI enriched fraction (1.16 mg L, 0.02 μΜ), the rKgp propeptides (40.0 mg/L), 1 mM cysteine, and DQ BSA (10 μί; 0.1 g/L), made up to a final volume of 200 μΐ with TC150 buffer. Na-p-tosyl-l-lysine chloromethylketone TLCK (1 mM) treated KgpcatAABMI proteases were used as a control. TLCK is a strong cysteine protease inhibitor known to inhibit both Rgp and Kgp activity (Fletcher et at., 1994; Pike et at., 1994). Leupeptin, an Rgp inhibitor was added to the assay to inhibit any Arg-gingipain activity that may be present (Kitano et at., 2001 ). The assay mixtures were incubated in the dark for 2 h at 37°C prior to measuring the fluorescence which indicates the degree of albumin degradation, using a fluorometer (PerkinElmer 1420 Multilabel Counter VICTOR3™). The fluorescence value obtained with the negative control (TLCK-treated) was subtracted from all values. All assays were performed in triplicate with 2-3 biological replicates unless stated otherwise, and the mean ± standard deviation was calculated.

Samples from each well were analysed for propeptide and protease hydrolysis using reversed phase-high performance liquid chromatography (RP-HPLC) and SDS-PAGE. 200 μΙ_ of each sample was analysed on an analytical Zorbax 300 SB-Ci _& reversed phase column (4.6 mm x 250 mm) connected to an Agilent Preparative 1100 HPLC instrument (Agilent Technologies) using a flow rate of 1 mL/min and a gradient of 0- 100% solvent B (90% acetonitrile- 0.1% (v/v) TFA in deionised water) in 30 min. For SDS-PAGE analysis, each assay sample (200 μΙ_) was centrifuged at 14,500 rpm for 5 min, then 50 μΙ_ of the supernatant was denatured with 5% (v v) 1 M DTT and 25% (v/v) 4x reducing sample buffer, heated for 10 min at 70°C and briefly microcentrifuged before being loaded onto a precast 8-12% gradient Bis-Tris gel. SeeBlue ^® Pre-Stained standard was used as a molecular marker and a potential difference of 150 V and MES buffer were used to run the gel. The gel was stained with Coomassie ^® Brilliant Blue (G250) overnight and destained in deionised water. The gel was scanned using an Epson Smart Panel scanner connected to a Proteineer SP system (Bruker Daltonics).

Statistical Analysis

Protease activity data were subjected to a single factor analysis of variance (ANOVA). When the ANOVA indicated statistical significant difference (p < 0.05) between the means of tested inhibitors, a modified Tukey test was performed on the data to identify which inhibitors were significantly different (Zar, 1984; Fowler and Cohen, 1997).

Molecular Modelling

The program Fugue (Shi et al., 2001) was used to identify possible structure motifs for the three gingipain propeptides against a curated protein database HOMSTRAD (Mizuguchi et al., 1998). The program PSI-BLAST was run concurrently to identify any other putative orthologs and paralogs.

Purification of Lys-gingipain (Kgp)

P. gingivalis ECR368 was grown anaerobicafly for 3 days and the culture supernatant harvested for Kgpc*&ABM1 by acetone precipitation and centrifugation. The acetone precipitated proteins were loaded onto a desalting column (Sephadex G25) and eluted by 50 mM sodium acetate pH 5.3 buffer. The first peak was collected and concentrated using a 10,000 MW cut-off membrane. This extract was subjected to anion exchange and cation exchange chromatography and finally size-exclusion chromatography (Figure 4) to separate the Kgpcat&ABM1 from other proteins in the supernatant. Samples from each purification step were analysed using SDS-PAGE gels for enzyme purity (Figure 5). The purity of the KgpcatAABMI increased with each subsequent purification step resulting in a KgpcatAABMI enriched fraction (lane 5; Figure 5).

Expression and Purification of Recombinant Kgp Propeptide (rKgp)

The rKgp propeptide was designed using a His-Tag sequence followed by a thrombin cleavage site, N-terminal to the propeptide. The rKgp propeptide was expressed in E. coii and extracted using Ni affinity chromatography of the cell lysate. To remove the His- tag, the E. coii cell lysate bound to the Ni-column was treated with thrombin which cleaved the propeptide leaving the His-tag attached to the Ni-column. The released propeptides were collected and applied to an open column with Benzamidine Sepharose to remove the thrombin protease followed by a gel filtration column to purify the rKgp propeptide (Figure 6A). The identity of the rKgp propeptide was determined using MALDI-TOF MS analysis (Figure 6B).

Spectrophotometric Determination of Protein Concentration

The concentrations of the Kgpcat&ABM1 enriched fraction (MW 50,114 Da, 454 aa) and the rKgp propeptides were determined using spectrophotometric means (Grimsley and Pace, 2003). The absorbance at 280 nm (A280nm) of the Kgp _ca tAABM1 enriched fraction was 0.033 and the extinction coefficient was 105,340 M ^"1 cm ^"1 ; therefore the concentration of the Kgp^ABMI enriched fraction was 0.0157 g/L Several batches of Kgp _cat AABM1 enriched fractions were analysed for its protein concentration by A280nm. However, the final concentration of KgpcatAABMI enriched fraction in each assay was set as 1.16 mg/L (0.02 μΜ).

The concentration of the rKgp propeptides was determined in the same manner. The A280nm of the rKgp propeptide (MW 23,403, 213 aa) was 0.1169, and has an extinction coefficient of 11,920 M~ ¹ cm ^*1 and therefore a concentration of 0.23 g/L. The final concentration of rKgp propeptide in the assays was 20.0 (0.85 μΜ) and 40.0 mg/L (1.71 μΜ).

Protease Inhibition Assay The inhibition of KgpcatAABMI by the rKgp propeptides was determined using chromogenic and fluorescent substrates. In the chromogenic substrate assay, the final concentrations of rKgp propeptides were 20.0 mg L (0.86 μΜ) and 40.0 mg/L (1.71 μ ) and the concentration of KgpcatAABMI enriched fraction was 1.16 mg L (0.02 μΜ). The control used was TLCK at a concentration of 1 mM. The rKgp propeptide exhibited -75% inhibition of KgpcatAABMI activity at a concentration of 40.0 mg L (1.71 μΜ) while 20.0 mg L (0.85 μ ) rKgp propeptide inhibited -60% KgpcatAABMI activity (Figure 7). The rate of substrate hydrolysis was linear throughout the assay (Figure 8).

Samples from these assays were collected and analysed using RP-HPLC to determine potential hydrolysis of the rKgp propeptide or the KgpcatAABMI The HPLC profiles indicated that the rKgp propeptide was still intact (Figure 9). The samples (200 μΐ) were centrifuged and 50 pL of the supernatant was treated with DTT and sample buffer and analysed by SDS-PAGE. The SDS-PAGE analysis demonstrated that the propeptide was still present at the expected molecular weight (Figure 10).

The inhibition kinetics of the rKgp propeptide against KgpcatAABMI determined using the chromogenic substrate GPKNa revealed non-competitive inhibition. The ΚΓ for Kgp propeptide was calculated to be 2.01 μΜ (Figure 11).

The fluorescent BSA substrate assays were performed within a 2 h incubation period. The rKgp propeptide exhibited -66% inhibition of KgpcatAABMI enriched fraction activity at a concentration of 10.0 mg L (0.45 μΜ) (Figure 12). However, this assay measures total protease activity, so the rKgp propeptide inhibition of KgpcatAABMI is underestimated due to the residual presence of RgpA that will cleave BSA.

Samples from the assays were collected and analysed by SDS-PAGE (Figure 13A). The control contains -0.03 pg KgpcatAABMI and -1 pg BSA. A pellet was observed in the centrifuged samples containing rKgp propeptide and KgpcatAABMI enriched fraction while no pellets were observed in the centrifuged samples just containing KgpcatAABMI enriched fraction (control). These pellets were resuspended in supernatant and applied to SDS gels. The SDS gels indicate that the KgpcatAABMI (MW -50,000) and the rKgp propeptides (MW -25,000) were still intact after the 2 h incubation period (Figure 13A & 13B). The presence of BSA (MW 62,000) and its cleaved products were also observed on the gel. The KgpcatAABMI cleaved all BSA into small peptides that were difficult to detect as those cleavage products most likely ran off the end of the gel (Figure 13A, lanes 2 and 3) while intact BSA was still present when rKgp propeptide was added to KgpcatAABMI (lanes 4 and 4) and the cleaved peptides were still relatively large, between about 14 and 3kDa, indicating an inhibition of Kgp protease activity by the propeptide. The TLCK controls indicate that the proteases were inhibited and no BSA degradation was observed (Figure 13B).

Example 2 - Preparation of RgpB Propeptide and inhibition of Rgp activity

Growth Conditions for P gingivalis HG66

Glycerol cultures of P. gingivalis strain HG66 were grown anaerobicaliy at 37°C in an anaerobe chamber, with an atmosphere of 10% CO2, 5% H2, 85% N ₂ , on horse blood agar (HBA; Oxoid). P. gingivalis cultures were maintained by passages weekly until 7- 10 passages were completed, after which a fresh culture was recovered from glycerol stocks. To grow P. gingivalis in broth culture, a starter culture was prepared by inoculation of several colonies (selected from a 5-7 day old plate) into 20 mL BHI broth (Brain Heart Infusion broth (37 g/L)), supplemented with haemin (5 mg/L), cysteine (0 5 g/L), vitamin K3 (menadione) (5 mg L) before being incubated overnight at 37°C. Culture purity was routinely assessed by Gram stain and observation of colony morphology on HBA plates.

Purification of Arg-gingipain (RgpB)

For harvesting and purification of the mature RgpB, 40 mL of starter culture was used to inoculate 2 L BHI broth which was then incubated over three-four days at 37°C. The P. gingivalis cells were removed by centrifugation at 17,700 g for 1 h at 4°C, after which the supernatant was collected and the pH adjusted to pH 5 3 with 50 mM Sodium Acetate then filtered through 0.8/0.2 μΜ filters to remove vesicles (contained in the pellets). The supernatant was poured off, collected and stored on an ice/salt mixture; chilled acetone was slowly added to the chilled supernatant in a 3:2 ratio v/v and the precipitated proteins collected by centrifugation (8,000 g for 30 min, -10°C). The supernatant was carefully discarded and the precipitate was redissolved in NaOAc pH 5.5 buffer. After centrifugation (8,000 g for 30 min, -10°C), the supernatant was filtered through a 0.22 μΜ filter. This extract was applied to a gel filtration column (Superdex G75, XK16 100) attached to an AKTA-Basic FPLC system, to separate the gingipains from the other proteins. The column was eluted with NaOAc pH 5.5 buffer at a flow rate of 0.5 m Urn in, with the eluate being monitored at 280, 254 and 215 nm and the resulting fractions collected and stored at -70°C.

Expression and Purification of Recombinant RgpB-propeptide

The genomic DNA encoding the propeptide of RgpB was amplified by polymerase chain reaction (PCR) using the genomic DNA of RgpB as a template. Primers 5' ACG CAG CAT ATG CAA AGC GCC AAG ATT AAG CTT GAT 3 ^* and 5' ACG CAG CTC GAG TCA TCT ATT GAA GAG CTG TTT ATA AGC 3' were used for PCR. These primers contained the Nde1 and Xhol restriction sites. An additional stop codon site was designed at the antisense position. The size of the DNA was checked by SDS-PAGE and the PCR product was cloned into pGEM-T Easy vector (Promega) using TA cloning kit (Invitrogen). The PCR insert was removed after cleavage with enzymes Nde1 and Xhol, purified by gel extraction then inserted into the PET-28b expression vector (Novagen). The insert was sequenced to verify correct amplification and ligation.

For expression in E. coii BL-21 (DE3) (Novagen), the PET-28b vector was transformed into the BL-21 (DE3) cells. Expression was induced by addition of 1 mM Isopropyl β-D- 1-thiogalactopyranoside (IPTG). After 20 h, 15 °C, of induced expression, the cells were harvested by centrifugation at 8,000 g for 20 min. The cells were suspended in lysis buffer (50 mM Na ₂ HP0 , 300 mM NaCI, 10 mM imidazole, pH 8.0) and then disrupted by sonication (20 min) and stirring (30 min, 4°C). The lysate was centrifuged and the resulting supernatant purified using Ni affinity chromatography to obtain purified recombinant propeptide.

A 50% Ni-NTA (Qiagen) slurry (4 mL) was added to the supernatant, stirred for 15 min at 4°C and loaded on an open column with a bed volume of 20 mL, the flow through was removed. The resin was washed twice with 10 mL purification buffer (50 mM potassium phosphate at pH 8.0, 150 mM NaCI, and 20 mM imidazole). Purification buffer (2 mL) containing 25 NIH units of thrombin (Sigma) was added to the slurry and incubated for 2 h at room temperature. The released propeptides and thrombin protease were washed from the column using 15 mL purification buffer, and this solution was loaded onto another column containing 1 mL of Benzamidine Sepharose resin (Pharmacia). The solution was left to react for 15 min at room temperature to enable the thrombin protease to bind to the Benzamidine Sepharose resin. Once the flow through fraction was collected, the Benzamidine Sepharose resin was then washed twice with 2.5 mL of wash buffer (5 mM potassium phosphate at pM 7.0, 50 mM NaCI), with each of the washes collected too. The flow through fraction was then combined with the two wash fractions, resulting in a 20 mL solution that was lyophilised. The redissolved extract was applied to a gel filtration column (Superdex G75, XK16 100) attached to an AKTA-Basic FPLC system and eluted with 50 mM NH4HCO3 at a flow rate of 1 mL min. The eluate was monitored at 280 and 215 nm. The eluate was collected, lyophilised and stored at -70°C.

Protease Inhibition Assay

The proteolytic activity of the RgpB was determined in an assay using a fluorescent DQ- BSA substrate. Fluorescence was measured over 11 hours at 37 °C with a reading taken every hour. Addition of 10 mg L (0.44 μΜ) or 20 mg/L (0.88 μΜ) RgpB propeptide resulted in near total inhibition of RgpB proteolytic activity over the entire length of the assay, demonstrating the sustained inhibition of the protease by the RgpB propeptide. The negative control was 1 mM TLCK (Figure 14).

A dose response of the RgpB propeptide was demonstrated within a 2 h incubation period where 1 mg L inhibited -50% of RgpB activity whilst 5 mg/L totally abolished activity (Figure 15). Inhibition kinetics of the RgpB propeptide were determined using the chromogenic substrate BapNA (Figure 16). The Ki' for non-competitive inhibition was calculated to be 11.8 nM. Propeptide Selectivity and Specificity

Both rRgpB and rKgp propeptides demonstrated selectivity for their cognate protease with no inhibition observed when rKgp propeptides were incubated with RgpB and wee versa (Table 1). The specificity of the propeptides was further examined using two examples of cysteine proteases. The clan CA protease papain, with a propeptide of 115 residues, was not significantly inhibited by rKgp and rRgpB propeptides (Table 1). The Clan CD protease caspase 3 that has structural homology with the RgpB and Kgp catalytic domains also was not inhibited by either rKgp or rRgpB propeptides. The noncompetitive inhibition mode demonstrated by both propeptides, coupled with the selectivity for the cognate proteases is suggestive of exosite binding by the propeptides. Table 1

Protease [Protease] Inhibitor [Inhibitor] (mg L) Substrate % Proteolytic

Activity

RgpB 0.0085 mg/ml Kgp-PP 50 BapNa 105

Kgp 0.0075 mg/ml RgpB-PP 50 GPKNa 118

Caspase 3 60 units Kgp-PP 100 Ac-DEVD- 121.1 ± 6.8

pNa (200

uM)

Caspase 3 60 units RgpB-PP 100 Ac-DEVD- 128.7±6.2

pNa (200

uM)

Papain 2.75 mg ml Kgp-PP 40 BapNa 88

Papain 2.75 mg ml RgpB-PP 40 BapNa 68

Whole cell 3.2x10 ⁷ cells Kgp-PP 40 GPKNa 65

W50

80 40

Whole cell 3.2x10' cells RgpB-PP 40 BapNa

Planktonic growth inhibition

P. gingivalis W50 was grown in a protein-based minimal medium and reached a

maximum cell density equivalent to an OD ₆₂ o _t im of 0.32 after 40 h of incubation. Both propeptides demonstrated a significant inhibitory effect on P. gingivalis W50 planktonic growth (Figure 17). The P. gingivalis triple protease mutant lacking the RgpA, RgpB and Kgp gingipains, did not grow in this minimal medium thus confirming that gingipain proteolytic activitiy is essential for the breakdown of the proteins BSA and haemoglobin to short peptides for subsequent uptake by the bacterium. Example 3 - Compositions and Formulations

To help illustrate compositions embodying aspects of the invention directed to treatment or prevention, the following sample formulations are provided.

The following is an example of a toothpaste formulation.

Ingredient % w w

Dicalcium phosphate dihydrate 50.0

Glycerol 20.0

Sodium carboxymethyl cellulose 1.0

Sodium lauryl sulphate 1.5

Sodium lauroyl sarconisate 0.5

Flavour 1.0

Sodium saccharin 0.1

Chlorhexidine gluconate 0.01

Dextranase 0.01

Compound, peptide or peptidomimetic of the invention 0.2

Water balance

The following is an example of a further toothpaste formulation.

Ingredient % w W Dicalcium phosphate dihydrate 50.0

Sorbitol 10.0

Glycerol 10.0

Sodium carboxymethyl cellulose 1.0

Lauroyl diethanolam ide 1.0

Sucrose monolaurate 2.0

Flavour 1.0 Sodium saccharin 0.1

Sodium monofluorophosphate 0.3

Chlorhexidine gluconate 0.01

Dextranase 0.01 Compound, peptide or peptidomimetic of the invention 0.1

Water balance

The following is an example of a further toothpaste formulation.

Ingredient % w w Sorbitol 22.0

Irish moss 1.0

Sodium Hydroxide (50%) 1.0

Gantrez 19.0

Water (deionised) 2.69 Sodium Monofluorophosphate 0.76

Sodium saccharine 0.3

Pyrophosphate 2.0

Hydrated alumina 48.0

Flavour oil 0.95 Compound, peptide or peptidomimetic of the invention 0.3 sodium lauryl sulphate 2.00

The following is an example of a liquid toothpaste formulation.

Ingredient % w/w Sodium polyacr late 50.0

Sorbitol 10.0 Glycerol 20.0 Flavour 1.0

Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Ethanol 3.0

Compound, peptide or peptidomimetic of the invention 0.2

Linolic acid 0.05

Water balance

The following is an example of a mouthwash formulation.

Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium saccharin 0.1

Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Lauroyl diethanolamide 0.3

Compound, peptide or peptidomimetic of the invention 0.2 Water balance

The following is an example of a further mouthwash formulation.

Ingredient % w/w

Gantrez® S-97 2.5 Glycerine 10.0 Flavour oil 0.4 Sodium monofluorophosphate 0.05

Chlorhexidine gluconate 0.01

Lauroyl diethanolamide 0.2

Compound, peptide or peptidomimetic of the invention 0.3 Water balance

The following is an example of a lozenge formulation.

Ingredient % w

Sugar 75-80 Corn syrup 1-20

Flavour oil 1-2

NaF 0.01-0.05

Compound, peptide or peptidomimetic of the invention 0.3

Mg stearate 1-5 Water balance

The following is an example of a gingival massage cream formulation.

Inaredient % w/w

White petrolatum 8.0

Propylene glycol 4.0

Stearyl alcohol 8.0

Polyethylene Glycol 4000 25.0

Polyethylene Glycol 400 37.0

Sucrose monostearate 0.5

Chlorhexidine gluconate 0.1

Compound, peptide or peptidomimetic of the invention 0.3 Water balance

The following is an example of a periodontal gel formulation.

Ingredient % w/w

Pluronic F127 (from BASF) 20.0

Stearyl alcohol 8.0

Compound, peptide or peptidomimetic of the invention 3.0

Colloidal silicon dioxide (such as Aerosil® 200™) 1.0

Chlorhexidine gluconate 0.1 Water balance

The following is an example of a chewing gum formulation.

Ingredient % w w

Gum base 30.0

Calcium carbonate 2.0

Crystalline sorbitol 53.0

Glycerine 0.5

Flavour oil 0.1

Compound, peptide or peptidomimetic of the invention 0.3

Water balance

A further illustration of the invention is as follows.

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism's cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingiva!is mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K, values of 2.04 μΜ and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism's cell surface cysteine proteinases, the Arg- and Lys- specific gingipains [1-2] have been implicated as major virulence factors that play an important role in colonisation and establishment of the bacterium as well as in the evasion of host defences [3-5]. Recent studies have demonstrated associations between periodontitis and systemic morbidities such as diabetes and cardiovascular disease [6], pre-term and low weight births [7], Alzheimer's disease [8], cancers [9], respiratory diseases [10] and rheumatoid arthritis [11]. The correlation between these systemic diseases and the entry of the bacterium and its gingipains into the circulation system are currently under investigation^ 2].

The gingipains RgpA, RgpB, and Kgp are encoded by three genes, rgpA, rgpB, and kgp respectively [13-15]. The gene rgpB encodes a single chain proteinase with a short 24 amino acid (aa) leader sequence, 205 aa propeptide, and a -500 aa catalytic domain [16]. In contrast, the longer rgpA and kgp genes each encode a leader sequence, propeptide, catalytic domain plus additional haemagglutinin-adhesin (HA) domains. Due to the importance of the gingipains in virulence [3-5] there is interest in the development of specific and safe inhibitors of the proteinases. Examination of the reported peptide-derived and non-peptide inhibitors of the gingipains in the literature reveals a surprising diversity of affinity, specificity and structural features. The inhibitors also display various modes of inhibition: competitive, non-competitive and uncompetitive [17-21]. To describe the specificity of proteases, a model of an active site composed of contiguous pockets termed subsites S1, S2 ... etc is used with substrate residues P1 , P2...etc occupying the corresponding subsites [22]. The residues in the substrate sequence are numbered consecutively outward from the cleavage site -P4-P3-P2- PI+PI'-PZ-PSW-, -S4-S3-S2-81*81 ^, -82 ^, -S3 ^, -S4'-. The scissile bond represented by the symbol + is located between the P1 and P1' positions, while the catalytic site is represented by the symbol *.

Bioinformatic analysis of known proteins and synthetic substrates cleaved by the gingipains reveals that although hydrophobic residues are frequently found at positions P4-P2 and P1'-P4', overall the size, charge, and shape preferences of substrates are not clear (unpublished, [23]). This may reflect the ability of the gingipain active site to accommodate various substrates with only a strong specificity for an Arg or Lys residue in the P1 position.

Recent studies have highlighted that protease propeptides are a promising source of inhibitors for the cognate protease [24-25]. Many cysteine proteases are synthesized as inactive forms or zymogens with N -terminal propeptide regions. These propeptides may have multiple functions including inhibiting the proteolytic activity of the mature enzyme, folding of the precursor enzyme, protecting the enzyme against denaturation in extreme pH conditions, transporting the precursor enzyme to Jysosomes, and mediating membrane association [26]. Typically the enzyme becomes activated upon removal of the propeptide by intra- or intermolecular proteolysis or in other cases by Ca ²⁺ binding or acidification [26]. Although cysteine protease propeptides range from 30-250 aa, most are less than 100 aa residues [26-28]. The gingipain catalytic-domain propeptides are unusually long, being -200 residues suggesting that the gingipain propeptides may have a more complex function than the shorter propeptides of other proteases.

The aim of this study was to characterize the inhibitory potential of recombinant Kgp and RgpB propeptides against their cognate catalytic domains purified from P. gingivalis. The specificity of recombinant^ expressed RgpB and Kgp propeptides for protease inhibition was determined as well as the interaction of the propeptides with both cognate and heterologous proteases and their effect on the growth of the bacterium.

Production of Recombinant Kgp Catalytic Domain

Plasmids and oligonucleotides used in the course of this work are listed in Table 2 and Table 3 respectively. Plasmids used were propagated in Escherichia coli a-Gold Select (Bioline Australia) or BL-21 (DE3) cells (Novagen). Allele exchange suicide plasmids (described below) were all linearised using Xbal restriction endonuclease (RE) digestion and transformed into electroporation-competent P. gingivalis cells [29] with transformants selected after anaerobic incubation at 37°C for up to ten days. EcoRV and Apal recognition sequences were engineered into plasmid pNS1 [30] upstream of the kgp promoter[31] using oligonucleotide primers EA-For and EA-Rev (Table 2) and the QuikChange li Site-directed Mutagenesis Kit (Stratagene) following manufacturer's instructions, generating pNS2. The Bacteroides fragilis cephalosporinase-coding gene cepA was amplified from a pEC474 template DNA [32] using oligonucleotides CepAf and CepAr and ligated into pGEM-T Easy (Promega) to generate pCS19. cepA was excised from pCS19 using EcolCRI/Apal RE digestion and ligated into pNS2 that had been digested with BstEII (Bll), end-filled then digested with Apal. The resultant plasmid pPC1 has cepA that is transcribed from its own promoter and replaces nucleotides (nt) of pNS2 that include the kgp promoter and kgp nt coding from Met1-Tyr748. P. gingivalis W50 was transformed with pPC1 to produce the Kgp-null strain ECR364. Plasmid pPC2 was produced by ligating ermF excised from pAL30 [33] using Apal and EcoRICRI RE digestion into pNS2 digested with Apal and EcoRV. The nt coding ABM1 at the C-terminus of the Kgp catalytic domain (Gly681-A710, GEPSPYQPVSNLTATTQGQKVTLKWEAPSA) were then deleted from pPC2 using a combination of splicing by overlap extension (SOE) PCR, RE digestion and ligation as follows. Primer pairs ABM1del_For1 plus ABM1del_Rev1 and ABM1del_For2 plus ABM1del_Rev2 were used to generate two PCR amplicons which were annealed, extended and amplified using ABM1del_For1 and ABM1del_Rev2 as primers. The SOEn amplicon was digested with SnaBI and BstEII and ligated to SnaBI-BstEII digested pPC2 to generate pPC3 (Table 2) that was linearised and electroporated into ECR364 to replace cepA generating P. gingivalis ECR368 that produces rKgp with the ABM1 (Gly ⁸⁸¹ - Ala ⁷¹⁰ ) deletion.

Bacterial Strains and Growth Conditions P. gingivalis W50, ECR368 producing rKgp, and strain HG66 [16] were grown at 37°C in a MACS MG500 anaerobe workstation (Don Whitely Scientific) with an atmosphere of 10% C0 ₂ , 5% H ₂ , 85% N ₂₎ on 10% horse blood agar (HBA; Oxoid), with erythromycin supplementation (10 μο/ιτιί) for ECR368. P. gingivalis was grown in batch planktonic culture in Brain Heart Infusion broth (BHI, 37 g/L), supplemented with haemin (5 mg/L), cysteine (0.5 g/L), and erythromycin (10 g/mL) for ECR368. Culture purity was routinely assessed by Gram stain and observation of colony morphology on HBA plates.

P. gingivalis W50 was grown in a minimal medium [34-35] for at least 6 passages and then stored at -80°C for subsequent growth experiments. The minimal medium was prepared as follows: basal buffer (10 mM NaH ₂ P0 ₄ , 10 mM KCI, and 10 mM MgCfe) was supplemented with haemoglobin (50 nM) and BSA (3% A-7906; Sigma- Aldrich Co.), pH 7.4, and filter sterilized (0.1 pm membrane filter Filtropur BT50, Sarstedt). The cells (10 ⁸ in 200 pL) were inoculated into each well of a 96-well microtitre plate (Greiner Bio-One 96-Well Cell Culture Plates) with 100 mg L of rKgp-propeptide (Kgp-PP), rRgpB-propeptide (RgpB-PP) or Kgp-PP plus RgpB-PP. The plate was sealed with a plateseal microtitre plate sealer (Perkin Elmer Life Sciences, Rowville, VIC, Australia) and incubated overnight at 37°C in the anaerobic chamber. The cell density of the culture was monitored at 620 nm for 50 h at 37°C, using a Multiskan Ascent mi crop I ate reader (Thermo Electron Corporation). The P. gingivalis W50 isogenic triple mutant lacking RgpA, RgpB, and Kgp W50ABK [36] was used as a negative control of growth in the minimal medium.

Purification of Kgp and RgpB

A procedure for the large scale purification of rKgp from the P. gingivalis strain ECR368 and RgpB from P. gingivalis HG66 was developed. Briefly, the bacteria were subcultured using a 1/100 v/v inoculum into 5-6 L BHI broth without additional haemin and incubated at 37°C for three days. The cells were pelleted by centrifugation (17,700 g, 60 min, 4°C) then the pH of the collected supernatant was lowered to pH 5.3 using acetic acid prior to filtration. The filtrate was concentrated using tangential flow filtration on a Sartorius Sartoflow alpha system with a 10,000 Da Molecular Weight Cut Off (MWCO) membrane, followed by diafiltration with 1 L 50 mM Na-acetate pH 5.3. The proteins were precipitated with chilled acetone added slowly to a final ratio of supernatant: acetone of 1:1.5, and separated by centrifugation (17,700 g, 30 min, - 10°C). The precipitate was solubilised in 50 mM Na-acetate pH 5.3 and centri uged (17,700 g, 30 min, -10°C). The resultant supernatant was filtered through a 0.22 m filter and desalted using Sephadex G-25 (200 mL) in 50 mM Na-acetate pH 5.3. The void volume was collected and then subjected to ion exchange chromatography using Q-sepharose (200 mL) equilibrated in 50 mM Na-acetate pH 5.3. After elution of the unbound fraction, a gradient of 0-1 M NaCI in 50 mM Na-acetate pH 5.3 was applied to elute the proteins containing Arg-protease activity and then remove the haemin.

The unbound fraction from the Q-sepharose, containing rKgp was diluted in 10 volumes of 50 mM Na-acetate pH 5.3 to reduce the ionic strength and loaded onto a 50 mL SP- sepharose column equilibrated in 10 mM Na-acetate pH 5.3. A gradient of 0-1 M NaCI in 50 mM Na-acetate pH 5.3, enabled the elution of the bound proteins that contained Lys-specific activity. The fractions were pooled, concentrated using 3,000 Da MWCO filters and subjected to size-exclusion chromatography using a 300 mL Superdex G75 column and the fraction containing rKgp was collected and stored at -70°C. Samples collected at each purification step were analysed for Lys- and Arg-protease activity, purity using SDS-PAGE, and protein estimation by absorbance at 280 nm, bicinchoninic acid (BCA) assay (Pierce, USA) and 2D Quant assay (GE Healthcare, Australia). The same protocol was used to purify RgpB from P. gingivalis HG66 culture supernatants. The concentrations of the RgpB (MW 55,636, 507 aa, , ε =54x103 M ^'1 cm-1) and rKgp (MW 50,114 Da, 454 aa, ε =105x103 M ^"1 cm-1) were determined by measuring the absorbance at 280 nm using a 96 well UV plate and a PerkinElmer 1420 Multilabel Counter VICTOR3™ reader. The purified rKgp and RgpB proteins were subjected to trypsin hydrolysis and then LC-MS MS analysis. The tryptic peptides were derived solely from the respective proteinase with no contamination by other proteins. The purified rKgp (0.66 U/mg) exhibited no Arg-X proteolytic activity and the purified RgpB (5 U/mg) exhibited no Lys-X proteolytic activity.

Production and Purification of Recombinant Kgp and RgpB Propeptides

Recombinant Kgp and RgpB propeptides were produced with an -terminal hexahistidine tag followed by the thrombin cleavage sequence to enable the binding to Ni-affinity resin with release following thrombin cleavage. DNA encoding the propeptide of P. gingivalis W50 Kgp (aa 20-228; O07442_PORGI)[30] or P. gingivalis W50 RgpB (aa 25-222; PG0506, CPG2_PORGI)[14] was amplified by PCR using the genomic DNA of strain W50 as a template and BIOTAQ DNA polymerase. Primer pair Kgp-PP-for and Kgp-PP-rev and primer pair Rgp-PP-for and Rgp-PP-rev, containing Ndel and Xhol RE sites and a stop codon in the antisense oligonucleotide were used for PGR of Kgp and Rgp propeptide coding DNAs respectively. The PCR products were ligated into pGEM-T Easy vector and the inserts sequenced. The plasm id inserts were then excised using Ndel and Xhol cleavage then ligated into Ndel/Xhol cleaved pET-28b expression vector (Novagen) and used to transform E. coii a-Gold Select cells. The recombinant plasmids were isolated and the insert was sequenced to verify correct amplification and ligation.

The recombinant pET-28b vectors were then transformed into E. coti BL-21 (DE3) (Novagen) and gene expression induced by addition of 1 mM isopropyl β-Ο-1- thiogalactopyranoside to cultures (ODeoo nm -0.5-0.7) growing in Luria-Bertani medium [37]. After 4 h of induced expression the cells were harvested by centrifugation (8,000 g. 20 min, 4°C), suspended in lysis buffer (50 mM a2HP0 , 300 mM NaCI, 10 mM imidazole, pH 8.0) and disrupted by sonication (4 s on, 8 s off, 32% amplitude, for 15 min with a tapered 6.5 mm microtip) and stirring (30 min, 4°C). The lysate was centrifuged at 15,000 g for 15 min and the recombinant propeptides purified from the supernatant using Ni affinity chromatography with a modification of the procedure of Hondoh et a/. (2006) [38]. Briefly, a 50% Ni-NTA (Qiagen) slurry (4 ml_) was added to the supernatant, which was then stirred for 15 min at 4°C. The mixture was loaded on an open column with a volume of 20 mL and the flow through was removed. The resin was washed twice with 10 mL of purification buffer (50 mM Na ₂ HP0 ₄ , 300 mM NaCI, 20 mM imidazole, pH 8.0). The column was stoppered and purification buffer (2 mL) containing 25 NIH units of thrombin (Sigma) was added to the slurry and incubated for 2 h at room temperature to cleave the propeptide His-tag and release propeptide from the nickel resin. The released propeptide and thrombin protease were then washed from the column using 15 mL of purification buffer and this solution was loaded onto a stoppered column containing 1 mL of Benzamidine Sepharose resin (Pharmacia). The solution was left to incubate for 15 min at room temperature to enable the thrombin protease to bind to the Benzamidine Sepharose resin. Once the flow through fraction was collected, the resin was washed twice with 2.5 mL of wash buffer (5 mM Na ₂ HP0 ₄ , 50 mM NaCI, at pH 8.0) and each of the washes collected. The flow through fraction was then combined with the two wash fractions, resulting in a 20 mL solution. The extract was concentrated through a 3 kDa MWCO filter (Amicon) and applied to a gel filtration column (HiLoad 26/600 Superdex 75) attached to an AKTA-Basic FPLC system and eluted with 50 mM Tris-HCI, 150 mM NaCI, at pH 8.0 at a flow rate of 2 mL/min. The eluate was monitored at 280 and 215 nm. The eluate was collected, concentrated with a 3,000 MWCO Amicon centrifugal filter unit and the concentrations of the rKgp propeptide (MW 23,403 Da, 213 aa, ε= 11 ,920 ^*1 cm-1) and the rRgpB propeptide (MW 23,204 Da, 209aa, ε= 10,430 M ^" cm-1 ) determined using absorbance at 280 nm.

MALDI-TOF MS Analysis

Peptides and proteins were identified using an Ultraflex MALDI TOF/TOF Mass Spectrometer (MS) (Bruker, Bremen, Germany) and LC-MS. The samples were co-crystallized (1 :1 v/v) on an MTP Anchorchip™ 800/384 TF plate with saturated 4- hydroxy-a-cyanocinnamic acid matrix in standard buffer (97% acetone, 3% 0.1% TFA). The samples were analysed using Bruker Daltonics FlexAnalysis 2.4 and Bruker Daltonics BioTools 3.0 software with fragmentation spectra matched to an in-house P. gingival® ^' database installed on a local MASCOT server.

In-gel Digestion and LC-MS Analysis

Protein bands were excised from the Coomassie® blue-stained SDS-PAGE gel, and analysed by LC-MS MS as published previously [39]. The tryptic digests were acidified with trtfluoroacetic acid (TFA) to 0.1 % before online LC-MS/MS (UltiMate 3000 system, Dionex) with a precolumn of PepMap C18, 300 mm (inner diameter) x 5 mm (Dionex) and an analytical column of PepMap C18, 180 mm (inner diameter) x 15 cm (Dionex). Buffer A was 2% (v/v) acetonitrile and 0.1% (v/v) formic acid in water and buffer B was 98% (v/v) acetonitrile and 0.1% (v/v) formic acid in water. Digested peptides (5 μΐ) were initially loaded and desalted on the precolumn in buffer A at a flow rate of 30 μ Urn in for 5 min. The peptides were eluted using a linear gradient of 0-40% buffer B for 35 min, followed by 40-100% buffer B for 5 min at a flow rate of 2 μϋιτίίη directly into the HCTultra ion trap mass spectrometer via a 50 mm ESI needle (Bruker Daltonics). The ion trap was operated in the positive ion mode at an MS scan speed of 8100 m/z/s over an m/z range of 200-2500 and a fast Ultra Scan of 26000 m/z/s for MS/MS analysis over an m/z range of 100-2800. The drying gas (N ₂ ) was set to 8-10 L/min and 300°C. The peptides were fragmented using auto-MS MS with the SmartFrag option on up to five precursor ions between m/z 400-1200 for each MS scan. Proteins were identified by MS/MS ion search using Mascot v 2.2 (Matrix Science) queried against the P. gingivalis database obtained from J. Craig Venter Institute (JCVI.ORG). Intact Protein Analysis

An accurate molecular weight mass of the protein was determined using an Agilent 6220 Q-TOF by direct infusion Electrospray Ionization (ESI Q-TOF). The mass spectrometer was operated in positive MS only mode and data were collected from 100 to 2500 m/z. Internal, reference masses of 121.0508 and 922.0097 were used throughout. Deconvolution of the mass spectra was carried out using the Agilent Mass Hunter Qualitative Analysis software (B.05) and protein masses were obtained using maximum entropy deconvolution.

Protease Inhibition Assays

Lys- and Arg-specific proteolytic activity was determined using the synthetic chromogenic substrates N-(p-tosyl)-Gly-Pro-Lys 4-nitroanilide acetate salt (GPKNA) and N-ben∑oyl-DL-arginine-4-nitroanilide hydrochloride (BapNA) (Sigma Aldrich), respectively. The protease assays were conducted as described previously [18]. The assay mixture contained P. gingivalis W50 whole cells (final cell density of -3 x 10 ⁷ cells/mL) or either the RgpB or rKgp (3.3 - 8.5 mg L) proteases, recombinant propeptides at various concentrations, 1-10 mM cysteine pH 8.0, 5 mM dithiothreitol (DTT) and 1 mM chromogenic substrate made up to a final volume of 200 μΐ with TC150 buffer (50 mM Tris-HCI, 150 mM NaCI, 5 mM CaCI ₂ , pH 8.0). Protease inhibitor, Afa-p-tosyl-l-lysine chloromethylketone (TLCK) (1 mM) treated rKgp and RgpB proteases and blank wells with no proteases were used as negative controls. The caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[0-methyl]- fluoromethylketone; Sigma USA) was used to selectively inhibit rKgp. Substrate cleavage was determined by measuring the absorbance at 405 nm at 10 s intervals for -20-30 min at 37°C using a PerkinElmer 1420 Multilabel Counter VICTOR3 ^w . The proteolytic activity of the W50 whole cells and the RgpB/ rKgp enriched fraction was determined as Units/10 ¹¹ cells and Units/mg respectively, where 1 unit is equivalent to 1 μιτιοΐθ p-nitroanilide released/min.

RgpB and rKgp protease activity was also determined using DQ™ Green bovine serum albumin (DQ-BSA) (Molecular Probes, USA) [18,40-41]. The assay mixture contained rKgp or RgpB (3.3 - 8.5 mg L), recombinant propeptides (various concentrations), 1-10 mM cysteine, 5 mM DTT and DQ BSA (10 L; 0.1 g L), made up to a final volume of 200 μΐ with TC150 buffer. Negative controls were prepared as described earlier. The assay mixtures were incubated in the dark for 3 h at 37°C prior to measuring fluorescence using a fluorometer (PerkinElmer 1420 Multilabel Counter VICTOR3™).

Samples from each well were analysed for propeptide and protease hydrolysis using SDS-PAGE. Each sample (3 x 200 μΐ) was concentrated using a 3 kDa MWCO Amicon centrifugal filter unit at 14,000 g for 5 min. The concentrate was denatured using 5% (v/v) 1 M DTT and 25% (v/v) x4 reducing sample buffer with heating for 10 min at 70°C unless otherwise stated. After microcentrifugation, 20-30 μΐ was loaded onto a precast 8-12% gradient Bis-Tris gel. SeeBlue* Pre-Stained standard was used as a molecular marker and a potential difference of 140 V and MES buffer (Life Technologies, Australia) were used to run the gel. The gel was stained with Coomassie* Brilliant Blue (G250) overnight and destained in deionised water.

Propeptide Specificity

The specificity of each propeptide for its cognate enzyme, was examined by incubating the propeptide (50 mg/L) with the other gingipain at 7.5-8.5 mg/L concentration. The cross-reactivity with papain (P3375, Sigma) (2.75 g/L) was examined using BapNA as a substrate. The cross-reactivity with 60 Units of caspase (BML-SE 169-5000, Enzo LifeSciences) was examined using (200 μΜ) Ac-DEVD-pNa (ALX-260-048-M005, Enzo LifeSciences) substrate.

Determination of Type of Inhibition and Inhibition Constants

Inhibition kinetics were determined using purified rKgp (7.5 mg/L) and RgpB (8.5 mg L) in the chromogenic substrate assay as described above, initial reaction rates were obtained at substrate (GPKNA/BapNA) concentrations of 0.125, 0.25, 0.5, 0.75, and 1 mM and inhibitor (DTT-stabilised monomer of rKgp rRgpB propeptide) concentrations of 0 to 200 mg L. The proteolysis by rKgp (3.3 mg/L) was also examined using the fluorescent BSA substrate with rKgp propeptide concentrations of 2.5 - 50 mg/L The initial rates of reaction were plotted against substrate concentrations. The curves were fitted individually by non-linear regression analysis to the Michaelis-Menten expression: v - d[P]] dt = Vmax[S]/(Kni + [S]) using the program Kaleidagraph (Synergy Software). The calculated Km and V _ma x parameters of the proteolytic assays with increasing inhibitor concentrations were not consistent with competitive inhibition. Subsequently, the Km value derived from the control experiment without inhibitor was fixed and used for all subsequent fitting of the data sets with increasing inhibitor concentrations. The reciprocal of the \/ _mm values derived from the fitted curves were plotted against the inhibitor concentrations. K| was obtained from the x-intercept value.

Statistical Analysis

Protease activity data were subjected to a single factor analysis of variance

(ANOVA). When the ANOVA indicated statistical significant difference (p < 0.05) between the means of tested inhibitors, a modified Tukey test was performed on the data [42-43].

Analysis of Proteinase Stability and Enzyme Kinetics

Both RgpB and rKgp were stable at 4°C at pH 5.3 for several months without loss of activity. The Km for RgpB with the substrate BapNA was 64 μΜ and activation was dependent on the cysteine concentration in the proteolytic assay. Similar to RgpB the level of rKgp activation was dependent on cysteine concentration in the proteolytic assay and glycyl-glycine at 10 mM enhanced rKgp hydrolysis of the substrate GPKNA two-fold. The K _m value for rKgp was 46 μΜ consistent with the Km value of 50 μΜ using the same substrate GPKNA, reported for Kgp isolated from P. gingivalis HG66 that releases the Lys-gingipain with associated adhesins into the culture fluid [44]. The Kcat was 4.5 s ^'1 and the I Km parameter representing the catalytic efficiency was 6.3 x 10 ^{4 "} s- ¹ . Since the synthetic small molecule chromogenic substrates are not the natural substrates in vivo, a fluorescently-labelled protein substrate, DQ-BSA with 23 arginines and 59 lysines was also used as a substrate to measure the proteolytic activity. Trypsin- iike proteases cleave the self-quenched DQ-BSA releasing peptides with an average length of less than 8 amino acids [45]. Since the DQ-BSA is a multisite substrate the observed K _m is an average over all sites. Based on the equation below, the time course data were fitted to the expression with the assumption that the total product formed P«. exactly equals So and where S < Km.

A = 5o[l - exp(- K*/K*)Eu)]

Using this assay with DQ-BSA as substrate the catalytic efficiency I m for rKgp was 5.00 x 10 ³ M ¹ s ^*1 and for RgpB was 7.75 x 10 ^{3 *1} s ^'

Expression and Purification of Kgp and RgpB Recombinant Propeptides The Kgp and RgpB recombinant propeptides were designed to contain His-tag sequences followed by a thrombin cleavage site that was N-terminal to the mature propeptide sequence (Figure 18). The recombinant propeptides were expressed in E coli and purified by binding the His-tagged propeptide to a nickel-sepharose affinity column, followed by thrombin cleavage to remove the His-tag and benzamidine- sepharose treatment to remove thrombin contamination. The purification of the Kgp propeptide is shown in Figure 19A. After size exclusion chromatography of the thrombin-cleaved recombinants, both Kgp and Rgp recombinant propeptides were deemed pure by SDS-PAGE and MS, with yields of 10-13 mg/L culture fluid. LC-MS analysis of tryptic peptides also confirmed the expected sequences of both propeptides. MS analysis using ESI Q-TOF showed a deconvolved protein mass of 23,407.8, corresponding to a molecular mass of 23,403 Da for the thrombin cleaved Kgp propeptide and a deconvolved protein mass of 23,205.3, corresponding to a molecular mass of 23,204 Da for the thrombin cleaved RgpB propeptide.

Example 4 - Dimerisation of Kgp Propeptide

Initial studies with the Kgp-propeptide yielded inconsistent inhibition results. The Kgp propeptide exhibited the propensity to dimerize at higher concentrations as found in the cell lysate, Ni-affinity column-bound and thrombin-free products (Figure 19) and as detected by the relative K _av during size exclusion chromatography (Figure 19B). The monomer-dimer equilibrium at room temperature was evident for both Kgp propeptide monomer and dimer fractions as observed from the SDS gel within 1 h of separation by chromatography. The involvement of the single cysteine residue within the propeptide amino acid sequence in this dimerisation was investigated. Size-exclusion chromatography of the eluted dimer fractions incubated with 5 mM DTT demonstrated release of monomer. SDS-PAGE of the dimer and monomer fractions with and without 5 mM DTT confirmed the involvement of the cysteine residue (Figure 19C). Following the DQ-BSA substrate assay with the Kgp propeptide monomer and dimer in equilibrium, post-assay contents revealed that precipitation occurred on standing, suggestive of enzyme propeptide interactions. However the precipitation was not observed in the assays with added 5 mM DTT using the Kgp propeptide DTT-stabilized monomer, lodoacetylation of the Kgp propeptide after DTT treatment prevented dimer formation based on Superdex G75 size-exclusion chromatography and non-reducing PAGE analysis. Reproducible inhibitory activity was achieved with the monomer purified in the presence of 5 mM DTT using size-exclusion chromatography, with additional 5 mM DTT plus 10 mM cysteine in the proteolytic assays. These assay conditions ensured that the protease rKgp was fully reduced thus producing higher activity of the mature enzyme and a reproducible dose inhibitory response in both assays using DTT-stabilized monomer Kgp-propeptide. In the proteolytic assay with the chromogenic substrate, activity of rKgp (0.15 μΜ) increased by 49 ± 1% with the addition of 5 mM DTT. In the DQ-BSA assay, addition of 5 mM DTT produced a 25 ± 4% enhancement of activity.

Propeptide Inhibition of Cognate Proteases

The inhibition of P. gingivalis W50 whole cell proteolytic activity by the Kgp and

RgpB recombinant propeptides was determined using chromogenic substrates. The rate of substrate hydrolysis was monitored for linearity, to ensure there was no sharp increase in absorbance during the assay which would indicate that the inhibitory peptides were being used as a preferred substrate. The Kgp propeptide exhibited -35% inhibition of P. gingivalis W50 whole cell Lys-protease activity at 80 mg/L, while the RgpB propeptide exhibited 41% inhibition of W50 whole cell Arg-protease activity at 80 mg/L (Table 4).

To establish targeted inhibition of the catalytic domain of the proteases, the propeptide was incubated with purified RgpB or rKgp. Using both chromogenic and fluorescent DQ-BSA assays, rKgp and RgpB were inhibited by their propeptides in a dose-dependent manner (Figures 20 and 21). The DTT-stabilized monomer at 100 mg L (4 μΜ) demonstrated 68% inhibition of 0.15 μΜ rKgp compared to negligible 0-5% inhibition by the dimer with GPKNA as substrate (Figure 27). Similarly, in the DQ-BSA assay the DTT-stabilized monomer at 100 mg L (4 μΜ) demonstrated 57% inhibition of 0.15 μΜ rKgp compared to negligible 0-4% inhibition by the equivalent dimer (Figure 27). The iodoacetylated monomer (100 mg/L) demonstrated 28 ± 5% inhibition in the proteolytic assay using DQ-BSA as substrate. The RgpB recombinant propeptide at a concentration of 10 mg/mL inhibited ~ 95% of RgpB activity.

The thrombin-like capability of the proteinases to cleave small molecule substrates while bound to inhibitors [46] was examined. The proteolysis assays with increasing concentrations of inhibitor were conducted with excess substrate. Fluorescence analysis of the 96-well plates 6-12 h after the proteolysis assay with DQ- BSA was consistent with the original inhibitor dose-response observed during the assay. In contrast the proteolysis assay using the small chromogenic substrates revealed that substrate consumption continued for a further 6-12 h irrespective of the presence and level of propeptide inhibitor. One interpretation for this observation is that the propeptide-protease interaction allowed small molecules to still have access to the active site, however larger substrates were blocked.

Propeptide Selectivity and Specificity

Both RgpB and Kgp propeptides demonstrated selectivity for their own cognate protease with no inhibition observed when Kgp propeptides were incubated with RgpB and vice versa (Table 4). The specificity of the propeptides was further examined using two examples of cysteine proteases. The cysteine protease papain (2.75 mg/mL). with a propeptide of 115 residues, was not significantly inhibited by Kgp nor RgpB propeptides at 50 mg/L concentrations (Table 4). The cysteine protease caspase 3 that has structural homology with the RgpB and Kgp catalytic domains also was not inhibited by either Kgp or RgpB propeptides.

Determination of Type of Inhibition and Inhibition Constants

In order to determine the inhibition constant of Kgp and RgpB propeptides and characterize inhibition mechanism, a kinetics analysis was performed with purified rKgp and RgpB. The dissociation constant Kj ^' , for non-competitive binding of the inhibitor Kgp propeptide to the enzyme rKgp, was 2.01 μΜ for the monomer. The inhibition kinetics were also analysed for the fluorescent multi-site substrate DQ-BSA and the derived K| ^' parameter was 2.04 μΜ. The RgpB propeptide also displayed non-competitive inhibition kinetics against RgpB with a Kj ^' of 12 nM (Figure 22).

Analysis of Propeptide Stability

The Kgp propeptide contains 13 Lys residues which could make the propeptide a potential substrate for Kgp proteolytic activity. To examine the fate of the Kgp recombinant propeptide in the presence of the proteases, the post-assay contents were analysed using SDS-PAGE and HPLC. The SDS-PAQE gels and HPLC chromatograms revealed intact Kgp and RgpB propeptides as well as degradation products that were then further analysed by LC-MS. Identification of the tryptic peptides coupled with the expected sizes of the Kgp propeptide fragments enabled a fragmentation pattern to be derived. Lys ¹¹⁰ was the most susceptible to cleavage by the proteinase. Lys residues 4, 41 , 69, 100, 129, 168 and 204 were also found to be susceptible to cleavage. In contrast, Lys residues 6, 22, 37, 84, and 116 were relatively resistant to proteolytic cleavage by Kgp. Kgp propeptide Arg residues at position 13, 146, and 149 were also observed to be relatively resistant to proteolysis by RgpB. The observation of Lys and Arg residues that are relatively proteolytically resistant to cleavage by Kgp and RgpB is indicative that the long propeptides have conformational preferences. To improve resistance of the propeptides (or fragments) to proteolysis, the non-structurally conserved, non-interacting lysines and arginines can be substituted by any natural or non-naturally occurring amino acid, preferably glutamine or asparagine thus maintaining polarity and alpha-helical propensity. For propeptides derived from or with similarity to rKgp-PP, substitution or modification can be at any one or more lysines namely 4, 110, 100, 129, 69, 168, and 41 or equivalent to account for minor variations and deletions between strains. For propeptides derived from or with similarity to rRgpB-PP, substitution can be at any one or more of Arg 102, Lys 97, Lys 121 , Arg 66, Arg 158, and Lys 39 or equivalent to account for minor variations and deletions between strains. For propeptides derived from or with similarity to rRgpA-PP, substitution or modification can be at any one or more of Arg 103, Lys 98, Lys 122, Arg 67, Arg 159, and Lys 40 or equivalent to account for minor variations and deletions between strains.

In vivo Processing of Secreted rKgp Precursor Forms

A culture of P. gingivalis ECR368 was examined at Days 1 (exponential growth) and 3 (stationary phase) after inoculation. A reducing SDS-gel of the cell free culture fluid revealed the presence of precursors with estimated sizes of -70 and 60 kDa designated Full-ProKgp and Half-ProKgp respectively (Figure 23). These are consistent with precursor forms of the gingipains reported previously [47-481. The ~60 kDa intermediate present at equivalent or greater abundance indicates that the sequential cleavage rates k ₂ <ki. The presence of an intra-molecular disulphide bond within the -60 kDa precursor form was investigated. A non-reducing SDS-gel (Figure 23) of the 60 kDa precursor revealed the presence of a higher molecular weight ~70 kDa form, indicating that in a small population the 1st half of the propeptide although cleaved was still covalently attached to the Kgp catalytic domain through a disulphide bridge. The stable intermediate precursors with extra 0 kDa or 20 kDa propeptide regions edited earlier than the mature Kgp as expected, from Superose 12 in 50 mM phosphate, 150 mM NaCI, pH 6. Propeptide-mediated Inhibition of P. gingivals Growth

P. gingivalis W50 was grown in a protein-based minimal medium and reached a maximum cell density equivalent to an OD 620nm of 0.32 after 40 h of incubation. The P. gingivalis triple gingipain mutant lacking RgpA, RgpB and Kgp does not grow in this defined protein-based minimal medium confirming that gingipain proteolytic activity is essential for the breakdown of the proteins (BSA and haemoglobin) in this medium. Both Kgp and RgpB propeptides demonstrated a significant inhibitory effect on P. gingivalis W50 growth in this protein-based minimal medium (Table 5).

Despite recognition that the traversal of the Arg- and Lys- gingipains from the cytosol to the final cell surface destination is accomplished without premature activation, the role of the gingipain propeptides has not been extensively investigated. This current study has demonstrated that Kgp and RgpB propeptides inhibit the proteolytic activity of the membrane bound proteinases of P. gingivalis W50 in whole cell assays. To demonstrate targeted inhibition, characterise the mode of inhibition, and investigate the inter-molecular proteinase-propeptide interaction, cognate catalytic domains were purified from strains HG66 (RgpB) and ECR368 (rKgp).

In contrast to the nanomolar K| estimated for the RgpB recombinant propeptide, a micromolar K| was calculated for the Kgp propeptide. This has been attributed to the tendency of the Kgp propeptide to form covalent dimers through a single cysteine residue. The inhibitory capability of the mixture of monomer/dimer rKgp propeptides added to the proteolytic assay was inconsistent. This was resolved after separation of the DTT stabilized monomers from the non-inhibitory dimers using size-exclusion chromatography in 5 mM DTT.

The recent report of the RgpB propeptide co-crystallized with the cognate RgpB catalytic domain indicates that the propeptide attaches laterally to the RgpB catalytic domain through a large concave surface. The RgpB propeptide adopts an overall "croissant " shape with a projecting " inhibitory" loop consisting of sixteen residues (Lys113- Glu128) that approaches the active-site cleft of RgpB on its non-primed side in a substrate-like manner [49].

Observation of the precursor ProKgp (-70 kDa) with the intermediate half-

ProKgp ( 60 kDa) by reducing SDS-PAGE, at equivalent or greater abundance in the culture fluid during exponential growth of the P. gingivalis mutant ECR 368 indicates that the second cleavage step is slower than the first cleavage step. Although precursor forms have been observed for both RgpB and Kgp [47-48], the presence of the isulphide bridge between the Kgp propeptide and catalytic domain in the precursor form has not been reported previously and may have resulted from oxidation during extraction. This observation can not be explained by the reported structure of the RgpB propeptide interacting with the RgpB catalytic domain [49]. The catalytic domain of Kgp has four cysteines: Cys ²⁰⁰ , Cys ²⁴⁸ , Cys ²⁴⁹ and Cys ²⁶⁰ (Figure 24A). The observed in vitro inhibition by the discrete Kgp propeptide is not dependent on the formation of a disulphide bridge between the propeptide and catalytic domain as the inhibition is retained both in a reducing environment and by the iodoactylated Kgp propeptide. However disulphide bridge formation within the precursor form does occur in a non- reducing environment.

From a model of Kgp (Figure 24A) based on the RgpB structure, the catalytic cysteine is the most exposed and hence most likely to form a disulphide bond. The effect of the propeptide cysteine forming a disulphide bridge with either the catalytic cysteine Cys ²⁴⁹ or the neighbouring Cys ²⁴⁸ would have the effect of abolishing Lys- protease activity in the 70 kDa precursor form. This would be consistent with the recent report that an active site probe, a biotinylated irreversible Kgp-specific inhibitor [50] did not bind to the active site of the 70 kDa precursor form under non-reducing conditions [48]. However it is also plausible that the cleaved N-terminal half of the Kgp propeptide forms a disulphide bridge with one of the other two cysteines within the mature Kgp protease: Cys ²⁰⁰ found only in Kgp, or Cys ²⁶⁰ , common to both RgpB and Kgp (Figure 24A). In the model of the mature proteinase, both these cysteines are less accessible for bridge formation; however, accessibility may be altered in the precursor form. To understand the observed strong selectivity of the propeptides for the cognate proteases, the sequence variation of the RgpA/B and Kgp propeptides and the catalytic domains from the P. gingivalis strains W50, W83, ATCC 33277, TDC60, 381 , W12 was examined. The RgpA/B and Kgp propeptides from the known P. gingivalis strains are all highly conserved with a calculated percentage identity (%ID) of 98-100% between the propeptide homologs. However sequence conservation is less between the RgpA and RgpB propeptide paralogs (75-76% ID ) and between the RgpA B and Kgp propeptide paralogs (20-22% ID). Similarly the sequences of the catalytic domains of RgpA B and Kgp are also highly conserved (94-100% ID) between the homologs with less conservation between the paralogs. This is consistent with the observed selectivity.

The specificity of the propeptides for the gingipains was examined using two examples of cysteine proteases. Since, the three gingipain propeptides range from 203 to 209 residues, significantly larger than the average propeptide lengths of ~40 residues observed in most cysteine proteases [26], the 212 residue papain that is inhibited by its own 115 residue propeptide was selected. Neither Kgp nor RgpB propeptides demonstrated any inhibition for papain consistent with the differences between the papain and gingipain catalytic domains and active site configurations.

The second example was selected based on the structural similarities of the catalytic domains. Caspase 3 (pdblpau) and RgpB structures (pdblcvr) [51-53] share a common "caspase-hemoglobinase" fold with similar active site pockets despite limited sequence similarity [54]. The mature caspase 3 enzyme and zymogen backbone structures can be superimposed to within 38 A over 106 residues. The current understanding of caspase activation and the caspase structure, presented a compelling argument to examine the effects of Kgp and RgpB propeptides on caspase activity. The absence of inhibition exhibited by both propeptides against caspase 3, highlights the specificities of the 200 residue propeptides.

Both RgpB and, by homology, Kgp catalytic domains have the appearance of two adjacent caspase sub-domains plus the C-terminal Ig-fold [54]. The RgpB active site cysteine and histidine occur in the second caspase sub-domain and their respective Ca atoms are within 6.3 A. In the first caspase sub-domain the RgpA/RgpB sequences have a cysteine (Cys ¹¹⁵ ) and histidine (His ⁷⁹ ) at topologically analogous positions (Figure 24B). The catalytic potential of these two residues in RgpB and RgpA has not been explored. However, this difference between Kgp and RgpB/RgpA may also account for the selectivity exhibited by the cognate propeptides.

To further understand the interaction between the conserved propeptides and the cognate proteases, the residues within the catalytic domains of RgpA, RgpB and Kgp that differ between the different strains of P. gingivalis were identified. These point mutated residues found within RgpA and RgpB were mapped against the crystal structure of RgpB. This revealed that the residues located on the first a-helix immediately C-terminal of the fifth β-strand and the N-terminal portion of the next a-helix are conserved. This surface-exposed, conserved patch is depicted between the position of the known N -terminal residue of the catalytic domain and the active site (Figure 25A). In the case of Kgp, 28 residues that differ between different strains of P. gingivalis were identified. Three residues within 10 A of the catalytic site were changed: A4 9S, L454S, and I478V. Interestingly, the A449S and L454S point mutations are found together in F5XB86 (TDC60), Q51817 (W83), and Q6Q4T4 (an un-named strain) making a small region close to the catalytic site of Kgp more hydrophilic in those strains. Mapping all the 28 point-mutated residues to the model of Kgp revealed an analogous surface- exposed, structurally identical, conserved patch in Kgp. The surface-exposed, conserved patches in RgpB and Kgp are predicted to be covered by the propeptide in the respective zymogens.

Models of both Kgp and the Kgp propeptide were produced using Orchestrar from within Sybyl-8.1 [55] and based on the X-ray crystal structure of RgpB (lcvr.pdb) [56] and chain A from the crystal structure of RgpB co-crystallized with its propeptide (4ief.pdb) [49] respectively. The Kgp propeptide model was validated by calculating the 'Fugue alignment' [57] between the Kgp and RgpB propeptides, which gave a Z-score of 0.72 classified as 'certain' with greater than 99% confidence. The model of the propeptide had an rms deviation of 1.28 A from the crystal coordinates after energy minimization to a maximum gradient of 0.5 kcal mol ^"1 A ^'1 using the AMBER force-field. A model of the Kgp propeptide docked with Kgp was then produced by independently aligning by least-squares the model of Kgp and the model Kgp propeptide against the B and A-chains respectively of the co-crystallized RgpB/RgpB propeptide (4ief.pdb). This alignment predicts that the Lys ¹¹⁰ of the inhibitory-loop of the Kgp propeptide will insert into the catalytic pocket of Kgp. A schematic representation of the inhibition of Kgp by its propeptide based on this model is shown in Figure 25B. From the model structure the cleavage of the propeptide at Lys ¹¹⁰ will leave a substantial protein domain still capable of allosterically blocking access to the catalytic site by large, substrate proteins. The bound orientation of the propeptides with their proteases is consistent with an interaction between the identified conserved patch (Fig 25A) and the propeptide. The schematic (Fig 25B) is also consistent with possible exosite binding that could explain the selectivity and specificity of the propeptides. Experimentally, the peptide bond C-terminal to Lys ¹¹⁰ was found to be susceptible to cleavage by Kgp. This is consistent with the location of Lys ¹¹⁰ being in a loop; the peptide bond is only protected from cleavage when the propeptide is bound to Kgp with the appropriate orientation.

it was interesting to examine the effects of the propeptides on growth of P. gingivals. The requirement of cell surface located proteinases for nutrient acquisition, tested using the triple mutant without the RgpA, RgpB and Kgp gingipains in a protein- based minimal medium was consistent with previous reports [41,58]. The observed retardation of the planktonic growth of P. gingivalis by the added propeptides highlights their potential for inhibition of P. gingivalis growth and virulence.

In summary the P. gingivalis cell surface gingipains are carefully regulated prior to activation by high-selectivity propeptides that are tailored to each proteinase. It is possible that the long propeptide has a role in propeptide-mediated folding as well as preventing proteinase premature activation throughout the multiple processing, propeptide detachment, and rearrangement events that occur to enable the cell surface assembly of the gingipain complexes.

It should be understood that while the invention has been described in detail herein, the examples are for illustrative purposes only. Other modifications of the embodiments of the present invention that are obvious to those skilled in the art of molecular biology, dental treatment, and related disciplines are intended to be within the scope of the invention.

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Table 2. Plasmids used in the course of this study.

Plasmids Description" Reference pEC474 pBR322::ce A [32] pCS19 pGEM-T Easy: :cep This study pUC18.:3521 nt BamHI fragment of P. gingivaiis W50

pNS1 [30]

encompassing the 3' of PG1842 and the 5' of kgp pNS1 with nucleotide T405C, A414G, T418C, A419C

pNS2 This study mutations to produce Apal and EcoRV recognition sites. pNS2::cepA cepA replaces nt that include the kgp promoter pPC1 This study and kgp nt coding from e^-Tyr ⁷⁴ * ermF ligated between the Apal and EcoRV sites of pNS2.

pPC2

ermF upstream of the kgp promoter. pPC3 pPC2 excluding kgp codons 681-710 This study pET-28b Expression vector Novagen pKgpPPI Insert in pGEM-T Easy codes Kgp propeptide, residues 20-228 This study pRgpPPI Insert in pGEM-T Easy codes Rgp propeptide, residues 25-222 This study

Insert from pKgpPPI in pET28b codes Kgp propeptide,

pKgpPP2 This study residues 20-228

Insert from pRgpPPI in pET28b codes Kgp propeptide,

pRgpPP2 This study residues 20-228 Table 3. Oligonucleotides used in the course of this study

Oligonucleotide Sequence*

EA-For GATTACAGTCGATATCTTGGCAAAGGGCCCATTGACAGCC

EA-Rev GGCTGTCAATGGGCCCTTTGCCAAGATATCGACTGTAATC

CepAf CGGATATAGGGACGTCAAAAGAG

CepAr GGCTACAGATACTGGACGTCTCAA

GCTTCTGCCGGTTCTTACGTAGC

ABM 1 del Rev2 ACAAGAACTGGTAACCCGTATTGTCTC

ABM 1 del Rev1 CTGC CTTCTTTACCTGAATTTGCTTGATCA

ABM 1 del For2 AATTCAGGTAAAGAAGGCAGAAGGTTCCCG

Kgp-PP^for AC GCAGC ATATGCAAAGCGCC AAGATTAAGCTTGAT

Kgp-PP rev ACGCAGCTCGAGtcaTCTATTGAAGAGCTGTTTATAAGC

Rgp-PP-for ACGCAGCATATGCAGCCGGCAGAGCGCGGTCGCAAC

Rgp-PP-rev ACGCAGCTCGAGtcaGCGCGTAGCTTCATAATTCATGAA

Restriction endonuclease sites are underlined and stop codons in lowercase

Table 4. Summary of the proteolytic activities of various purified proteases and P. gingivalis whole cell preparations in the presence of ^

o

RgpB and Kgp propeptides ©

Protease [Protease] Inhibitor [Inhibitor] (mg/L) Substrate % Proteolytic Activity g

RgpB 0.0085 mg/mL Kgp-PP 50 BapNA 105±3

0.0075 mg/mL RgpB-PP 50 GPKNA 118±11

Caspase 3 60 Units Kgp-PP 100 Ac-DEVD-pNa (200 μΜ)

Caspase 3 60 Units RgpB-PP 100 Ac-DEVD-pNa (200 μΜ)

Papain 2.75 mg/L Kgp-PP 100 BapNA 102±10

Papain 2.75 mg/L RgpB-PP 100 BapNA 103±17

Whole cell W50 3.2x 10 ⁷ cells Kgp-PP 40 80 GPKNA 92±23 65±29

H

Whole cell W50 3.2x 10 ⁷ cells RgpB-PP 40 80 BapNA 68±32 59±38 i

Table 5. Relative growth inhibition of P. gingivals in a protein-based minimal medium (MM) by RgB-propeptide (PP) and Kgp-propeptide (PP).

Percentage of growth

MM 100%

MM + RgpB-PP 100 mg/L 55±19%

MM + Kgp-PP 100 mg L 45±22%

MM + RgpB-PP 100 mg/L+ rKgp-PP 100

mg L 60±12%