USES OF HUMAN PEPTIDE DEFORMYLASE This application claims benefit of U. S. Serial Nos.

60/618,205, Filed October 13,2004 and 60/518,002, Filed November 5,2003. The contents of these preceeding applications are hereby incorporated in their entirties by reference into this application.

The invention disclosed herein was supported in part by National Institute of Health Training Grant (CA062948) and NIH Grant (CA55349). Accordingly, the United States Government may have certain rights in this invention.

Throughout this application, various publications are referenced and full citations for these publications may be found in the text where they are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION Investigators have previous reported that uncertainties remain in using gene-targeted therapies which uses nucleic acid based molecules or"antisense molecules"to inhibit gene expression at either the transcriptional or post- transcriptional level [51, 52]. These uncertainties are created by the randomness in which the antisense molecules modulate the expression of their intended targets [51,52].

Peptide deformylase (PDF) activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new

peptides are initiated with an N-formylated methionine.

This allows methionine aminopeptidase, an essential activity in organisms, to process non-N-formylated polypeptides [1-31.

Because eukaryotic cytoplasmic protein synthesis does not involve N-formylation and therefore has no need of deformylase activity, it was thought that E. coli PDF (EcPDF) would be an ideal target for the development of antibiotics with little or no mammalian toxicity [4-7]. However, genome database searches have revealed many eukaryotic PDF-like sequences in parasites, plants and mammals [8], and so far, two plant deformylases [8-10] and a parasitic deformylase [11] have been characterized.

The first eukaryotic peptide deformylase to be identified was in the higher plant, Arabidopsis thaliana. Researchers showed that the two deformylase-like genes in this plant code for functional eukaryotic PDFs [8]. These enzymes appear to be localized in the mitochondria and plastids [8, 9], both having evolutionary ties to prokaryotes. Another eukaryotic peptide deformylase was later identified and characterized in Plasmodium falciparum, the primary parasite responsible for malaria in humans [11]. This P. falciparum deformylase (PfPDF) is inhibited by actinonin and other known PDF inhibitors, suggesting PfPDF as a potential target for anti-malarial therapies [11, 12].

Sequence homology and database searches identified an open reading frame for a human PDF-like protein (HsPDF) [8]. It is composed of two exons on chromosome 16 and is homologous to bacterial, insect and plant PDFs. The cDNA sequence has been shown to be expressed at the same level in all human tissues [8]. Therefore, the data suggest that N-terminal protein processing is evolutionarily conserved and may be important in the chloroplasts or mitochondria of at least some eukaryotic organisms.

Because of the interest in various PDFs as targets for anti- bacterial and anti-malarial drugs, the possibility that HsPDF may be a functional enzyme was investigated and thus relevant to the development of human therapeutic strategies for bacteria, parasites or cancer. In addition, a known inhibitor of EcPDF, actinonin, has previously exhibited anti-leukemia activity in vitro and in vivo [13, 14]. The kinetic properties of HsPDF, which is active enzymatically and appears to differ in substrate specificity and kinetics in comparison to other previously described peptide deformylases. In addition, it is shown that actinonin inhibits HsPDF activity, suggesting a possible explanation for actinonin's cytotoxicity against various tumor cell lines.

SUMMARY OF THE INVENTION This invention provides a substance derived naturally or manufactured through chemical or genetic engineering that is capable of inhibiting the removal of N-formyl group from peptides by peptide deformylase. This substance capable of inhibiting the activity of peptide deformylase includes but is not limited to polypeptide, small molecules, and a fragment of human peptide deformylase. An ordinary skilled artisan may modify the sequence of the human peptide deformylase for inhibiting the peptide deformylase. The substance may simply include the enzymatic domain of the peptide deformylase.

This invention also provides a nucleic acid molecule encoding the above fragment of human peptide deformylase or its functional equivalent. This invention provides an isolated human peptide deformylase or its functional equivalent. The functional equivalents are compound which are capable of performing substantial enzymatic activity as the human peptide deformylase.

This invention provides methods of identifying (1) a compound that inhibits peptide deformylase or its functional equivalent, and (2) a compound that inhibits tumor growth.

The above methods each comprises of the following steps: (a) contacting the compound with peptide deformylase or its functional equivalent and an appropriate substrate which produces a primary aliphatic amine upon the action of the deformylase or its functional equivalent; (b) reacting the produced aliphatic amines with an appropriate reagent to yield a detectable marker; (c) measuring the amount of marker yielded; and (d) comparing that with the marker yielded when the compound was not present, a decrease in the

amount yielded indicating that the compound is capable of inhibiting the peptide deformylase.

This invention also provides methods for identifying (1) a compound capable of inducing apoptosis of cells, (2) a compound capable of inducing necrosis of cells, (3) a compound capable of inducing death of cells by causing denaturation of the mitochondrial proteins leading to apoptosis of cells. Each of the said method comprises of the following steps: (a) contacting peptide deformylase or its functional equivalent with said compound under conditions permitting the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed or showing inhibition of the enzyme's function.

The invention further provides a method of identifying a compound that inhibits tumor growth comprising comparison of the inhibitory concentration or binding affinity of the compound to a bacterial peptide deformylase with a human peptide deformylase, and this inhibitory concentration or binding of the compound to human peptide deformylase indicates a better inhibition of tumor growth.

This invention provides both (1) a composition comprising an effective amount of the compound identified by the above methods and a suitable carrier for inhibiting peptide deformylase and (2) a composition comprising an effective amount of the compound identified by the above methods and a suitable carrier for inhibiting tumor growth.

This invention also provides a method for preventing or treating tumor in a subject comprising administration to the subject an effective amount of the compound identified by the above methods.

This invention provides two effective siRNAs, referred to herein as"siRNA HsPDF 581-601"and"siRNA HsPDF 659-679", which are able to significantly inhibit tumor cell proliferation at a dose as low as 10 nM (See Figure 22D)- well below the concentration for nonspecific effects reported in the literature. There was no concentration- dependent effect with the target-specific siRNAs. The sequence of siRNA HsPDF 581-601 is 5'- ACCCCAAUGGAGAACAGGUTTTTUGGGGUUACCUCUUGUCCA-5'and the sequence of siRNA HsPDF 659-679 is 5'- AGGGCUGCCUGUUUAUUGATTTTUCCCGACGGACAAAUAACU-5'.

This invention will be better understood from Examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

DETAILED DESCRIPTION OF THE FIGURES Figure 1. HsPDF activity with metal cation substitution The enzymatic activity of HsPDF with different metal substitutions was measured by the FDH-coupled deformylase assay. CoCl2 (o), NiCl2 (A), or ZnSO4 (D) were added during HsPDF protein induction to make the respective metal cation substituted enzymes. In order to determine if additional metal cations in the PDF assay buffer would improve HsPDF enzymatic activity, 200AM CoCl2 (@), 200yM NiCl2 (A), or 200yM ZnSO4 (d) was added to their respective substituted enzymes. A control measurement was made without any HsPDF enzyme (x) to account for any background absorbance changes.

The graph is representative of three independent experiments.

Inset, Western blot probed with anti-His antibody demonstrating the purity of the truncated HsPDF histidine tagged fusion protein used in these studies.

Figure 2. Kinetic analysis of HsPDF activity.

(A) HsPDF activity was measured as a function of substrate formyl-Met-Ala-Ser (fMAS) using the FDH-coupled assay. (B) HsPDF activity was measured as a function of substrate formyl-Met-Leu-p-nitroanilide (fML-pNA) in the AAP assay.

All determinations are representative of at least two independent experiments. Kinetic curves were generated by . the Kaleidograph computer program using data points not reflecting substrate inhibition. See Figure 3 for the respective kinetic values derived from these curve fits.

Figure 3. Kinetic parameters for HsPDF with a comparison to other peptide deformylases.

Kinetic values for HsPDF were determined using methods described in Lee et al. , 2003. Kinetic values for PDFs from other organisms were referenced from previously published materials.

Figure 4. Actinonin inhibition of HsPDF activity.

(A) Actinonin inhibits HsPDF activity with an IC50 of 43 nM when measured using the FDH-coupled deformylase assay, and data points represent the average + sem of six independent experiments. Inset A, Structure of actinonin, a known EcPDF inhibitor. (B) Lineweaver-Burke plot of actinonin inhibition. Deformylase activity was measured using the solid phase fluorescent deformylase assay in the presence of OnM (x), 50nM (d), 100nM (A), and 250nM (A) actinonin.

Figure 5. Actinonin inhibition of human tumor cell lines Cytotoxicity was determined by measurement of inhibition of (3H)-thymidine incorporation. Actinonin is cytotoxic to APN- positive HL60 cells (@) with an IC50 of 8. 8 AM and APN- negative Daudi cells (o) with an IC50 of 5. 3 AM. The graph is representative of data from at least eight independent experiments.

Figure 6. ClustalW alignment of the deduced HsPDF amino acid sequence with bacterial, plant and parasitic PDF sequences from the NCBI database using the MacVector program Hs, H. Sapiens (NP071736); Pf, P. falciparum (NP704619); Ec, E. coli (NP417745); At, A. thaliana (AAG33973 and AAG33980).

Figure 7. Coomassie stained 12% SDS-PAGE gel showing affinity purified fractions containing HsPDF protein The protein was induced in bacterial cells harboring constructs designed to produce the N-terminally truncated protein with an additional C-terminal 6xhistidine tag. The soluble fraction was purified on a nickel-charged HisBind resin column. The fractions were electrophoresed and the proteins were transferred to PVDF membrane and detected with anti-His antibodies. Lane 1, soluble proteins; lane 2, initial column void volumn fraction; lane 3, binding buffer

eluted fraction; lane 4, wash buffer eluted fraction; lane 5, imidazole eluted protein. (MW=molecular weight markers) Figure 8. Kinetic analysis of HsPDF with different N- formylated peptide substrates (A) HsPDF activity was measured as a function of substrate formyl-Met-Ala-Ser (f-MAS) for 30 minutes in the FDH-coupled assay. (B) HsPDF activity was measured as a function of substrate formyl-Met-Leu-p-nitroanilide (f-ML-p-NA) for 30 minutes in the AAP coupled assay. (C) HsPDF activity was measured as a function of substrate formyl-Met-Ala-His-Ala (f-MAHA) for 30 minutes in the FDH coupled assay. (D) HsPDF activity was measured as a function of substrate formyl-Met- Thr-Met-His (f-MTMH) for 30 minutes in the FDH coupled assay.

All determinations are representative of at least two independent experiments. Kinetic curves were generated by the Kaleidograph computer program using data points not reflecting substrate inhibition Figure 9. Confocal microscopy images of HeLa cells transiently transfected with full length HsPDF and EYFP fusion protein Row 1 : First panel shows localization of HsPDF, second panel shows localization of a mitochondrial marker, and third panel shows the overlay of panels one and two. Row 2: All panels are identical to the first row panels and show another set of HeLa cell images from the same cell culture dish. Overlay images show that HsPDF co-localizes with mitochondria and confirm that the protein is expressed in the mitochondria.

Figure 10. Actinonin in vitro cytotoxicity against human tumor cell lines Cell lines are grouped according to tissue. IC50 values were determined for actinonin treatment by measurement of

either thymidine incorporation, leucine incorporation, XTT or MTT metabolism, or trypan blue exclusion.

Figure 11. Antitumor effect of actinonin in human xenograft mouse models (A) Actinonin given intraperitoneally or orally at the MTD against CWR22 human prostate tumor in nude mice. (B) Actinonin given intraperitoneally at 150 mg/kg to nude mice bearing A549 human non-small cell lung carcinoma (NSCLC).

Figure 12. Summary table of actinonin analog activity (uM) Compounds were tested against various cell lines (CWR22, TSU, Daudi, HL60) for cytotoxicity and against various enzymes (APN, EcPDF, HsPDF) for enzymatic inhibition.

Figure 13. Reaction involved in the direct fluorometric assay for peptide deformylase.

Figure 14. (A) Time course for the formation of 3 as a function of substrate concentration. For each substrate concentration the total amount of enzyme in reaction was 30 jUg into 50 yL and the fluorescamine assay was performed on 10 UL aliquots of this mixture for each timepoint. (B) Time course for the formation of 3 as a function of the amount of enzyme put into reaction. For each amount used the total reaction volume was 50 yL and the fluorescamine assay was performed on 10 AL aliquots for each timepoint. The substrate concentration was 4 mM.

Figure 15. (A) Fluorescamine standard curve with Met- Ala-Ser. (B) Met-Ala-Ser standard curve linear fit.

Figure 16. Velocity versus substrate concentration plot for deformylation of fMAS by Co2+-HsPDF beads after 45 minutes at 20°C and pH 7.2.

Figure 17. pH dependence of the HsPDF-beads activity with 4mM fMAS.

Figure 18. (A) Lineweaver-Burk plot for the inhibition of HsPDF by actinonin. (B) Lineweaver-Burk plot for the inhibition of EcPDF by actinonin.

Figure 19. Kinetic analysis of HsPDF activity against mitochondrial peptides (A) HsPDF activity was measured as a function of substrate formyl-Met-Ala-His-Ala (fMAHA) using the formate dehydrogenase-coupled PDF assay. (B) HsPDF activity was measured as a function of substrate formyl-Met-Thr-Met-His (fMTMH) using the formate dehydrogenase-coupled PDF assay.

All determinations are representative of at least two independent experiments. Kinetic curves were generated by the Kaleidograph computer program. Kinetic values derived from these curves are displayed in Table 1.

Figure 20. The N-terminus targets HsPDF to the mitochondria HeLa cells were transfected with full length or N-terminally truncated (aa 64-244) HsPDF tagged at the C-terminus with YFP and imaged alive by laser scanning confocal microscopy 24 hours after transfection and 30 minutes after adding MitoTrackerTM Red CMX Ros to the media. Bar represents 10 gm.

Figure 21. Chemical structures of actinonin and selected analogs with lowest IC50 against HsPDF (See Table 3).

Figure 22. siRNAs designed to target HsPDF inhibit protein expression and tumor cell proliferation. (A) siRNAs were designed to target HsPDF. The two sequences, siRNA HsPDF 581-601 and siRNA HsPDF 659-679, were the siRNAs that were effective. The numbers depicted above the sequences refer to the nucleotide number relative to the start codon of human Pdf mRNA. (B) HeLa cells stably expressing HsPDF-YFP fusion protein were transfected with 100 nM of control or HsPDF-specific siRNAs. Mean peak fluorescence determined 48 hours after transfection show that the target specific siRNAs significantly (*p<0.001) reduced protein expression.

(C) HeLa cells were transfected with 100nM of control or HsPDF-specific siRNAs. Cell proliferation was determined by tritiated thymidine incorporation over a period of 3 days following transfection. The control siRNA (&commat;) group showed no significant (p>0.05) changes in cell proliferation when compared to the no siRNA control (o) group. siRNA HsPDF 581-601 (A) and siRNA HsPDF 659-679 (t) caused significant (*p<0.05) decreases in cell proliferation after 1 and 2 days post-transfection. (D) HeLa cells were transfected with 10, 20,50 and 100 nM of control or HsPDF specific siRNAs. Cell proliferation was determined by tritiated thymidine

incorporation 2 days following transfection. The HsPDF specific siRNAs significantly (*p&lt;0. 001) inhibited cell proliferation at all four doses when compared to both the no siRNA group and the corresponding dose of control siRNA, while significant (+p<0. 001) inhibition was seen only at the higher doses (50 and 100 nM) of the control group.

Figure 23. Actinonin selectively causes mitochondrial membrane depolarization in a time-and dose-dependent manner.

RL lymphoma cells (bottom three curves) were incubated with actinonin at 10 u. g/mL (*), 20 ag/mL (A), or 100 Fg/mL (F), treated with JC-1 dye, and analyzed by flow cytometry.

Actinonin treatment led to a time-and dose-dependent depolarization of the mitochondrial membrane, as evidenced by the significant (*p<0.05) decrease in the fluorescent red to green ratio. CCCP (+) was used as a positive control and showed depolarization after 10 minutes of treatment. Normal peripheral blood lymphocytes (top three curves) were incubated with actinonin at 10 pg/mL (o), 20 pg/mL (A), or 100 Fg/mL (T), treated with JC-1 dye, and analyzed by flow cytometry. There was no time-or dose-dependent depolarization of the mitochondrial membrane with actinonin treatment, although the CCCP (0) control was effective at 10 minutes. All graphed values (mean + SD) represent a normalized fluorescence ratio (red/green) calculated by dividing the ratio for each timepoint by the ratio at time 0

(RL cells at timeo = 1. 853 + 0.079, normal cells at timeo 0.349 + 0.106).

Figure 24. Actinonin inhibits human tumor growth in xenograft mouse models. A: Athymic nude mice bearing CWR22 human prostate tumor xenografts were treated with vehicle control (o), actinonin at 250 mg/kg intraperitoneally (A), or actinonin at 500 mg/kg orally (N) twice a day for 2 weeks (excluding weekends) as soon as subcutaneous tumors were palpable. Tumor size was measured every 3-5 days with calipers. Actinonin administered either intraperitoneally or orally was able to significantly (*p<0.005) inhibit the growth of the human prostate tumor xenografts. B: Athymic nude mice bearing A549 human non small cell lung cancer xenografts were treated with vehicle control (o) or actinonin at 150 mg/kg intraperitoneally (A) once a day for 2 weeks (excluding weekends) as soon as subcutaneous tumors were palpable. Tumor size was measured every 3-5 days with calipers. Actinonin significantly (*p<0.01) inhibited the growth of the human lung cancer xenograft.

DETAILED DESCRIPTION OF THE INVENTION This invention provides a substance capable of competing against the enzymatic activity of human peptide deformylase.

In an embodiment, the substance is a polypeptide or a polypeptide derived from the naturally-occurring human peptide deformylase, a nucleic acid molecule, or a fragment of human peptide deformylase.

This invention provides an isolated human peptide deformylase or its functional equivalent.

This invention provides a nucleic acid molecule encoding the above-described polypeptide, a small interfering RNA that specifically targets human peptide deformylase mRNA or a nucleic acid molecule of at least 12 nucleotides hybridizing with the nucleic acid molecule encoding the human peptide deformylase. In an embodiment, the nucleic acid sequence is <BR> <BR> <BR> <BR> <BR> 5'-ACCCCAAUGGAGAACAGGUTT<BR> <BR> <BR> <BR> <BR> <BR> TTUGGGGUUACCUCUUGUCCA-5'or 5'-AGGGCUGCCUGUUUAUUGATT<BR> <BR> <BR> <BR> TTUCCCGACGGACAAAUAACU-5'.

This invention provides a vector comprising the above- described nucleic acid molecule.

This invention provides a small interfering RNA that specifically targets human peptide deformylase mRNA or its duplexes. In an embodiment, the small interfering RNA targets human peptide deformylase between bases 580 and 680.

This invention provides a nucleic acid molecule of at least 12 nucleotides hybridizing with the nucleic acid molecule encoding the human peptide deformylase. In an embodiment, the nucleic acid sequence is 5'-ACCCCAAUGGAGAACAGGUTT TTUGGGGUUACCUCUUGUCCA-5'or 5'-AGGGCUGCCUGUUUAUUGATT TTUCCCGACGGACAAAUAACU-5'.

This invention provides a cell comprising the above- described vector, small interfering RNA, or nucleic acid molecule. In an embodiment, the above-described substance, polypeptide or the nucleic acid molecule is linked to a solid matrix.

This invention provides a method of identifying a compound that inhibits peptide deformylase comprising steps of: (a) contacting the compound with peptide deformylase or its functional equivalent and an appropriate substrate which produces a primary aliphatic amine upon the action of the deformylase or its functional equivalent; (b) reacting the produced aliphatic amines with an appropriate reagent to yield a detectable marker; (c) measuring the amount of marker yielded; and (d) comparing that with the marker yielded when the compound was not present, a decrease in the amount yielded indicating that the compound is capable of inhibiting the peptide deformylase.

This invention provides a method of identifying a compound that inhibits tumor growth comprising steps of: (a) contacting the compound with peptide deformylase or its functional equivalent and an appropriate substrate produces a primary aliphatic amine upon the action of the deformylase or its functional equivalent; (b) reacting the produced aliphatic amines with an appropriate reagent to yield a detectable marker; (c) measuring the amount of marker

yielded; and (d) comparing that with the marker yielded when the compound was not present, a decrease in the amount yield indicating that the compound is capable of inhibiting the peptide deformylase, thereby inhibiting the tumor growth.

This invention provides a method for identifying a compound capable of inhibiting tumor growth, comprising steps of: (a) contacting peptide deformylase or its functional equivalent with said compound under conditions permitting. the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed.

This invention provides a method for identifying a compound capable of inducing apoptosis of cells, comprising steps of: (a) contacting peptide deformylase or its functional equivalent with said compound under conditions permitting the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed or showing inhibition of the enzyme's function.

This invention provides a method for identifying a compound capable of inducing necrosis of cells, comprising steps of: (a) contacting peptide deformylase or its functional equivalent with said compound under conditions permitting the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed or showing inhibition of the enzyme's function.

This invention provides a method for identifying a compound capable of inducing death of cells by causing denaturation of the mitochondrial proteins leading to apoptosis of cells, comprising steps of: (a) contacting peptide deformylase or

its functional equivalent with said compound under conditions permitting the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed or showing inhibition of the enzyme's function.

In an embodiment, the above-describe methods further comprise contacting effective concentration of the compound with tumor cells. As used herein, the functional equivalent is a substance capable of competing against the activity of peptide deformylase. In another embodiment, the peptide deformylase or human peptide deformylase or its functional equivalent is linked to a matrix. In a further embodiment, the above-described methods further comprise comparing with the result with a bacterial peptide deformylase.

This invention provides a method of identifying a compound that inhibits tumor growth comprising comparison of the inhibitory concentration or binding affinity of the compound to a. bacterial peptide deformylase with a human peptide deformylase, and this inhibitory concentration or binding of the compound to human peptide deformylase indicates a better inhibition of tumor growth. In an embodiment, the inhibitory concentration or binding affinity of the compound to human peptide deformylase, is at least two times better than the inhibitory concentration or binding to bacterial peptide deformylase. In another embodiment, the inhibitory concentration or binding affinity of the compound to human peptide deformylase, is at least five times better than the inhibitory concentration or binding to bacterial peptide deformylase. In a further embodiment, the inhibitory concentration or binding affinity of the compound to human peptide deformylase, is at least ten times better than the inhibitory concentration or binding to bacterial peptide deformylase.

This invention provides a compound identified by the above- described methods which is not previously known.

This invention provides a composition comprising an effective amount of the above-described substance, small interfering RNA, nucleic acid molecule or compound identified by the above-described methods and a suitable carrier for inhibiting peptide deformylase.

This invention provides a composition comprising an effective amount of the above-described substance, small interfering RNA, nucleic acid molecule or compound identified by the above-described methods and a suitable carrier for inhibiting tumor growth.

In an embodiment, deformylase activity in the above- described methods is detected at picomolar level. In another embodiment, the marker is a flourophore. In a further embodiment, the appropriate reagent which yields the flourophore is a flourescamine.

This invention provides a method for inhibiting growth of tumor cell comprising contacting the said tumor cell the above-described substance, small interfering RNA, nucleic acid molecule or the compound identified by the above- described methods.

This invention provides a method for preventing or treating tumor in a subject comprising administration to the subject an effective amount of the above-described substance, small interfering RNA, nucleic acid molecule or the compound identified by the above-described methods.

This invention provides a domain of human peptide deformylase or peptide whose activity is capable of being inhibited by Actinonin.

This invention provides a peptide or polypeptide comprising a domain of human peptide deformylase whose activity is capable of being inhibited by Actinonin.

This invention provides a nucleic acid molecule encoding a domain of human peptide deformylase or peptide whose activity is capable of being inhibited by Actinonin.

This invention provides a vector comprising the above- described nucleic acid molecule.

This invention provides a cell comprising the above- described vector.

Substance Capable of Inhibiting Peptide Deformylase This invention provides a substance derived naturally or manufactured through chemical or genetic engineering that is capable of inhibiting the removal of N-formyl group from peptides by peptide deformylase.

The above compound may be linked to a matrix. In an embodiment, the matrix is a solid matrix. In a further embodiment, the matrixes are beads.

As used herein, peptide deformylase is defined as enyzymes which are capable of removal of N-formyl group from peptide, which include but not limited to mammal, human, bacterial, parasite or plant peptide deformylase.

As used herein, a peptide is a molecule consisting of two (2) or more amino acids. Peptides are smaller than proteins,

which are also longer chains of amino acids. Molecules small enough to be synthesized from the constituent amino acids are, by convention, called peptides rather than proteins. The dividing line between peptide and protein is about 50 amino acids.

As used herein, a polypeptide consists of a long chain (10- 100) of amino acids linked by peptide bonds.

This substance capable of inhibiting the activity of peptide deformylase includes but is not limited to polypeptide, small molecules, and a fragment of human peptide deformylase.

An ordinary skilled artisan may modify the sequence of the human peptide deformylase for inhibiting the peptide deformylase. Said modification may include addition, deletion or mutation of certain amino acid sequence in the fragment.

The substance may simply include the enzymatic domain of the peptide deformylase. Specific antibodies against this domain may also be the substance capable of inhibiting the enzyme. As encompassed by this invention, the agent is capable of binding to peptide deformylase. As peptide deformylase is a known antigen, it would be within the skill of ordinary artisan to produce antibodies capable of binding to peptide deformylase. See Using Antibodies: A Laboratory Manual: Portable Protocol No. 1 by Ed. Harlow (1998).

This invention also provides a nucleic acid molecule encoding the above fragment of human peptide deformylase or its functional equivalent.

This invention also provides a vector of the nucleic acid molecule encoding the above fragment of human peptide deformylase or its functional equivalent. This invention

also provides an expression system comprising the above vector.

Vectors are well known in this field. Said vectors could be plasmids. See e. g. Graupner, U. S. Patent No. 6,337, 208 entitled Cloning Vector, issued January 8,2002. See also Schumacher et. al. U. S. Patent No. 6,190, 906 entitled Expression Vector for the Regulatable Expression of Foreign Genes in Prokaryotes, issued February 20,2001.

The cell which containing the vector of the nucleic acid molecule encoding the above fragment of human peptide deformylase or its functional equivalent. This invention also provides an expression system to produce said fragment.

This invention provides an isolated human peptide deformylase or its functional equivalent. The functional equivalents are compounds which are capable of performing substantial enzymatic activity as the human peptide deformylase.

In a. further embodiment, the cell of the vector of the nucleic acid molecule encoding the above peptide deformylase.

Uses of Peptide Deformylase for Screening This isolated peptide deformylase may be used for screening compounds for anti-tumor or other activities. The screening method may be a competitive assay.

This invention provides a method of identifying a compound that inhibits peptide deformylase or its functional equivalent. Said method comprises of the following steps: (a) contacting the compound with peptide deformylase or its functional equivalent and an appropriate substrate which

produces a primary aliphatic amine upon the action of the deformylase or its functional equivalent; (b) reacting the produced aliphatic amines with an appropriate reagent to yield a detectable marker; (c) measuring the amount of marker yielded; and (d) comparing that with the marker yielded when the compound was not present, a decrease in the amount yielded indicating that the compound is capable of inhibiting the peptide deformylase.

This invention also provides a method of identifying a compound that inhibits tumor growth. Said method comprises of the following steps: (a) contacting the compound with peptide deformylase or its functional equivalent and an appropriate substrate produces a primary aliphatic amine upon the action of the deformylase or its functional equivalent; (b) reacting the produced aliphatic amines with an appropriate reagent to yield a detectable marker; (c) measuring the amount of marker yielded; and (d) comparing that with the marker yielded when the compound was not present, a decrease in the amount yield indicating that the compound is capable of inhibiting the peptide deformylase, thereby inhibiting the tumor growth.

This invention also provides a method for identifying a compound capable of inhibiting tumor growth, comprising steps of: (a) contacting peptide deformylase or its functional equivalent with said compound under conditions permitting the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed.

This invention also provides a method for identifying a compound capable of inducing apoptosis of cells, comprising steps of: (a) contacting peptide deformylase or its functional equivalent with said compound under conditions

permitting the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed or showing inhibition of the enzyme's function.

This invention also provides a method for identifying a compound capable of inducing necrosis of cells, comprising steps of: (a) contacting peptide deformylase or its functional equivalent with said compound under conditions permitting the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed or showing inhibition of the enzyme's function.

This invention also provides a method for identifying a compound capable of inducing death of cells by causing denaturation of the mitochondrial proteins leading to apoptosis of cells, comprising steps of: (a) contacting peptide deformylase or its functional equivalent with said compound under conditions permitting the formation of a complex of the peptide deformylase or its functional equivalent and the compound; and (b) detecting the complex formed or showing inhibition of the enzyme's function.

As used herein, the methods above further comprising contacting effective concentration of the compound with tumor. cells.

As used herein in the methods above, (1) the functional equivalent is a substance capable of competing against the activity of peptide deformylase ; (2) the peptide deformylase or its functional equivalent is linked to a matrix ; and (3) the peptide deformylase is a human peptide deformylase.

Differential Inhibition The invention further provides a method of identifying a compound that inhibits tumor growth comprising comparison of the inhibitory concentration or binding affinity of the compound to a bacterial peptide deformylase with a human peptide deformylase, and this inhibitory concentration or binding of the compound to human peptide deformylase indicates a better inhibition of tumor growth.

This method, wherein the inhibitory concentration or binding affinity of the compound to human peptide deformylase, is at least two times, five times, and ten times better than the inhibitory concentration or binding to bacterial peptide deformylase.

High-throughput Assay ) As encompassed by this invention, high-throughput assay can be utilized here to screen compounds capable of inhibiting the activity of the peptide deformylase, thereby obtaining compounds capable of inhibiting tumor growth. High- throughput assays are known methods to screen compounds for enzyme inhibition, or receptor or other target binding. See e. g. Burbaum et. al. U. S. Patent No. 5,876, 946 (1999). See also Parce et. al. U. S. Patent No. 6,413, 782 (2002) describing methods of manufacturing high-through put screening system that screen large numbers of different compounds for their effects on a variety of chemical and biochemical systems.

The deformylase activity mentioned in the above methods is detected at picomolar level and the appropriate reagent which yields the flourophore is a flourescamine.

The marker mentioned in the above methods is a flourophore.

This invention further provides a method for inhibiting growth of tumor cell comprising contacting the said tumor cell with the compound identified by the above methods.

Effective inhibitory concentrations could be determined by incubating different doses of the compound with different tumor cells.

Compounds Resulting from Screening This invention provides compounds which are identified by the above methods but not previously known. This invention provides both (1) a composition comprising an effective amount of the compound identified by the above methods and a suitable carrier for inhibiting peptide deformylase and (2) a composition comprising an effective amount of the compound identified by the above methods and a suitable carrier for inhibiting tumor growth.

This invention provides a pharmaceutical composition comprising the above-identified compound and a pharmaceutically acceptable carrier.

For the purposes of this invention, a"pharmaceutically acceptable carrier"means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include but are not limited to any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.

Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods.

This invention also provides a method for preventing or treating tumor in a subject comprising administration to the subject an effective amount of the compound identified by the above methods.

This invention provides a domain of human peptide deformylase or peptide whose activity is capable of being inhibited by Actinonin.

This invention provides a peptide or polypeptide comprising a domain of human peptide deformylase whose activity is capable of being inhibited by Actinonin.

This invention provides a nucleic acid molecule encoding a domain of human peptide deformylase or peptide whose activity is capable of being inhibited by Actinonin.

This invention provides a vector comprising the nucleic acid molecule which encodes a domain of human peptide deformylase or peptide whose activity is capable of being inhibited by Actinonin.

This invention provides a cell comprising the vector comprising the nucleic acid molecule which encodes a domain of human peptide deformylase or peptide which activity is capable of being inhibited by Actinonin.

Finally, this invention provides an expression system to produce the domain of human peptide deformylase or peptide which activity is capable of being inhibited by actinonin.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS First Series of Experiments: Human mitochondrial peptide deformylase, a new anti-cancer target of actinonin based antibiotics The N'terminal methionine excision (NME) pathway is an essential mechanism in all organisms (Al, A2). Although N- formyl groups modify the first methionine of all newly synthesized proteins in the cytoplasm of prokaryotes and organelles of eukaryotes, this formyl-methionine is often not retained and is cleaved from the mature protein as a part of a post-translational modification that may be linked to the N-end rule governing the half-lives of proteins (A3).

Two enzyme families are involved in this NME pathway, peptide deformylase (PDF) and methionine aminopeptidase (MAP). PDF removes all N-formyl groups and unmasks the amino group of the first methionine, which is a prerequisite for the subsequent action of MAP (A2).

Although most of the initial work characterizing PDFs has focused on Escherichia coli, there is an increasing interest in PDF as a target for developing therapies against other pathogens. These include Thermus thermophilus and Bacillus stearthermophilus (A4), Staphylococcus aureus (A5), Haemophilus influenzae, Streptococcus pneumoniae (A6), Leptospira interrogans (A7), and Mycobacterium tuberculosis (A8). In fact, numerous PDF inhibitors have been shown to have in vitro activities against several of these pathogens, and it has been speculated that these same molecules could be used as broad spectrum antibiotics against a variety of infectious diseases (A9).

The development of antimicrobial agents targeting PDF could encounter a potential hurdle. While PDF was originally thought to be a prokaryotic enzyme, recent genome database

searches have revealed eukaryotic PDF-like sequences in parasites, plants and mammals (A10), and recent studies have shown that these eukaryotic PDFs are active in vitro and in vivo (All to A15). There are three classes of PDFs based on structural and sequence analyses (A16, A17). Type 1 is divided into two subclasses: PDF1A includes plant and mammalian mitochondrial PDFs, whereas PDF1B includes enzymes found in gram-negative bacteria, some gram-positive bacteria and plants. The eukaryotic PDF1B enzymes are targeted to both plastids and mitochondria (A10). Type 2 and type 3 PDFs are found only in gram-positive bacteria, however type 3 PDFs have no associated deformylase activity.

The first eukaryotic peptide deformylase to be identified was in the higher plant, Arabidopsis thaliana. The two deformylase-like genes in this plant code for functional eukaryotic PDFs (A10). These enzymes appear to be localized in the mitochondria and plastids (A10, A12), two organelles of prokaryotic origin. Another eukaryotic peptide deformylase was later identified and characterized in Plasmodium falciparum, the primary parasite responsible for malaria in humans (All). This catalytically active deformylase (PfPDF) is inhibited by actinonin and other known PDF inhibitors, suggesting PfPDF as a potential target for anti-malarial therapies (All, A18).

The human Pdf gene contains two exons on chromosome 16 and the gene product is homologous to other characterized PDFs, albeit with some significant differences. The human Pdf mRNA has been reported to be expressed at the same level in all types of human tissues (A10). More recently, investigators have shown that a recombinantly expressed human peptide deformylase (HsPDF) is active in vitro (A13 to A15). Therefore, the data suggest that N-terminal protein processing is evolutionarily conserved and may be important in the chloroplasts or mitochondria of at least some

eukaryotic organisms, although its role in mammals is controversial (A8, A14).

Previous laboratory work showed that actinonin, a naturally occurring antibiotic derived from a Streptomyces species (A19) that inhibits aminopeptidase N (APN or CD13), has antiproliferative effects on human leukemia and lymphoma cells in vitro and anti-tumor activity in a syngeneic AKR leukemia mouse model (A20, A21). However, the antiproliferative effects were seen in APN-negative cells, and therefore the activity could not be mediated by APN inhibition. Therefore, another target of actinonin that might explain its selective anti-cancer activity was sought.

Actinonin's potent inhibition of bacterial, plant and parasitic peptide deformylases (All, A12, A22), prompted an investigation of the possibility that HsPDF may be a new cancer cell target if it were a functionally important human mitochondrial enzyme. HsPDF is a functional mitochondrial enzyme necessary for cell growth and proliferation.

Moreover, a class of new inhibitors, based on actinonin, which selectively inhibit the growth of a broad variety of human cancer cells in vitro and human tumors in vivo in mice was designed and synthesized. And, a model for actinonin's mechanism of action has been proposed. These findings have implications for the development of various therapeutic strategies for bacteria, mycobacteria, parasites or cancer that target this enzyme.

Results HsPDF activity against human mitochondrial substrates The ClustalW (A23) sequence alignment (Figure 6) of amino acid residues of HsPDF compared to other forms of PDF shows that the human protein has significant homology (30-40%) to the other catalytically active proteins. In addition, the

critical metal binding residues (C172, H214, H218) and the catalytic residue (E215) are conserved (A18, A24, A25). The expression cloning and purification of HsPDF for enzymatic studies was previously described (A13). N-terminal truncation allowed expression of a soluble, active enzyme.

All enzymatic analyses reported here were conducted with the cobalt-substituted N-terminal truncation mutant extended with a C-terminus 6x-histidine tag for purification.

The suitability of different formylated peptides as potential substrates were examined (Figures 19A and B).

HsPDF was able to deformylate two formylated peptides, formyl-Met-Ala-His-Ala (fMAHA) and formyl-Met-Thr-Met-His (fMTMH), which were designed to mimic the N-terminal amino acid sequence of two different human mitochondrially-encoded proteins, cytochrome c oxidase II and NADH dehydrogenase subunit V, respectively. The Kcat/Km values (Table 1) for these mitochondrial peptides are 5 to 10 fold higher than those for the generic substrate, formyl-Met-Ala-Ser (fMAS), which does not correlate with any mitochondrially-encoded proteins. This increase in Kcat/Km, sometimes referred to as the specificity constant', suggests a higher catalytic efficiency towards the mitochondrial peptides.

Interestingly, HsPDF was not active against the bacterial peptide, formyl-Met-Leu-Phe (fMLP/fMLF) (A13).

Table 1. Kinetic values for HsPDF activity with mitochondrial substrate Substrate VmaX, (pmoles/min/mg) Km (M) KCat (s-l) KCat/Km (M-ls-1)' fMAS B 0. 023 + 0.002 11,000 + 1600 0.0081 0.74 fMAHA 0.044 + 0.003 1700 + 330 0.016 9.2 fMTMH 0.0068 + 0.0001 450 + 56 0.0024 5.4 A Kcat/Kmis defined as the"specificity constant".

B Data taken from (ref. A13) Localization of HsPDF in the mitochondria of living cells

The full length amino acid sequence of HsPDF contains an N- terminal sequence that is predicted to serve as a mitochondrial targeting motif (All). To test the hypothesis that HsPDF is targeted to the mitochondria, HeLa cells with HsPDF-YFP (yellow fluorescent protein) fusion constructs was transfected. Live cell confocal microscopy showed that the full length HsPDF co-localizes with mitochondria that are stained with MitoTrackerTM Red (Figure 20). As a control, YFP was tagged with an N-terminal truncation mutant of HsPDF (aa 64-244) that lacks the mitochondrial signal sequence.

The truncated HsPDF-YFP fusion protein failed to co-localize with the mitochondrial marker and was instead homogenously distributed in the cytosol and nucleoplasm, a pattern indistinguishable from that of YFP expressed alone.

Actinonin inhibits cell growth in various human tumor cell lines It has been previously shown that actinonin has antiproliferative effects on both HL60 leukemia cells and Daudi lymphoma cells, and that antiproliferative activity does not correlate with APN expression. Therefore, it is hypothesized that HsPDF is a target of actinonin in tumor cells. As HsPDF is expressed diversely in human tissues (A10, A26), whether actinonin inhibits the growth and proliferation of a variety of human tumor cell lines was investigated. Actinonin had antiproliferative activity against 16 of 17 human tumor cell lines tested; representing nine different cell lineages (Table 2). Only HEK-293 renal cells were resistant. In comparison, a number of"normal" cell lines were found to be resistant to actinonin, as well.

These were the WI-38 and NIH-3T3 cell lines (human and mouse normal fibroblast cell lines, respectively), and hPBMCs (normal human peripheral blood mononuclear cells). The actinonin-sensitive mouse fibroblast cell line, AL67, differs from the NIH-3T3 parental cells in that it has been transformed by ras. Actinonin may have some myelosuppressive activities as evidenced by decreased colony formation of human bone marrow cells (A20). However, the dose required for 50% growth inhibition of these normal cells was more than 5 times the IC50 required for the tumorigenic hematopoietic cell lines tested here.

Table 2. The antiproliferative effect of actinonin on a panel of human and mouse cell lines Tissue type Cell line Description ICso Assay (µM ~ SD) Hematopoietic Daudi human B lymphoblast Burkitt's lymphoma 5. 2 + 2. 2 Thymidine incorporation HL60 human acute promyelocytic leukemia 9. 3 + 2. 9 Thymidine incorporation NB4 human acute promyelocytic leukemia 5. 0 ~ 1. 0 Trypan blue exclusion Raji human B lymphoblast Burkitt's lymphoma 4. 0 + 1. 0 Trypan blue exclusion RL human non-Hodgkin's B cell lymphoma 5. 9 + 0. 6 Thymidine incorporation hPBMC human peripheral blood mononuclear cells >500 XTT Breast MDA-MB-468 human breast adenocarcinoma 6. 9 + 0. 3 XTT SK-BR-3 human breast adenocarcinoma (metastatic) 14. 0 + 5. 1 XTT Prostate CWR22 human prostate carcinoma 32. 3 + 2. 3 Thymidine incorporation TSU-PRI human prostate carcinoma 60 + 7. 1 XTT DU145 human prostate carcinoma (brain metastasis) 28. 5 + 3. 7 XTT PC3 human prostate carcinoma (bone metastasis) 12. 8 + 3. 5 XTT Lung SK-LC-8 human lung carcinoma 20. 6~0. 2 XTT SK-LC-16 human lung carcinoma (non small cell) 16. 6 + 0. 4 XTT Ovary/Cervix A2780 human ovarian carcinoma 12. 5 + 0. 0 XTT HeLa human cervical adenocarcinoma 27. 4~4. 6 Thymidine incorporation Kidney HEK293 human embryonic kidney cells > 250 Thymidine incorporation Sarcoma HT-1080 human fibrosarcoma 15. 7~0. 2 XTT Fibroblast WI-38 human fibroblast (normal, lung tissue) >500 XTT NIH-3T3 mouse fibroblast (normal, embryo) >500 XTT AL67 mouse fibroblast (transformed by ras) 49.3 29.9 Thymidine incorporation AData taken from (A20) Actinonin analogs reveal link between HsPDF inhibition and antiproliferative activity

Thirty-three analogs of actinonin were synthesized to determine whether there was a correlation between inhibition of HsPDF activity and human tumor cell growth and to find more potent and specific compounds. Actinonin and its

analogs for both HsPDF enzyme inhibition and human tumor cell growth inhibition as measured by XTT (tetrazolium salt) metabolism and tritiated thymidine incorporation was screened (Table 3 and Figure 21). The cell lines used for the screen were CWR22Rvl and TSU-PRI (human prostate cancer), Daudi (human Burkitt's lymphoma) and HL60 (human acute myeloid leukemia). The two prostate cancer cell lines (APN- negative) were chosen as representative solid tumor cell lines, with and without androgen resistance characteristics.

As actinonin can also inhibit APN, two hematopoietic cell lines were chosen, one with and one without, APN expression.

All nine compounds that inhibited HsPDF with an IC50 of less than 0.1 WM were also potent antiproliferatives (IC50&lt;50 gM on at least one cell line). Of the sixteen compounds that inhibited HsPDF with an IC50 of 0. 2 tM or less, all were also antiproliferatives. Moreover, 6 of the 7 compounds with no measurable HsPDF inhibition exhibited no inhibition of cell growth. These data show that HsPDF inhibition is consistently associated with antiproliferative activity against human tumor cells. Conversely, lack of activity against HsPDF is generally associated with inability to inhibit the growth of tumor cells. The observation that nine compounds displaying weak HsPDF inhibition were antiproliferative suggests that these compounds inhibit cell growth via HsPDF-independent mechanisms.

Table 3. Effect of actinonin and analogs (ICsos) on human tumor cell lines and HsPDF activity XTT assay Thymidine incorporation PDF Assay Compound CWR22Rvl TSU Daudi HL60 HsPDF (µM) (µM) (µM) (µM) (µM) Actinonin 22 60 5 9 0.043 SKI-AC-111111 3 11 3 3 0.069 SKI-AC-1 59 105 13 77 0.072 SKI-AC-11101 63 37 32 10 0.076 SKI-AC-10 10 25 3 12 0.078 SKI-AC-11117 10 21 6 8 0.086 SKI-AC-11198 10 13 8 3 0.090 SKI-AC-8 17 43 13 16 0.093 SKI-AC-6 11 40 5 9 0.097 SKI-AC-11118 7 14 4 5 0.115 SKI-AC-11188 14 13 8 4 0.120 SKI-AC-11119 37 76 21 21 0.134 SKI-AC-9 60 146 22 30 0.151 SKI-AC-111113 96 67 18 13 0.177 SKI-AC-11168 270 102 23 3 0.192 SKI-AC-11178 48 90 99 20 0.200 SKI-AC-111112 81 12 48 15 0.234 SKI-AC-1-19 ND ND 28 69 0.239 SKI-AC-11114 180 320 193 500 0.276 S-AC-111114 460 230 119 105 0.300 SKI-AC-11116 10 34 8 17 0.335 SKI-AC-11112 2 11 1 7 0.365 SKI-AC-5 35 115 19 19 0.400 SKI-AC-I-18 ND 240 18 26 0.404 SKI-AC-111110 12 43 10 12 0.427 S-AC-11115 14 53 12 20 0.473 S-AC-11128 30 72 17 28 0.522 SKI-AC-51121 ND 750 217 196 >0.400 SKI-AC-3 500 610 326 183 >0.500 SKI-AC-11113 ND ND 500 500 >0.500 SKI-AC-11138 ND ND 102 200 >0.500 SKI-AC-11158 ND 120 >250 229 >0.500 SKI-AC-111101 ND ND 159 143 >0.600 SKI-AC-11148 ND 120 26 23 >0 600 ND, not determined

Knockdown of HsPDF by RNAi leads to inhibition of cell proliferation If the anti-tumor effect of actinonin is mediated by inhibition of HsPDF, then cell growth must depend to some extent on HsPDF activity. To test this hypothesis, the effect of silencing the human Pdf gene on tumor cell growth was examined. Transfection of HeLa cells with small interfering RNA (siRNA) duplexes that specifically target human Pdf mRNA (Figure 23A) led to a significant decrease in both Pdf mRNA (data not shown) and protein expression (Figure 22B) as well as a decrease in cell proliferation as evidenced by the tritiated thymidine incorporation assay (Figure 22C). Control siRNAs (non-specific duplexes) had no effect on mRNA levels, protein expression or proliferation.

The target specific siRNAs did not reduce expression of the fluorescent protein control (data not shown). Kinetic analysis revealed that the substantial decrease of both protein expression and cell proliferation was not observed until 48 hours after transfection, consistent with the requirement for catabolism of preformed protein to observe the functional effects of gene silencing. The siRNA sequences that showed significant inhibition of cell proliferation targeted the region between bases 580 and 680.

Several ineffective siRNA sequences targeted other regions of the mRNA consistent with the general observation that siRNA is effective for some but not all sequences within a message.

In order to address concerns regarding off-target and concentration-dependent effects on mammalian gene expression (A27 to A29), a range of doses for our effective siRNAs was tested. It has been reported that a characteristic feature of nonspecific effects on gene expression is dependence on siRNA concentration and that nonspecific effects occur at siRNA concentrations of 100 nM but not at 20 nM (A29). It

is found that the two effective siRNAs were able to significantly inhibit tumor cell proliferation at a dose as low as 10 nM (Figure 22D), which is well below the concentration for nonspecific effects reported in the literature. Moreover, there was no concentration-dependent effect with the target-specific siRNAs, only with the control siRNAs. These data confirm that HsPDF is important for cancer cell growth and survival.

Thus, the antiproliferative effects of actinonin and numerous analogs on a wide variety of human tumor cell lines, in combination with the requirement of HsPDF for cell growth, provide compelling evidence that HsPDF is a critical target of actinonin and that the inhibition of this protein in the mitochondria leads to cell death in tumor cells.

Actinonin causes selective mitochondrial membrane depolarization The evidence that actinonin targets HsPDF, a mitochondrial protein, suggests that actinonin may mediate its antiproliferative action via disruption of mitochondrial function. A key early indicator of mitochondrial toxicity is the loss of potential across the mitochondrial membrane (A30) : Mitochondrial membrane potential was monitor by using a fluorescent dye, JC-1, which exhibits a membrane potential-dependent accumulation in mitochondria. JC-1 emits at 525 nm (green) in monomeric form; in the presence of high mitochondrial membrane potential, JC-1 forms red (590 nm) fluorescent aggregates. Thus, mitochondrial depolarization can be quantitated by a decrease in the red/green fluorescence intensity ratio. It is shown that actinonin treatment of RL cells (human B-cell lymphoma line) causes a significant time-and dose-dependent depolarization of the mitochondrial membrane (Figure 23). The magnitude of the depolarization at the highest dose (100 pg/mL) is equal

to that of the positive control, CCCP (carbonyl cyanide m- chlorophenylhydrazone), a proton ionophore that irreversibly uncouples respiration (A31). The kinetics of the actinonin effect were far slower than for CCCP, which directly disrupts the electron transport chain, consistent with an expected indirect effect of actinonin on this pathway. In addition, when cells were treated with actinonin for 24 hours and then removed from treatment, the mitochondrial membrane potential recovered to the cells'normal resting potential after approximately 8 hours (data not shown).

This actinonin-dependent depolarization and subsequent recovery with actinonin wash-out was also seen with HeLa (cervical adenocarcinoma) and HL60 (myeloid leukemia) cell lines. In contrast, normal peripheral blood lymphocytes incubated with actinonin at the same three doses did not exhibit a depolarization over time (Figure 23).

ATP depletion might be a possible consequence of actinonin- induced mitochondrial dysfunction. To further examine this question, ATP levels in Daudi cells treated with varying doses of actinonin (5,10, and 20 pg/mL) was measured and found that it depleted ATP levels in a time-and dose- dependent manner. At the highest dose of 20 pg/mB, actinonin depleted ATP levels by 56. 0% at 12 hours, 67. 3% at 24 hours, and 95. 8% at 36 hours. Preliminary experiments to examine actinonin-induced apoptosis was conducted. It has been reported (A20) that actinonin induces a low level of apoptosis (approximately 10%) in non-APN expressing cells, based on the TUNEL assay. This low level of apoptosis using annexin-V staining was confirmed and no more than 7% annexin-V-positive, propidium iodide-negative Daudi cells in response to the same dose of actinonin was found.

Actinonin inhibits human tumor xenograft growth in mice Although previous work has shown that actinonin was effective in a syngeneic AKR leukemia mouse model, the broad anti-cancer activity seen here in vitro prompted an investigation of whether actinonin would be effective against mouse xenograft models of human solid tumors.

Actinonin has been safely administered to mice as an antibiotic at doses up to 400 mg/kg (A19). Therefore, it does not appear to have significant toxicity to normal tissues despite its antitumor activity in vitro. Remarkably, actinonin exhibited significant antitumor activity when given intraperitoneally (IP) or orally (PO) in a CWR22 human prostate tumor xenograft model in nude mice (Figure 24A).

During treatment, the animals showed no signs of clinical toxicity. The 250 mg/kg IP regimen and the 500 mg/kg PO regimen were similarly effective at inhibiting tumor growth; this suggests that actinonin is orally bioavailable.

Similar antitumor activity was seen in two other human tumor xenograft models in nude mice bearing A549 human non-small cell lung cancer (Figure 24B) and PC-3 human prostate cancer (data not shown). Therefore, it appears that actinonin has significant activity in vivo against human tumors with tolerable toxicity.

Discussion The biochemistry of protein deformylation in various non- mammalian organisms is of considerable interest because of the potential for use of the enzymes involved as targets for therapeutic interventions. Bacterial PDF has been studied extensively and is currently considered a target for the development of antibacterial drugs (A16, A17, A32 to A38).

The goal of this work was to determine if the newly discovered human mitochondrial deformylase was the target of actinonin, and if actinonin had broad cancer therapeutic

activity. Aminopeptidase N was originally considered the relevant target of actinonin in mammalian cells (A39, A40).

However, previous work in our laboratory (A20, A21) demonstrated that the anti-leukemic activity of actinonin, a peptidomimetic antibiotic, is not mediated by APN. This finding, in combination with actinonin's known bactericidal effects that are due to its inhibition of PDF (A5, A6, A22), led us to examine the possibility that HsPDF may, be an important enzyme in human cells and thus, the potential target of actinonin in tumor cells. Therefore, the possibility that a functional human peptide-deformylase protein might be a potential target for novel cancer therapeutics was explored.

It is shown that HsPDF is expressed in human mitochondria and capable of selectively deformylating model human mitochondrial N-methionyl formylated proteins. Moreover, it is shown that actinonin and numerous newly designed chemical analogs, which are all capable of potent HsPDF inhibition, all result in potent antiproliferative effects on tumor cells. In addition, siRNA reduction of HsPDF mRNA and protein results in reduction in tumor cell proliferation.

Finally, actinonin is an active and tolerable anti-cancer agent in human solid tumor xenograft models in mice.

Genome database searches had revealed that a human Pdf homologue exists (A10) and that the deduced amino acid sequence exhibits significant sequence similarities to Escherichia coli peptide deformylase (EcPDF), including key catalytic residues. The encoded human protein, HsPDF, was found to be enzymatically active in the presence of various N-formylated peptides (A13 to A15). Our results confirmed that HsPDF is enzymatically active for generic N-formylated peptides, and furthermore, has even greater activity for peptides that mimic the N-terminus of human mitochondrially encoded proteins. It is also shown that HsPDF is localized

in the mitochondria by virtue of an N-terminal targeting sequence.

The complete function and relevance of HsPDF in human cells remains to be determined. Recently, Nguyen et al. (A14) suggested that HsPDF is likely to be an evolutionary remnant without any functional role in protein formylation/deformylation, based on HsPDF's weak catalytic activity. In contrast, Serero et al. (A15) conclude that human mitochondria have a functional human PDF that is involved in the conserved N-terminal methionine excision pathway. Our data support the conclusion that HsPDF is a functional enzyme located in the mitochondria. It is shown that HsPDF is important for tumor cell growth and survival and is the target of a new class of small molecules.

These conclusions are consistent with data from plant and bacterial systems. Plant and parasitic PDFs have been shown to be expressed in organelles such as plastids and mitochondria (A10 to A12, A41, A42). HsPDF is nuclear- encoded but predicted to be localized in the mitochondria (All, A43). This prediction was based on the realization that the only nascent proteins requiring deformylation in eukaryotic cells are those synthesized on mitochondrial ribosomes. This mitochondrial localization is now confirmed.

Moreover, HsPDF's higher specificity towards two formylated peptides (fMAHA and fMTMH) designed to mimic the N-terminal sequence of two human mitochondrially-encoded proteins, support the idea that HsPDF evolved to deformylate proteins that are synthesized in mammalian mitochondria. This subset of cellular proteins is well defined; the mitochondrial genome codes for only 13 polypeptides that are subunits of the various complexes comprising the respiratory chain (A44, A45). Thus, inhibition of HsPDF in the mitochondria could presumably lead to disruption of protein complexes in the mitochondrial respiratory chain and possibly even cell death.

This hypothesis is consistent with evidence of an analogous role for plant PDF's deformylation of chloroplast encoded proteins, which are crucial components of photosystem II (A42). Actinonin, which causes bleaching of the plant, inhibits plant PDF and therefore, leads to destabilization of specific proteins of photosystem II. The antibacterial and anti-plant activities of actinonin would lead to the supposition that actinonin is inhibiting the growth of tumor cells via inhibition of HsPDF. Evidence to support this hypothesis has been provided here. Numerous analogs that potently inhibit HsPDF enzymatic activity, all potently inhibit cell growth. Nearly all analogs that have no anti- i PDF activity are not antiproliferative. In addition, siRNA knockdown of HsPDF confirm that its presence is necessary for cell proliferation.

It has been previously reported that aminopeptidase inhibitors, including actinonin and bestatin, inhibit cell proliferation of leukemia cells via suppression of serine phosphorylation of both MAPK and GSK-3beta (A46). However, this mechanistic description refers to cellular events in response to APN inhibition and is limited to APN expressing cells. Furthermore, bestatin does not inhibit HsPDF (data not shown) suggesting that actinonin's inhibition of HsPDF and subsequent inhibition of APN-negative cell proliferation relies on a different mechanism. Actinonin provides an interesting mechanistic model for its effects because it is inhibiting post-translational modifications of proteins.

HsPDF is thought to be involved in the deformylation and processing of mitochondrially encoded proteins. These proteins comprise the subunits of 4 out of the 5 complexes in the electron transport chain. When HsPDF is inhibited by actinonin, there would be an accumulation of unprocessed proteins which could lead to two possible outcomes. The reduction in properly assembled electron transport chain

complexes would lead to a reduction in proton gradient, and consequently reduced ATP synthesis. This loss of the proton gradient would result in a depolarization of the mitochondrial membrane, and actinonin not only causes mitochondrial membrane depolarization but also ATP depletion.

In addition, the accumulation of unfolded proteins in the mitochondria may induce a mitochondrial specific stress response, which reportedly results in an increase in the level of a transcription factor called CHOP or C/EBP homology protein (A47). Preliminary genechip analysis (data not shown) suggests that actinonin upregulates the expression of CHOP in human cancer cells, and CHOP has been implicated in programmed cell death (A48). Under either mechanism, prolonged actinonin inhibition of HsPDF leading to membrane depolarization and/or a mitochondrial stress response would ultimately lead to cell death.

The mitochondria play a pivotal role in cell death and apoptosis (A49 to A51). These organelles produces the bulk of the cell's ATP, which is an endogenous inhibitor of the permeability transition pore complex or PTPC (A52, A53).

ATP depletion, which could occur if the mitochondrial respiratory chain was inhibited, might facilitate PTPC opening and mitochondrial membrane permeabilization. Thus, it is conceivable that inhibition of HsPDF could lead to inhibition of the respiratory chain and subsequent ATP depletion and thus, facilitate mitochondrial membrane permeabilization, a critical event for apoptosis. Actinonin has already been shown to induce G1 arrest and apoptosis in leukemia and lymphoma cell lines (A20). However, the low level of apoptosis seen in non-APN expressing cells does not support a substantial role for apoptosis as the primary mechanism by which actinonin kills cells.

Interestingly, it is also shown that withdrawal of actinonin after a 24 hour incubation leads to a recovery of the

mitochondrial membrane depolarization reported here. Thus, if HsPDF activity is transiently inhibited by actinonin, the unfolded proteins should still be available as substrates for HsPDF when actinonin is removed, leading to the recovery of a functional electron transport chain. This effect is in contrast to that seen with uncouplers of respiration such as CCCP, which irreversibly uncouples respiration.

Despite the inhibition of cell free HsPDF and antiproliferative activity against all but one human tumor cell line tested (n=17), actinonin has been shown to be safe at high doses in mice (A19) and it is shown that it has little antiproliferative effects on normal cells in culture, as well. Remarkably, actinonin had significant antitumor effects in human xenograft mouse models of prostate and lung cancer, as both an oral and parenteral agent. In addition, preliminary results with SKI-AC-111111 suggest that the analog is not only a more potent antiproliferative agent in vitro compared to actinonin, but it is also approximately three times more potent as an in vivo antitumor agent in a human prostate tumor xenograft model (data not shown).

However, the mechanisms for the apparently selective anti- cancer in vivo activity are not clear. There has been recent attention to mitochondria as potential targets of anticancer therapy. Evidence suggests that the mitochondria of tumor cells are sufficiently different from normal cells (A44, A54), such that tumor cells may be more sensitive to mitochondrial insult. MKT-077, a cationic rhodacyanine dye, is an example of a drug that is selectively toxic to carcinoma cells (ASS) and a suggested explanation for its mechanism of action is the inhibition of mitochondrial respiration. Recently, Fantin et al. (A56) showed that a mitochondriotoxic small molecule can selectively inhibit tumor cell growth. Thus, actinonin's inhibition of HsPDF, resulting in mitochondrial disruption, could have similar

tumor specificity. It is shown here that actinonin does not cause mitochondrial membrane depolarization of normal circulating lymphocytes, indicating tumor cell selectivity.

Human bone marrow colony growth is reduced by actinonin in vitro (A20); however, in the cancer therapeutic studies in mice described here, effective anti-tumor doses of actinonin did not give rise to obvious toxic side effects.

Therefore, HsPDF is a new human mitochondrial enzyme that may provide a novel and tumor-selective target for the development of anti-cancer therapies.

Methods Reagents N-formylated peptide substrates used in the deformylase assay were formyl-Met-Ala-Ser (fMAS), formyl-Met-Ala-His-Ala (fMAHA), and formyl-Met-Thr-Met-His (fMTMH). All substrates were either purchased or custom synthesized from Bachem Bioscience Inc. Actinonin and CCCP (carbonyl cyanide m- chlorophenylhydrazone) were purchased from Sigma-Aldrich.

JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'- tetraethylbenzimidazolylcarbocyanine iodide) was purchased from Molecular Probes. Analogs of actinonin were synthesized by the Organic Synthesis Core Facility at Memorial Sloan Kettering Cancer Center.

Peptide deformylase assay The spectrophotometric assay is based on the method described by Lazennec and Meinnel (A57) and is a formate dehydrogenase-coupled assay for peptide deformylase activity.

Our measurements were conducted at 25oC in polystyrene cuvettes containing 50 mM HEPES (pH 7.4), 10 mM NaCl, 0.2 mg/ml BSA, 2.4 mM NAD+ (Roche), 1 U formate dehydrogenase (Sigma-Aldrich), and 0 to 32 mM N-formylated peptide (Bachem

Bioscience, Inc.). The reaction was initiated with the addition of 20 to 100 pg of HsPDF enzyme. The rate of NADH production was measured by monitoring the increase in absorbance at 340 nm using a Spectronic Genesys 2 spectrophotometer (Spectronic Instruments).

Localization of HsPDF in living cells The full coding sequence or a truncation of the nucleotides encoding the first 63 amino acids of the human Pdf cDNA were amplified by polymerase chain reaction (PCR) and cloned in- frame (primer sequences and restriction sites available upon request) into pEYFP-N1 (BD Biosciences Clontech). HeLa cells (American Type Culture Collection) were maintained in 5% C02 in DMEM supplemented with 4 mM L-glutamine, 4.5 g/L glucose and 10% FBS (Cellgro). HeLa cells to be examined by fluorescence microscopy were plated at 2x105 per plate into 35-mm dishes containing a glass coverslip-covered 15-mm cutout (MatTek Corporation) and transfected the next day using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. To stain mitochondria, MitoTrackerTM Red CMX Ros (Molecular Probes) was added to cell cultures 30 minutes prior to imaging. Living cells were imaged with a Zeiss 510 inverted laser scanning confocal microscope (LSM). Representative images were processed with Adobe Photoshop 7.0. siRNA preparation and transfection Prior to transfection (24 h), HeLa cells were trypsinized, diluted in fresh media without antibiotics, and transferred to 96-well plates at a density of 10,000 cells per well. On the day of transfection, cells (50-70% confluent) were washed with PBS and the media was replaced with Opti-MEM I reduced serum medium (Invitrogen). Transfections of siRNA were carried out using Oligofectamine (Invitrogen) according

to the manufacturer's instructions. Target-specific siRNA duplexes were designed using an in-house algorithm (MSKCC Core Facility) and provided at a concentration of 10 pM.

The human Pdf (NCBI accession no. NM_022341)-specific siRNAs were positioned at 581-601 and 659-679 relative to the start codon and were compared with sequences in the human genome database to confirm that no other genes were targeted. A non-specific duplex control pool (Dharmacon) was used as a control and was comprised of 4 duplexes containing 33% GC content. All experiments were carried out two times, in triplicate each time. HeLa cell growth was determined via tritiated thymidine incorporation on Days 1,2, and 3 after transfection.

Determination of HsPDF mRNA levels and protein expression To confirm knockdown of HsPDF mRNA, HeLa cell RNA was harvested 24, 48 and 72 hours after siRNA transfection.

DNase-treated total RNA (5 µg) was used to prepare cDNA for subsequent Real-Time PCR. The Thermoscript RT-PCR System, (Invitrogen) was used according to the manufacturer's instructions. The cDNA samples were diluted 1: 5 with 5 mM Tris-HCl, pH 8.5 and 3 jul was used for a 25 pl reaction in a MicroAmp optical 96 well reaction plate on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The master mixes contained 2X TaqMan Universal Master Mix, RNase-DNase free water and the respective primers and probe. The HsPDF master mix contained primers at 700 mM and HsPDF probe at 200 mM. The beta-actin master mix (housekeeping reference gene) contained primers at 500 mM and beta-actin probe at 150 mM. The sequences for both primers (GeneLink) and probes (Applied Biosystems) are as follows: PDF forward primer 5'-GGGCAGCCCGCATCA-3', PDF reverse primer 5'- TGCTGTCCATTTTGTCAATAAACA-3', B-actin forward primer 5'- CTGGCACCCAGCACAATG-3', B-actin reverse primer 5'-

GCCGATCCACACGGAGTACT-3', PDF probe 6FAM- CAGCACGAGATGGACCACCTGCAG-TAMRA, and B-actin probe 6FAM- TCAAGATCATTGCTCCTCCTGAGCGC-TAMRA. For final calculations, each sample's Ct value was subtracted from its respective beta-actin value.

For HsPDF protein expression analysis, HeLa cells stably expressing the same full length HsPDF-YFP construct used in the confocal imaging studies was used. As a control, HeLa cells stably expressing a fluorescent vector control was used. Twenty-four and 48 hours after siRNA transfection, the cells were analyzed by flow cytometry (Beckman Coulter Cytomics FC500) and mean peak fluorescence was determined by a flow cytometry analysis program (FlowJo v4.5).

The data regarding siRNA knockdown of mRNA, protein and proliferation is represented as the mean + standard deviation and was analyzed for statistical significance by an ANOVA with a Newman-Keuls post test using Prism 3.0.

Cell proliferation assays A variety of human and mouse cell lines were used to assess the antiproliferative activity of actinonin. Human tumor cell lines included: hematopoietic (Daudi, HL60, NB4, Raji, RL), breast cancer (MDA-MB-468, SK-BR-3), prostate cancer (CWR22Rvl, TSU-PRI, DU145, PC3), lung cancer (SK-LC-8, SK- LC-16), ovarian cancer (A2780), cervical cancer (HeLa), and other : (HEK293, HT-1080). Human peripheral blood mononuclear cells (hPBMC), human fibroblasts (WI-38), mouse fibroblasts (NIH-3T3), and a mouse fibroblast cell line, NIH-3T3, transformed with ras (AL67) was used. To quantitate the effect of actinonin on cancer cell proliferation, two different assays were used. It is shown previously that assays of cell viability, such as XTT metabolism and trypan blue exclusion, directly correlate with tritiated thymidine

incorporation results from cells treated with actinonin.

Assay I was the tritiated thymidine incorporation assay in which an aliquot of 200 all of cells (10,000 cells/well) was plated and incubated at 37oC in 96-well plates in the presence or absence of actinonin. Serial dilutions of actinonin were made in complete media. After 1-5 days of incubation, 50 pl of 10 Ci/mL tritiated (3H)-thymidine (PerkinElmer) was added to each well and allowed to incorporate for 5 hours. Plates were frozen at-80oC overnight and cells were harvested onto filtermats (Wallac) using a semi-automatic harvester (Skatron). Filtermats were counted in a 1205 Betaplate liquid scintillation counter (Wallac). Assay II was the XTT assay in which cells in log phase growth were plated in a 96 well plate at a certain density. Actinonin was added to the plate at various concentrations made from serial dilutions in complete media.

When the cells in the control wells reached confluence, 50 je. l of the tetrazolium salt, XTT (dissolved at 1 mg/ml in 37oC serum free medium) was added with PMS (phenazine methosulphate, an electron transfer reagent) to each well of the plate, and the cells were incubated again for 2-4 hours at 37°C. Absorbances were read at a dual wavelength of 450/630 nm using a plate reader.

Determination of mitochondrial membrane depolarization RL cells (human B-cell lymphoma) were cultured in RPMI supplemented with 20% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L NaHC03, 2 mM L-glutamine and 1% penicillin/streptomycin. Cells were incubated with varying concentrations of actinonin (0-100 pg/mL) at a density of 1x106 cells/mL. Maximal dissipation of mitochondrial membrane potential (ate) was determined by incubation with the proton ionophore, CCCP, at a final concentration of 100 nM for 10 minutes prior to data acquisition. Mitochondrial

mass and membrane potential were assessed by incubating cells with JC-1 for 5 minutes at 37OC. Cells were washed 3 times in PBS and resuspended in media supplemented with actinonin at the original concentration. Flow cytometry was performed using a FACSCalibur (Becton Dickinson) running the Cell Quest software. 50,000 events were acquired in listmode for each data point and analyzed with Flow Jo software (Tree Star). Mitochondrial mass was measured in FL-1 ("green"-mean excitation emission at 525nm). Negative mitochondrial membrane potential was measured in FL-2 ("red"-mean excitation emission at 590nm). Mitochondrial membrane potential was expressed as a ratio of the negative membrane potential to the mass (red to green ratio). All measurements were performed in triplicate and are expressed as the mean + standard deviation. Statistical significance was determined by an ANOVA with a Newman-Keuls post test using Prism 3.0.

Measurement of ATP depletion Daudi (human B cell lymphoma) cells were cultured in RPMI media supplemented with 1 mM L-glutamine and 10% FBS. Cells were treated with three doses of actinonin (5,10 and 20 pg/mL) for varying periods of time (1 to 36 hours). At each timepoint, cells were counted and resuspended to a final concentration of 100,000 cells/mL. ATP was quantitated in 1000'cells using an ATP assay kit (EMD Biosciences) following the manufacturer's instructions. Immediate luminescence detection over 1 minute was performed with a Berthold Lumat LB9501 luminometer. ATP determinations were calculated using an ATP standard curve, and results were expressed as grams ATP/1000 cells.

Annexin-V staining Daudi (human B cell lymphoma) cells were cultured in RPMI media supplemented with 1 mM L-glutamine and 10% FBS. Cells were treated with three doses of actinonin (5,10 and 20 VLg/mL) for varying periods of time (1 to 96 hours). At the each timepoint, 1x106 cells were rinsed with binding buffer and stained with annexin V and propidium iodide (control) as part of the BD ApoAlert kit (BD Biosciences Clontech) according to the manufacturer's instructions. Cells were analyzed by flow cytometry (Beckman Coulter Cytomics FC500) and percent annexin-V-positive, propidium iodide-negative, was determined by a flow cytometry analysis program (FlowJo v4.5).

Human tumor xenograft models in mice Eight to ten week old athymic-NCr-nu mice were inoculated subcutaneously in the flank with minced CWR22 (prostate), A549 (lung) or PC3 (prostate) tumor cells mixed with Matrigel (Becton Dickinson). Once the tumors became palpable, the mice were randomized into control and treatment groups with 3-5 animals per group. Actinonin was administered intraperitoneally (IP) or orally (PO) daily for two weeks except for weekends. The doses of actinonin chosen for these studies were based on a preliminary toxicity study in which weight loss was the limiting criteria for calculation of the MTD or maximal tolerated dose. Mice in the control group were given vehicle alone.

Tumors were measured every 3-4 days with calipers. Tumor volumes were calculated by the formula 4/3 x n x [ (larger diameter + smaller diameter) divided by 4] 3. The data is represented as the mean + standard deviation and was analyzed for statistical significance by non-parametric Wilcoxon Rank Sum Test using SAS 8.2. All animal studies

were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) at Sloan Kettering Institute.

Screening of actinonin analogs for HsPDF inhibition and antiproliferative activity All actinonin analogs were dissolved in 10-50% DMSO and subsequent dilution in the enzymatic and antiproliferation assays brought the final DMSO concentration to below 1%. In addition, DMSO controls were used to calculate percent inhibition by analogs. CWR22Rvl and TSU-PRI cancer cell lines were used to assess the antiproliferative efficacy of analogs in the XTT assay. Details of the XTT assay have been described in a previous section. Daudi and HL60 cancer cell lines were used in the tritiated thymidine incorporation assay, as previously described. HsPDF inhibition by analogs was determined with the formate- dehydrogenase coupled PDF assay as described in a previous section. IC50 values represent the concentration at which the compound inhibits 50% of cell proliferation or enzymatic activity when compared to controls.

Second Series of Experiments: A New Human Peptide Deformylase Inhibitable by Actinonin Materials and Methods Reagents. Molecular biology chemicals were purchased from commercial suppliers: GeneLink (Hawthorne, NY), Invitrogen (Carlsbad, CA), Qiagen (Valencia, CA), New England Biolabs (Beverly, MA), Novagen (Madison, WI). N-formylated peptide substrates used in the deformylase assays were formyl-Met- Ala-Ser (fMAS), formyl-Mel-Leu-p-nitroaniline (fML-pNA), formyl-Met-Leu-Phe (fMLF or the more common misnomer fMLP) and were purchased from Bachem Bioscience Inc. (King of

Prussia, PA). Actinonin was purchased from Sigma Chemical Company (St. Louis, MO).

Cloning of HsPDF cDNA. Based on the annotated cDNA sequence found in the genome database, primers to reverse transcribe the mRNA from human acute myeloid leukemia cells (HL60) and to amplify the cDNAs encoding the full length putative HsPDF protein, as well as a 63 amino acid N-terminally truncated protein was designed. The primers (GeneLink) with the following sequences, 5'-GGAATTCCATATGGCCCGGCTGTGGGGCGCGCTG- 3'and 5'-CCGCTCGAGGTCATTCACCTTCATCCAATAGACG-3', were used for the full length protein. 5'- GGAATTCCATATGTCATTCTCGCACGTGTGCCAAGTCGGG-3'and the same 3' primer above were used for the truncated protein. Reverse transcription was carried out using HL60 mRNA as the template and SuperscriptT II RT as specified by the high-GC content protocol from Invitrogen. The resulting cDNA was used in PCR reactions containing 300yM dNTPs, 1mM MgS04, 0.3uM of each primer, lx PCR Enhancer Solution, and 2.5 units Platinum Pfx DNA polymerase (Invitrogen). The following protocol was used for amplification: 95°C for 3 min, 35 cycles of (95°C for 1. 5min/59-69°C for 45sec/72°C for 2.5min), and 72°C for 7 min. The PCR products (0.75 and 0.56 kb) were gel purified on QIAquick columns (Qiagen), and cloned into ZeroBlunt TOPO vector (Invitrogen) for a diagnostic restriction digest. Appropriate clones were then digested with restriction endonucleases NdeI and XhoI (New England Biolabs) and cloned into a bacterial expression vector pET-29b (Novagen). The resulting clones contained a C-terminal 6x-histidine tag for purification purposes. All clones were sequence verified and compared to the annotated genome database.

Expression and purification of HsPDF. The expression vectors encoding the HsPDF cDNAs were transformed into BL21 (DE3) pLysS cells (Novagen) and grown in LB media containing 50 yg/ml kanamycin and 34 yg/ml chloramphenicol at 37°C until OD600 reached 0.4. Cells were then induced with 0.5mM IPTG and incubated at 30°C for 18 hours. At this time, each culture was supplemented with 100yM of eitherCoCl2, NiCl2, or ZnS04. The cells were harvested and lysed in BugBuster/Benzonase reagent (Novagen) plus 100pM of either CoCl2, NiCl2, or ZnS04. The N-terminus of the recombinant proteins was sequenced by Edman degradative sequencing (Microchemistry Core Facility, MSKCC) to confirm the identity of the protein. Soluble HsPDF was affinity purified using His-Bind Resin (Novagen) columns according to the manufacturer's instructions. Prior to any enzymatic analyses, the imidazole used to elute the recombinant protein was removed by dialysis. The dialysis buffers contained 20mM Hepes, pH 7.4, lOmM NaCl, plus 100AM of either CoCl2, NiCl2, or ZnS04. Dialyzed protein fractions were concentrated to approximately 1.5 ml using an Amicon° Ultra-15 centrifugal filter device (Millipore, Bedford MA).

All purification, dialysis, and concentration procedures were conducted at 4°C.

Peptide deformylase assays. Three different assays were used to study the kinetic properties of HsPDF. Method I is an indirect spectrophotometric assay [15] that couples peptide deformylase activity with formate dehydrogenase (FDH) activity. Our measurements were conducted at 25°C in polystyrene cuvettes containing 50mM Hepes (pH 7.4), lOmM NaCl, 0.2 mg/ml BSA, 2.4mM NAD+ (Roche, Indianapolis IN), 1 unit formate dehydrogenase (Sigma), and 0 to 32mM N- formylated peptide (Bachem). The reaction was initiated with the addition of 20 to 100 Ag of HsPDF enzyme. The rate of NADH production was measured by monitoring the increase

in absorbance at 340nm using a Spectronic Genesys 2 spectrophotometer (Spectronic Instruments, Rochester NY).

Method II is a continuous spectrophotometric assay for peptide deformylase activity [16] that uses fML-pNA as the substrate, which is first deformylated and then subsequently processed by Aeromonas proteolytica aminopeptidase (AAP) to release p-nitroaniline. These measurements were conducted at 25°C in polystyrene cuvettes containing 50mM potassium phosphate (pH 7), 100yM EGTA, 0 to 800/iM peptide substrate N-formyl-Met-Leu-p-nitroanilide (Bachem), and 0.8 unit of Aeromonas proteolytica aminopeptidase (Sigma). The reaction was initiated with the addition of 70 mg of HsPDF enzyme.

The release of p-nitroanilide was measured by monitoring the increase in absorbance at 405nm. This assay was used only for kinetic studies and not for inhibition studies because actinonin also inhibits aminopeptidases. Method III is a direct solid phase fluorescent assay to measure peptide deformylase activity and is based on a previously published method using fluorescamine to detect the formation of the new amino group of the N-formylated substrate after deformylation [17]. This method was modified to be on a solid support and this method has been used for some of the enzyme analyses reported here. The substrate used in this direct PDF assay was formyl-Met-Ala-Ser (fMAS). pH, temperature and ionic strength profiles. The effects of pH, temperature, and KCl ionic strength on HsPDF activity were determined using either the assay coupled to formate dehydrogenase (for temperature and KCl measurements) or the direct solid phase fluorescamine assay (for pH measurements).

HsPDF. activity at pHs of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 in buffers composed of 0. 05M sodium acetate/0. 15M sodium chloride for pH 4.0-5. 0; 0.05M Mes or (N- morpholino) ethanesulfonic acid for pH 6.0 ; phosphate buffered saline for pH 7.0 ; 0. 1M sodium borate for pH 8.0 ;

and 0. 1M Ches or 2- (cyclohexylamino) ethanesulfonic acid for pH 9.0-10. 0 was test. Enzymatic activity at temperatures of 4°C, 25°C, 37°C, 50°C, and 65°C, as well as KCl ionic strength concentrations of OM, 0.25M, 0.5M, 0.75M and 1M [KCl] was also tested.

Actinonin inhibition of HsPDF. Affinity-purified HsPDF (10- 70ug) was pre-incubated for 3 to 60 minutes with 0 to 500nM actinonin prior to initiating the reaction in the formate dehydrogenase-coupled assay or the direct fluorescamine assay. The substrate used for both the inhibition and competition assays was formyl-Met-Ala-Ser (fMAS).

Cell culture conditions. HL60 (human acute myeloid leukemia, APN/CD13 positive) and Daudi (human B-lineage Burkitt's lymphoma, APN/CD13 negative) cells were obtained from ATCC and maintained in culture using RPMI 1640 supplemented with 10% heat-inactivated FBS (Omega Scientific, Inc. , Tarzana, CA) and 1% L-glutamine (Gibco/Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% CO2. Cell viability was higher than 90%, and cells were free of mycoplasma contamination.

Actinonin inhibition of tumor cell growth. Inhibition of the incorporation of (3H)-thymidine into tumor cells was used as an indicator of cytotoxicity. An aliquot of 200/-tl of Daudi or HL60 human cancer cells (10,000 cells/well) was incubated at 37°C in 96-well plates in the presence or absence of varying concentrations of actinonin. After 5 day incubation, 50/il of 10 Ci/mL (3H)-thymidine (PerkinElmer, Boston, MA) was added to each well and allowed to incorporate for 5 hours. Plates were frozen at-80°C overnight and lysed cells were harvested onto filtermats (Wallac, Finland) using a semi-automatic harvester (Skatron,

Sterling, VA). Filtermats were counted in a 1205 BetaplateTM liquid scintillation counter (Wallac, Finland).

Results Expression cloning and purification of recombinant HsPDF The full length cDNA was cloned by reverse transcription- polymerase chain reaction (RT-PCR) of mRNA extracted from human acute myeloid leukemia cells (HL60), which are sensitive to actinonin. Based on published reports that full-length PfPDF and AtPDFs could not be expressed in active form in E. coli [8, 11], a truncation mutant lacking the first 63 amino acids of the N-terminus but retaining the catalytic domain was constructed and it was overexpressed in bacterial cells. The protein, expressed with a C-terminus 6xhistidine tag, was purified to apparent homogeneity by nickel-affinity chromatography (Figure 1, inset). N- terminal sequence analysis showed an amino acid sequence of *FSHV (C) QVG confirming the presence of the expected truncated protein.

Peptide deformylase activity Due to the reported sensitivity of native EcPDF (containing Fe2+) to oxidation and its subsequent inactivation [18, 19], the recombinant HsPDF with various other metal cations in order to improve the likelihood that the protein would exhibit enzymatic activity was expressed. This activity was measured with formyl-Met-Ala-Ser as the substrate in the formate dehydrogenase-coupled assay [15]. The Co2+- containing form of HsPDF appeared to have enzymatic activity, while the Ni2'and Zn2+ forms of the enzyme were inactive (Figure 1). While addition of cobalt in the protein induction and purification buffers was necessary for

activity, once purified, the addition of the metal cations in the assay buffer did not have any significant effect on activity. The activity of the Co2+-HsPDF was measured for up to 60 minutes without noting a change in the slope of the reaction curve, thus the activity of HsPDF appeared to be stable.

Kinetic parameters of HsPDF In order to examine some standard enzymatic parameters, cobalt-containing form of HsPDF to conduct basic kinetic studies was used. We examined the use of different formylated peptides as potential substrates and found that HsPDF was able to deformylate fMAS, fML-pNA, but not fMLP (Figure 2 and Figure 3). We found that at concentrations above 20mM of fMAS, the substrate inhibited the human enzyme, an observation that has been noted previously with EcPDF [20]. The Kcat/Km values for HsPDF were significantly lower than the published EcPDF, AtPDFIA and PfPDF values [9,11, 16, 21]. This suggests that perhaps the substrates tested or the assay conditions used were not optimal for human enzyme, or that the N-terminal truncated form of HsPDF with cobalt-substituted metal is not fully functional. However, these data show that the cloned cDNA for HsPDF encodes a functional deformylase protein In further experiments with the catalytically active cobalt form of HsPDF, we found that deformylase activity was optimal at a pH between 6.0 and 7.2 and at temperatures between 25°C and 50°C. In comparison, the pH profile for HsPDF is similar to the one previously published for EcPDF [22]. Increasing KC1 concentration from 0 to 1000 mM reduced HsPDF activity by approximately 70% in a direct linear relationship, which is different from the large KC1 dependent increase in activity observed with EcPDF [20].

Actinonin inhibition of HsPDF and tumor cell growth Actinonin (Figure 4A, inset) is a naturally occurring inhibitor of bacterial peptide deformylase with an IC50 value of 0.8 to 90nM, depending on the metal cation form of the enzyme [23]. We found that actinonin inhibits HsPDF activity with an IC50 of 43 nM (Figure 4A). This inhibition appears to be competitive (Figure 4B), however, substrate inhibition was observed as well and at high inhibitor concentrations (lOOnM), some non-competitive behavior was also seen.

Actinonin was also tested for its ability to inhibit tumor cell growth in vitro. We chose to test HL60 cells, since those were the tumor cells from which the HsPDF cDNA was cloned, however, we also tested Daudi cells because they do not express aminopeptidase N (APN or CD13) and APN is the other known target of actinonin [24, 25]. We found that actinonin inhibited (3H)-thymidine incorporation in HL60 cells (IC50 of lops) and even more potently in the APN negative Daudi cells (IC50 of Sum) (Figure 5). Cell death measurements were confirmed by 3H-leucine incorporation, XTT metabolism, and trypan blue exclusion.

Discussion The biochemistry of protein deformylation in various non- mammalian organisms is of considerable interest because of the potential for use of the enzymes involved as targets for therapeutic interventions. Bacterial PDF has been studied extensively and is currently considered a novel target for the development of antibacterial drugs [4-7, 26-30]. More recent literature has shown that eukaryotic peptide deformylases, previously thought not to exist, are active

enzymatically [8,9, 11]. These newly discovered enzymes may play an important role in the normal function of eukaryotic organisms, and therefore may provide a novel target for drug design.

Previous work in our laboratory [13,14] has shown that actinonin's antitumor activity is not mediated via aminopeptidase N, an originally described target of this molecule [24, 25]. This finding, in combination with actinonin's known bactericidal effects that are due to its inhibition of EcPDF [23,31, 32], have led us to examine the possibility that HsPDF may be an active enzyme in human cells and thus, a potential therapeutic target in tumor cells. Therefore, we explored the possibility that a functional human peptide-deformylase protein might exist.

Genome database searches have revealed that a human PDF homologue does exist [8] and the deduced amino acid sequence exhibits significant sequence similarities to EcPDF. The putative human peptide deformylase-like protein also contains an N-terminal targeting sequence that predicts mitochondrial localization [11]. The ClustalW [33] sequence alignment of amino acid residues of HsPDF compared to other forms of PDF shows that the human protein has significant homology (30-40%) to the other catalytically active proteins.

In addition, the key metal binding residues (C172, H214, H218) and the key catalytic residue (E215) are conserved [12, 34, 35]. To determine if the putative HsPDF was functional, we cloned the HsPDF cDNA and expressed a recombinant truncated form of the protein that retains the catalytic domain. Full length protein was unable to be adequately expressed in soluble form, which is similar to observations by other investigators studying the plant and parasitic PDFs [8, 11].

The encoded truncated human protein was found to be enzymatically active in the presence of various N-formylated peptides; however, there are key differences when compared to EcPDF. First, we found that only the cobalt form of HsPDF was active, whereas the Ni2+, Zn2+ and Co2+ forms of other PDF enzymes have been shown to be active, albeit to varying degrees [9,11, 18, 21]. Although it is possible that our method for substituting Ni2+ and Zn2+ was not optimal and that any of the three metal cations could be the native one, it is unlikely that HsPDF is an iron metalloenzyme like EcPDF, because HsPDF is in the PDF1A class of deformylases [8]. Data from other investigators [10] suggest that the metal cation in the PDF1A class is oxidation-resistant and unlikely to be a ferrous ion. These authors [10] concluded that these PDF1A deformylases were probably zinc enzymes, however, our data does not support this conclusion for HsPDF.

One reason for this discrepancy is that these investigators were studying PDFs from higher plants, and it is reasonable to assume that the human enzyme may contain a different metal cation.

Second, the HsPDF enzyme is much slower catalytically toward the same formylated substrates tested with EcPDF. There is supporting evidence that eukaryotic PDFs may be generally less active, since the plant and parasitic PDFs have also been found to be less active than EcPDF [9, 11], although the HsPDF values reported here are even lower. However, this may be due to the lack of optimal substrates for eukaryotic PDFs, or that the organellar environment where these PDFs presumably function may be involved in either enhancing the catalytic reaction or protecting it from regulatory inhibition. Notably, eukaryotes contain few formylated proteins, unlike prokaryotes, which may need more robust activity.

Third, we found that HsPDF is unable to deformylate fMLP, while EcPDF is quite active against this substrate (unpublished observation). This is of interest because fMLP is a chemotactic peptide produced by bacteria [36]. One would expect that the bacterial enzyme, not necessarily the human enzyme, would recognize this substrate, as it is unlikely to appear inside human cells. This difference in activity towards fMLP supports the idea that HsPDF is a distinctly different enzyme from EcPDF and is active towards different N-formylated substrates. These data also raise the question as to whether fMLP might act as an inhibitor of HsPDF. However, we found that the human enzyme was able to deformylate fMAS normally, even in the presence of excess amounts of fMLP (data not shown).

The physiological function of HsPDF remains unclear, however.

The plant and parasitic PDFs have been shown to be located in organelles such as chloroplasts, plastids and mitochondria [8,9, 11,37, 38]. HsPDF, although nuclear- encoded, is predicted to be localized in the mitochondria [11, 39]. Moreover, formylated proteins in human cells are only found in the mitochondria, which would suggest that the role of HsPDF is to deformylate proteins that are encoded by the mitochondrial genome and synthesized in the mitochondrial matrix. This subset of cellular proteins is well defined and include ribosomal proteins, tRNAs, and 13 additional proteins involved in various complexes of the respiratory chain [40]. Thus, inhibition of HsPDF in the mitochondria would presumably lead to disruption of protein complexes in the mitochondrial respiratory chain and possibly even cell death. Our hypothesis is consistent with evidence of an analogous role for plant PDF's deformylation of chloroplast-encoded proteins, which are crucial components of photosystem II [38]. They report that actinonin inhibits the plant PDF enzyme, which leads to the

destabilization of specific proteins of photosystem II, thus explaining the bleached phenotype seen with actinonin treatment. Studies in our laboratory are currently underway to better determine the physiological function and localization of HsPDF, as well as its relevance to actinonin's tumor cell cytotoxicity.

Third Series of Experiments: Actinonin and Analogs Target a Human Mitochondrial Peptide Deformylase Peptide deformylase (PDF) activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine.

This allows methionine aminopeptidase, an essential activity in living organisms, to process non-N-formylated polypeptides [1-3]. Because eukaryotic cytoplasmic protein synthesis does not involve N-formylation and therefore has no need of deformylase activity, it was thought that bacterial PDF (EcPDF) would be an ideal target for the development of antibiotics with little or no mammalian toxicity [4-7]. However, genome database searches have revealed many eukaryotic PDF-like sequences in parasites, plants and mammals [8], and thus far, two plant deformylases [8-10], a parasitic deformylase [11], and a human deformylase [43,48, 46] have been characterized.

The first eukaryotic peptide deformylase to be identified was in the higher plant, Arabidopsis thaliana. Researchers showed that the two deformylase-like genes in the this plant code for functional eukaryotic PDFs [8]. These enzymes appear to be localized in the mitochondria and plastids [8, 9], both having evolutionary ties to prokaryotes. Another eukaryotic peptide deformylase was later identified and chararcterized in Plasmodium falciparum, the primary

parasite responsible for malaria in humans [11]. This catalytically active deformylase (PfPDF) is inhibited by actinonin and other known PDF inhibitors, suggesting PfPDF as a potential target for anti-malarial therapies [11, 12].

Sequence homology and database searches identified an open reading frame for a human PDF-like protein (HsPDF) [8]. It is composed of two exons on chromosome 16 and is homologous to bacterial, insect and plant PDFs. The cDNA sequence has been shown to be expressed at the same level in all human tissues [8]. Therefore, the data suggest that N-terminal protein processing is evolutionarily conserved and may be important in the chloroplasts or mitochondria of at least some eukaryotic organisms.

Previous work in our laboratory showed that actinonin, a naturally occurring antibiotic derived from a Streptomyces species [49], has antiproliferative effects on human leukemia and lymphoma cells in vitro and in a syngeneic AKR leukemia mouse model [13, 14]. However, this antiproliferative effect is not mediated by aminopeptidase N inhibition. Thus, we focused our research on finding another target for actinonin that might explain its anti- tumor activity. Because of previously published reports that actinonin is a potent inhibitor of bacterial peptide deformylase [23], we investigated the possibility that HsPDF may be a functional enzyme with a significant role in the mitochondria and thus relevant to the development of various human, therapeutic strategies for bacteria, parasites or cancer.

Materials and Methods Reagents. Molecular biology chemicals were obtained from commercial suppliers: GeneLink (Hawthorne, NY), Invitrogen

(Carlsbad, CA), Qiagen (Valencia, CA), New England Biolabs (Beverly, MA), Novagen (Madison, WI). N-formylated peptide substrates used in the deformylase assays were formyl-Met- Ala-Ser (fMAS), formyl-Mel-Leu-g-nitroaniline (fML-. pNA), formyl-Met-Leu-Phe (fMLF or the more common misnomer fMLP), formyl-Met-Ala-His-Ala (fMAHA), and formyl-Met-Thr-Met-His (fMTMH). All substrates were either purchased or custom synthesized from Bachem Bioscience Inc. (King of Prussia, PA). Actinonin was purchased from Sigma Chemical Company (St. Louis, MO).

Cloning of hsPDF cDNA. Based on the annotated cDNA sequence found in the genome database, we designed primers to reverse transcribe the mRNA from human acute myeloid leukemia cells (HL60) and to amplify the cDNAs encoding the full length putative HsPDF protein, as well as a 63 amino acid N- terminally truncated protein. The primers (GeneLink) with the following sequences, 5'- <BR> <BR> <BR> <BR> GGAATTCCATATGGCCCGGCTGTGGGGCGCGCTG-3'and 5/- CCGCTCGAGGTCATTCACCTTCATCCAATAGACG-3', were used for the full length protein. 5'- GGAATTCCATATGTCATTCTCGCACGTGTGCCAAGTCGGG-3'and the same 3' primer above were used for the truncated protein. Reverse transcription was carried out using HL60 mRNA as the template and Superscript II RT as specified by the high-GC content protocol from Invitrogen. The resulting cDNA was used in PCR reactions containing 300AM dNTPs, 1mM MgS04, 0.3uM of each primer, Ix PCR Enhancer Solution, and 2.5 units Platinum Pfx DNA polymerase (Invitrogen). The following protocol was used for amplification: 95°C for 3 min, 35 cycles of (95°C for 1. 5min/59-69°C for 45sec/72°C for 2.5min), and 72°C for 7 min. The PCR products (0.75 and 0.56 kb) were gel purified on QIAquick columns (Qiagen), and cloned into ZeroBlunt TOPO vector (Invitrogen) for a

diagnostic restriction digest. Appropriate clones were then digested with restriction endonucleases NdeI and XhoI (New England Biolabs) and cloned into a bacterial expression vector pET-29b (Novagen). The resulting clones contained a C-terminal 6x-histidine tag for purification purposes. All clones were sequence verified and compared to the annotated genome database.

Protein expression and purification. The expression vectors encoding the HsPDF cDNAs were transformed into BL21 (DE3) pLysS cells (Novagen) and grown in LB media containing 50 ßg/ml kanamycin and 34 Hg/ml chloramphenicol at 37°C until OD6oo reached 0.4. Cells were then induced with 0.5mM IPTG and incubated at 30°C for 18 hours. At this time, each culture was supplemented with 100µM of either CoC12, NiCl2, or ZnSO4. The cells were harvested and lysed in BugBuster/Benzonase reagent (Novagen) plus 100yM of either CoCl2, NiCl2, or ZnSO4. The N-terminus of the recombinant proteins were sequenced by Edman degradative sequencing (Microchemistry Core Facility, MSKCC) to confirm the identity of the protein. The soluble HsPDF was affinity purified using His-Bind Resin (Novagen) columns according to the manufacturer's instructions. Prior to any enzymatic analyses, the imidazole used to elute the proteins was removed by dialysis. The dialysis buffers contained 20mM Hepes, pH 7.4, lOmM NaCl, plus IOOAM of either CoCl2, NiCl2, or ZnS04. Dialyzed protein fractions were concentrated to approximately 1.5 ml using an Amicons Ultra-15 centrifugal filter device (Millipore, Bedford MA). All purification, dialysis, and concentration procedures were conducted at 4°C.

Peptide deformylase assay. The spectrophotometric assay is based on the method described by Lazennec and Meinnel [34] and is a formate dehydrogenase-coupled assay for peptide deformylase activity. Our measurements were conducted at 25°C in polystyrene cuvettes containing 50mM Hepes (pH 7.4),

lOmM NaCl, 0.2 mg/ml BSA, 2.4mM NAD+ (Roche, Indianapolis IN), 1 unit formate dehydrogenase (Sigma), and 0 to 32mM N- formylated peptide (Bachem). The reaction was initiated with the addition of 20 to 100 yg of HsPDF enzyme.} The rate of NADH production was measured by monitoring the increase in absorbance at 340nm using a Spectronic Genesys 2 spectrophotometer (Spectronic Instruments, Rochester NY).

Cell Culture Conditions. All human tumor cell lines were obtained either from ATCC or as gifts from other investigators. Cells were cultured in the recommended media with appropriate supplements and maintained at 37°C in a humidified atmosphere of 5% C02. Cell viability was higher than 90%, and cells were free of mycoplasma contamination.

Inhibition of Tritiated Thymidine Incorporation. An aliquot of 200 ul of Daudi or HL60 cells (10,000 cells/well) was washed and incubated at 37°C in 96-well plates in the presence or absence of actinonin. Serial dilutions were made in complete media. After 5 days of incubation, 50 ul of 10 uCi/mL tritiated (3H)-thymidine (PerkinElmer, Boston, MA) was added to each well and allowed to incorporate for 5 hours. Plates were frozen at-80°C overnight and cells were harvested onto filtermats (Wallac, Finland) using a semi- automatic harvester (Skatron, Sterling, VA). Filtermats were counted in a 1205 BetaplateTM liquid scintillation counter (Wallac, Finland).

Inhibition of XTT metabolism. Tetrazolium salts such as MTT and XTT have long been used to quantify oxidation/reduction reactions. Reduction of the tetrazolium salt by enzymes in metabolically active cells generates a colored signal that is proportional to the number of cells present in the reaction. The assay we used is based on the conversion of

XTT (sodium 3, 3'-{1-[(phenylamino) carbonyl]-3, 4- tetrazolium} bis (4-methoxy-6-nitro) benzene sulfonic acid) from an oxidized tetrazole to a reduced formazan. Both forms are water soluble which allows for direct spectrophotometric detection of the colored product at 475nm.

For our experiments, we dispensed 100ul of cells with 100ul of actinonin/analog dilution into each well of a 96-well plate. After 6 to 24 hours of incubation, 40ul of the XTT or ProCheck reagent (Intergen Co. , NY) was added to each well. After 4 hour incubation, the plate was read by a spectrophotometric plate reader at 490nm.

Animal xenograft model methods. Nude mice were implanted with xenografts of various human tumor cells. In these experiments, we used CWR22 (human prostate tumor), A549 (human non-small cell lung cancer), and PC-3 (human prostate cancer) cell lines for xenografts. Animals bearing tumors were then treated using various doses and dosing regimens of actinonin. Treatment began 3 days after tumor implantation and continued for 2 weeks. Individual doses and regimens are shown on the graphs of the data. Tumor xenografts were measured for size every 3-5 days.

Confocal microscopy methods. Full length HsPDF and the 63 amino acid N-terminal truncated version were transiently transfected into HeLa and COS-1 cells. The constructs were composed of the HsPDF sequence with a C-terminal EYFP fusion protein. Cells were grown on glass dishes and confocal images were taken 24-72 hours after transfection.

Mitotracker Red was used to stain mitochondria in the cells.

Co-localization of HsPDF and mitochondria was determined by overlay of green (HsPDF-YFP) and red (mitochondria).,

Results Cloning, expression and purification of recombinant HsPDF The putative human peptide deformylase-like protein contains an N-terminal targeting sequence that predicts mitochondrial localization [11]. The ClustalW [33] sequence alignment (Figure 6) of amino acid residues of HsPDF compared to other forms of PDF shows that the human protein has significant homology (30-40%) to the other catalytically active proteins.

In addition, the key metal binding residues (C172, H214, H218) and the key catalytic residue (E215) are conserved [12, 34, 35]. Therefore, we cloned the full length cDNA by reverse transcription-polymerase chain reaction (RT-PCR) of mRNA extracted from human acute myeloid leukemia cells (HL60), which are sensitive to actinonin. Based on published reports that full-length PfPDF and AtPDFs could not be expressed in active form in E. coli [8, 11], we constructed a truncation mutant lacking the first 63 amino acids of the N-terminus but retaining the catalytic domain and overexpressed it in bacterial cells. The protein, expressed with a C-terminus 6xhistidine tag, was purified to apparent homogeneity by nickel-affinity chromatography (Figure 7). N-terminal sequence analysis showed an amino acid sequence of *FSHV (C) QVG confirming the presence of the expected truncated protein. Due to the reported sensitivity of native EcPDF (containing Fe2+) to oxidation and its subsequent inactivation [19, 20], we expressed the recombinant HsPDF in the presence of Ni2+, Zn2+, and Co2+ to stabilize the protein for activity measurements. Only the cobalt substituted form of HsPDF showed any measurable activity [46].

Catalystic properties of HsPDF In order to examine some catalytic properties, we used the cobalt-containing HsPDF to conduct the following enzymatic studies. We examined the use of different formylated peptides as potential substrates (Figure 8) and found that HsPDF was able to deformylate two formylated peptides, fMAHA and fMTMH, which were designed to mimic the N-terminal amino acid sequence of two different mitochondrially-encoded proteins, cytochrome c oxidase II and NADH dehydrogenase subunit V, respectively. The Km/Kcat values for these mitochondrial peptides are 5 to 10 fold higher than that for the standard substrate, fMAS, which does not correlate with any mitochondrially-encoded proteins [46].

Localization of HsPDF by confocal microscopy To confirm our hypothesis that HsPDF plays a role in the mitochondria, we transfected COS-1 and HeLa cells with HsPDF-YFP fusion constructs. Confocal microscopy (Figure 9) showed that the full length HsPDF co-localizes with mitochondria, which are stained with Mitotracker Red. As a control, we made a construct with the N-terminally truncated version of HsPDF (used for protein expresssion and kinetic studies) that essentially deletes the signal sequence that normally targets the protein to the mitochondria. The truncated HsPDF-YFP fusion protein did not co-localize with the mitochondria and instead demonstrates an expression pattern similar to YFP alone.

Actinonin inhibits cell growth in various tumor cell lines Actinonin was tested for its ability to inhibit tumor cell growth in vitro. We have previously shown that actinonin is

cytotoxic to both HL60 leukemia cells and Daudi lymphoma cells, and the cytotoxicity does not correlate with aminopeptidase N expression. To investigate whether this antiproliferative effect was limited to leukemia and lymphoma cell lines, we tested actinonin against a panel of other tumor cell lines (Figure 10). The results indicate that actinonin is cytotoxic to all tumor cell lines tested, except for HEK-293 and COS-1 cells. This is likely due to the fact that kidney cells normally express high levels of p-glycoprotein, the MDR1 gene product that not only contributes to chemotherapeutic drug resistance in tumors but is also expressed in normal tissues such as liver, kidney and intestine [47]. Our explanation for the apparent resistance of HEK-293 and COS-1 cells is that both are transformed kidney cell lines that are likely to be overexpressing p-glycoprotein, and thus actinonin is unable to enter the cell and reach its intracellular target of HsPDF.

Actinonin inhibits tumor xenograft growth in mouse models Although previous work showed that actinonin was effective in a syngeneic AKR leukemia mouse model, we investigated whether actinonin would be effective in human solid tumor xenograft models. We demonstrate that actinonin exhibits antitumor activity when given intraperitoneally or orally in a CWR22 human prostate tumor xenograft model in nude mice (Figure 11A). During the treatment, the animals showed no signs of toxicity. In addition, we tested actinonin in two other xenograft models in nude mice bearing A549 human non- small cell lung cancer (Figure 11B) and PC-3 human prostate cancer (data not shown). The results of actinonin treatment for the other two models were similar to that seen with the CWR22 prostate tumor. Therefore, it appears that actinonin has antitumor activity against a variety of human tumors.

Actinonin analogs A number of analogs to actinonin were synthesized to help us determine whether there was a correlation between HsPDF inhibition and tumor cell cytotoxicity. We screened over 30 analogs (Figure 12) for enzyme inhibition (APN, EcPDF, and HsPDF) and tumor cell cytotoxicity (CWR22, TSU, Daudi, and HL60). Based on the compiled data, we were able to make a few observations. Compound #9, #1-18, #11113 and #11114 were fairly potent EcPDF inhibitors, but not very potent HsPDF inhibitors, and subsequently those compounds were not very potent cytotoxics. In general, the good HsPDF inhibitors (IC50s of less than 100nM) were fairly consistent cytotoxic compounds as well. However, there were many compounds that were not good HsPDF inhibitors, but very potent cytotoxics, which may suggest that there is another target protein mediating these cytotoxic effects in addition to our proposed target, HsPDF.

Discussion The biochemistry of protein deformylation in various non- mammalian organisms is of considerable interest because of the potential for use of the enzymes involved as targets for therapeutic interventions. Bacterial PDF has been studied extensively and is currently considered a novel target for the development of antibacterial drugs [4-7, 26-30]. More recent literature has shown that eukaryotic peptide deformylases, previously thought not to exist, are active enzymatically [8,9, 11]. These newly discovered enzymes may play an important role in the normal function of eukaryotic organisms, and therefore may provide a novel target for drug design.

Previous work in our laboratory [13, 14] has shown that actinonin's antitumor activity is not mediated by aminopeptidase N, an originally described target of this molecule [24, 25]. This finding, in combination with actinonin's known bactericidal effects that are due to its inhibition of EcPDF [23,31, 32], have led us to examine the possibility that HsPDF may be an active enzyme in human cells and thus, a potential therapeutic target in tumor cells. Therefore, we explored the possibility that a functional human peptide-deformylase protein might be a potential cancer target.

Recent genome searches have revealed that a human PDF homologue does exist [8] and the deduced amino acid sequence exhibits significant sequence similarities to EcPDF, including key catalytic residues (Figure 6). The encoded human protein was found to be enzymatically active in the presence of various N-formylated peptides [43,48, 46]. Our data suggest that HsPDF can deformylate mitochondrial peptides and it is localized in the mitochondria.

As for the function and relevance of HsPDF in human cells, there is still much speculation. A recently published article [43] suggests that HsPDF is likely to be an evolutional remnant without any functional role in protein formylation/deformylation. They base their conclusions on the fact that HsPDF has weak catalytic activity, and that there is an apparent lack of deformylation in mammalian mitochondria. However, our data suggest that HsPDF is a functional enzyme located in the mitochondria and possibly linked to actinonin's antitumor activity. Previously characterized plant and parasitic PDFs have been shown to be expressed in organelles such as plastids and mitochondria [8, 9,11, 37, 38]. HsPDF, although nuclear-encoded, is also predicted to be localized in the mitochondria [11, 39],

since that is where formylated proteins are synthesized in human cells. Our confocal microscopy data support this theory. Additionally, the fact that we have shown that HsPDF has a slightly higher specificity for two formylated peptides (fMAHA and fMTMH), designed to mimic the N-terminal sequence of two mitochondrially-encoded proteins, would support the idea that the role of HsPDF is to deformylate proteins, that are synthesized in the mitochondria. This subset of cellular proteins is well defined and include ribosomal proteins, tRNAs, and 13 additional proteins involved in various complexes of the respiratory chain [40].

Thus, inhibition of HsPDF in the mitochondria would presumably lead to disruption of protein complexes in the mitochondrial respiratory chain and possibly even cell death.

This hypothesis is consistent with evidence of an analogous role for plant PDF's deformylation of chloroplast encoded proteins, which are crucial components of photosystem II [38]. They report that actinonin, which causes bleaching of the plant, inhibits the PDF enzyme and therefore, leads to destabilization of specific proteins of photosystem II.

To address the findings from Nguyen at al. [43] concerning the lack of cytotoxicity seen in their cell lines, we would suggest that their PDF inhibitors may not be reaching the intracellular space and thus not inhibiting HsPDF in vitro.

Our in vitro cell line data suggests that actinonin, also a potent PDF inhibitor, is cytotoxic to all tumor cell lines tested, except those derived from kidney cells, and one of those (HEK-293) was one of the cell lines they tested.

Additionally, our in vivo data showing actinonin's antitumor effect in human xenograft mouse models combined with the correlation between cytotoxicity and enzyme inhibition, provides some reasonable evidence that there is a link between HsPDF inhibition and tumor cytotoxicity. In order to more definitively show the relevance of HsPDF to tumor

cells, we need to determine if the expression of HsPDF is vital to tumor cell growth and if HsPDF is the intracellular target of actinonin. These studies are currently, under investigation in our lab.

Fourth Series of Experiments: A Direct Fluorometric Assay for Peptide Deformylase Ribosomal protein synthesis in prokaryotes and in a rare subset of eukaryotic proteins is initiated with an N- formylated methionine. In bacteria the N-formyl group is subsequently removed from most of the peptides by peptide deformylase (PDF). This activity allows further processing of the N-terminal methionine by methionine aminopeptidase and other enzymes to yield mature proteins. PDF has been shown to be essential to bacterial growth [4,41]. Until recently, PDF was thought to be absent from eukaryotic systems, making it an attractive target for the development of new antibiotics [42]. However, genome database searches have revealed eukaryotic PDF-like sequences in parasites, plants and mammals [8], and recently the human peptide deformylase (HsPDF) has been cloned and characterized by our team as well as by others [43].

Actinonin is an antibiotic and CD13/aminopeptidase N (APN) inhibitor, and has been shown to be cytotoxic to leukemia and lymphoma cell lines in vitro and in vivo [14]. However, only part of or none of the antitumor effect is likely to be mediated by APN [14]. Actinonin is known to inhibit bacterial PDF (EcPDF) and we recently showed that actinonin also inhibits HsPDF activity. This inhibition may provide a mechanism of action to explain its antitumor activity. HsPDF therefore becomes an attractive target for the development of a new class of antitumor drugs.

To allow the mechanistic study of PDF and to screen inhibitors, several methods have been developed to measure PDF activity. The most commonly used is based on the release of formate, which is monitored through a coupled reaction involving formate dehydrogenase [7, 15]. The drawback of this assay is the poor specific activity of the dehydrogenase.

Moreover since HsPDF is much slower catalytically than EcPDF, large amounts of both formate dehydrogenase and HsPDF are needed when using this assay, making it inappropriate for HsPDF study and inhibitor screening. Another assay employs a dipeptide substrate, N-formylmethionyl-leucyl-p-nitroanilide and APN, which hydrolyses the product of the reaction Met- Leu-p-nitroanilide [16]. But since many PDF inhibitors also inhibit APN, this method cannot be used for screening. A direct assay has then been developed, which does not use any coupling enzyme, utilizing N-formyl- (P-thiaphenylalanine)- containing substrates that eliminate a thiol upon PDF activity. The thiol is then monitored spectrophotometrically using Ellman's reagent, 5, 5'-dithio-bis (2-nitrobenzoic acid) [44]. This assay is more attractive for high throughput screening, yet requires the use of non commercially available substrates.

Therefore we decided to design a direct, sensitive assay that would allow HsPDF study with natural substrates, and that would also be suitable for the screening of inhibitors.

The assay we developed is based on the detection of the primary amine formed during deformylation of the substrate N-formyl-Met-Ala-Ser (fMAS) (1) using fluorescamine (2) (Figure 13). To keep a low background and allow sensitive and accurate monitoring of the free amino groups formed, we immobilized PDF on resin beads to remove it from solution before performing the fluorescamine assay. Here we report the application of this approach to HsPDF and EcPDF activity measurement. This method proved to be highly sensitive and

accurate, and can be used for the characterization and comparison of PDF inhibitors.

Materials and Methods Materials. The peptides N-formyl-Met-Ala-Ser and Met-Ala-Ser were purchased from Bachem Bioscience Inc. (King of Prussia, PA). Actinonin and formate dehydrogenase were obtained from Sigma Chemical Co. (St Louis, MO). NAD+ was from Roche (Indianapolis, IN).

EcPDF expression. An expression vector encoding the E. coli peptide deformylase (pET-22b-def) was kindly given to us by Dr. Dehua Pei at Ohio State University. The plasmid was transformed into BL21-CodonPlus&commat; (DE3) -RP competent cells (Stratagene) and grown in LB media containing 50 yg/ml ampicillin and 25 Hg/ml chloramphenicol at 37°C with shaking for 12 hours until the OD600 reached 0.5 to 0.6. Cells were then induced with 200 AM IPTG and incubated at 37°C with shaking for an additional 2 hours. The cells were harvested, frozen at-80°C overnight and then lysed in B-Per reagent (Pierce) containing 100 ug/ml catalase and 10 mM TCEP to preserve enzymatic activity. The suspension was centrifuged at 15, 800xg for 20 minutes at 4°C. The soluble fraction (supernatant) was frozen at-80°C in aliquots and used for all subsequent enzymatic analyses.

HsPDF cloning. Based on the annotated cDNA sequence found in the genome database, we designed primers (GeneLink) with the following sequences, 5'- <BR> GGAATTCCATATGTCATTCTCGCACGTGTGCCAAGTCGGG-3'and 5'- CCGCTCGAGGTCATTCACCTTCATCCAATAGACG-3'to reverse transcribe the mRNA from human acute myeloid leukemia cells (HL60) and to amplify the cDNA encoding a 63 amino acid N-terminally truncated HsPDF protein. Reverse transcription was carried

out using HL60 mRNA as the template and SuperscriptTM II RT as specified by the high-GC content protocol from Invitrogen.

The resulting cDNA was used in PCR reactions containing 300AM dNTPs, 1mM MgSO4, 0.3uM of each primer, lx PCR Enhancer Solution, and 2.5 units Platinum Pfx DNA polymerase (Invitrogen). The following protocol was used for amplification: 95°C for 3 min, 35 cycles of (95°C for 1. 5min/59-69°C for 45sec/72°C for 2.5min), and 72°C for 7 min.

The PCR products (0.75 and 0.56 kb) were gel purified on QIAquick columns (Qiagen), and cloned into ZeroBlunt TOPO vector (Invitrogen) for a diagnostic restriction digest.

Appropriate clones were then digested with restriction endonucleases NdeI and XhoI (New England Biolabs) and cloned into a bacterial expression vector pET-29b (Novagen). The resulting clones contained a C-terminal 6x-histidine tag for purification purposes.

HsPDF expression and purification. The expression vector encoding the HsPDF truncated cDNA was transformed into BL21 (DE3) pLysS cells (Novagen) and grown in LB media containing 50 yg/ml kanamycin and 34 g/ml chloramphenicol at 37°C until OD600 reached 0.4. Cells were then induced with 0. 5mM, IPTG and incubated at 30°C for 18 hours. At this time, the bacterial culture was also supplemented with 100AM CoCl2.

The cells were harvested and lysed in BugBuster/Benzonase reagent (Novagen) plus IOOAM CoCl2. The N-terminus of the recombinant proteins were sequenced by Edman degradative sequencing (Microchemistry Core Facility, MSKCC) to confirm the identity of the protein. The soluble HsPDF was affinity purified using His-Bind Resin (Novagen) columns according to the manufacturer's instructions. Prior to any enzymatic analyses, the imidazole used to elute the proteins was removed by dialysis. The dialysis buffers contained 20mM Hepes, pH 7.4, lOmM NaCl, 100yM CoCl2. Dialyzed protein fractions were concentrated to approximately 1.5 ml using an Amicon) Ultra-15 centrifugal filter device (Millipore, Bedford MA). All purification, dialysis, and concentration procedures were conducted at 4°C.

Enzyme immobilization. HsPDF was bound to the His-Bind resin (Novagen, Inc. , Madison, WI) through its poly-histidin tag according to the manufacturer's batch method. Typically 400 AL of slurry was mixed with 800 Ag enzyme (200: 1 Ni: HsPDF molar ratio). EcPDF was bound to NHS-activated Sepharose 4 Fast Flow beads (Amersham Biosciences Corp., Piscataway, NJ) according to the manufacturer's protocol.

Molar ratios from 100 to 275 : 1 were used. The concentration of the PDF-beads suspensions obtained was adjusted to 1.5 mg/mL (activity equivalent) in PBS.

Formate dehydrogenase coupled PDF assay. To evaluate the amount of active enzyme linked to the beads we initially used the spectrophotometric assay coupled to formate dehydrogenase (FDH) [22, 15]. Typically 20 HL of bead suspension was added to polystyrene cuvettes containing 50 mM Hepes pH 7.2, 10 mM NaCl, 0.2 mg/mL BSA, 2.4 mM NAD+, 1 unit FDH and 4 mM N-formyl-Met-Ala-Ser in a 500 pL total volume. The suspension was agitated at 25°C and the rate of NADH production was measured by monitoring the increase in absorbance at 340 nm using a Spectronic Genesys 2 spectrophotometer (Spectronics Instruments, Rochester NY) after sedimentation of the beads.

HsPDF-beads time course assays. We employed N-formyl-Met- Ala-Ser (fMAS) as a substrate. For each condition (one substrate concentration or one enzyme amount) a 50 AL reaction mixture was set up in PBS in a round-bottomed microplate (Costar, Corning Incorporated, NY). The plate was agitated at room temperature. At each timepoint the plate was briefly centrifuged and aliquots of 10 pL supernatant

were used for the fluorescamine assay. The data presented are from a representative experiment.

HsPDF-beads pH profile analysis. 4 pL of the HsPDF beads suspension (5 yg HsPDF activity equivalent) were mixed with 2.5 yL fMAS 40 mM to reach a 4 mM final substrate concentration in a 25 AL total volume buffer. The suspension was agitated during 30 min at 25°C. The plate was then briefly centrifuged and aliquots of 10 AL were used for the fluorescamine assay. The different buffers used were 50 mM sodium acetate/150 mM sodium chloride for pH 4.0 and 5.0, 50 mM MES for pH 6.0, PBS for pH 7.2, 100 mM borate for pH 8.0, and 100 mM CHES for pH 9.0 and 10.0. The blank for each pH consisted in a 10, AL aliquot of each buffer that were used for the fluorescamine assay. This was used to eliminate the small variations observed that were probably due to the slight modification of the pH in the fluorescamine assay.

Two independant experiments gave a similar profile and the data presented are from one of them.

Inhibition assays. In a 50 AL total volume of PBS either EcPDF-beads (0.75 Ag activity equivalent) or HsPDF-beads (6. 4 jUg activity equivalents) were incubated with inhibitors.

Fluorescamine assay. We used the optimized procedure previously described for fluorescamine derivatization [45].

An aliquot of 10 yL from the supernatant of the quickly centrifuged PDF reaction mixtures is transferred to the wells of a flat-bottomed microplate (96 FluoroNunc white plate). Then 90 iLL of 0. 1% (w/v) fluorescamine dissolved in acetonitrile, 15 AL of 0.1 M borate buffer, pH 8.0 and 185 AL of water were added to each well in that specific order.

Fluorescence was read on a Fmax microplate reader (Molecular Devices Corp. , CA) using an excitation wavelength of 355 nm and an absorption wavelength of 460 nm.

The fluorescamine standard curve was built using increasing amounts of the peptide MAS, product of the enzymatic reaction between fMAS and PDF. Dilutions of a stock solution of MAS in water were added to the fluorescamine assay discribed above.

Results and Discussion In order to develop a direct, sensitive assay for PDF activity using natural substrates, we considered the use of fluorescamine. Fluorescamine (2) is intrinsically nonfluorescent, but reacts within seconds at room temperature with primary aliphatic amines to yield highly fluorescent derivatives like 3 (AeX = 381 nm, Aem = 470 nm) (Figure 13). This fluorophore therefore allows the detection of primary amines with a high sensitivity and could be used to monitor the deformylation reaction of the N-terminal residues on the PDF substrates even at low concentrations.

However PDF itself bears primary amines through its lysine residues. The presence of PDF in the fluorescamine assay would therefore induce a high background which would constitute an obstacle to a sensitive and reliable assay. In order to be able to easily remove PDF from the reaction mixture before proceeding to the fluorescamine derivatization we built PDF-resin conjugates.

PDF immobilization We initially chose N-hydroxysuccinimide (NHS) ester- activated resin beads as a mean to covalently attach the enzyme to beads through its lysine residues. NHS esters react with primary amines to form a stable amide covalent bond. Conjugating HsPDF to the NHS ester-activated resin inactivated the PDF when measured using the FDH coupled

assay. The reaction mixture supernatant had little or no activity (0 to 20% initial activity) after the conjugation process, whereas a control HsPDF incubated in the same conditions except for the presence of beads retained its activity (data not shown). Moreover the beads resulting from the coupling used in an affinity chromatography experiment failed to bind antibodies against HsPDF. This suggested that the NHS attack on the PDF lysines rendered it inactive and denaturated it. We confirmed this hypothesis in a control experiment in which HsPDF precipitated when mixed in the same conditions with NHS ester-activated biotin without beads. In contrast when this method was applied to EcPDF this conjugation strategy produced beads bearing PDF activity with yields ranging from 7 to 9%. In each case more than 90% of the EcPDF activity initially put into reaction was found in the supernatant at the end of the conjugation process. This result indicates that although the reaction yield between the EcPDF amines and the NHS ester moiety of the beads is poor, the process didn't inactivate the enzyme.

The difference observed in this coupling reaction between the bacterial and the human enzyme might be explained by the large difference in the lysine content between the two enzyme forms: 2 for HsPDF and 11 for EcPDF. The two lysines on HsPDF might be essential for its activity or conformation.

Therefore, in order to conjugate HsPDF into solide phase, we eventually took advantage of the poly-histidine tag expressed on the HsPDF. We could therefore use this ploy- histidine tail to link the enzyme to a Ni2+-immobilized resin with a 70% yield. The stability of this non-covalent bond was satisfactory since after 20 days at 4°C the beads kept 20% of their original activity. This loss was not significantly different from the inactivation observed with the plain enzyme stored in solution at 4°C (data not shown).

HsPDF assay optimization In order to determine the optimal conditions for the assay, we first tested the HsPDF-beads activity with increasing concentrations of substrate in presence of excess enzyme (Figure 14A). Enzyme activity was substrate concentration dependent. A linear increase in absorbance at 460 nm after excitation at 355 nm is observed over time for each concentration. Increasing the substrate concentration amplifies the signal up to the saturation observed at 4 mM.

We therefore chose this concentration for the assay. The assay was enzyme concentration dependant as well (Figure 14B).

In order to obtain an adequate signal to noise ratio, at least 1.5 jig enzyme for 4 timepoints must be used with an incubation time of 30 minutes. The background remained low, typically around 5 to 10 fluorescence units in those conditions. The sensitivity of the assay could probably be further optimized by using optimal excitation and absorption wavelengths, which were not possible due to limitations of our fluorimeter. The assay allows considerable economy of HsPDF. since only 1.5 Hg are needed for 4 measurements where 25 yg were typically used in the FDH coupled assay.

A standard curve for the fluorescamine assay with the peptide MAS was constructed in order to correlate the fluorescence read out to the number of moles of converted substrate. We determined that the assay was directly proportional to the amount of MAS within the range 0.5 pmol to 10 nmol (Figure 15). As anticipated, the use of fluorescamine allowed great sensitivity and accuracy.

HsPDF characterization We used this newly developed assay to characterize the cobalt form of HsPDF. The catalytic constants of the enzyme bound to the beads towards fMAS were determined from the

data of the optimization experiment previously described (Fig. 14). When the velocity was plotted against the substrate concentration we could determine a Vmax of 0.015 ymol/min/mg and a Km of 2.5 mM (Figure 16). This result suggests the human PDF is significantly less active than the bacterial enzyme [21, 16]. The constants are similar to the ones obtained with HsPDF using other assays [46, 43].

The HsPDF activity as a function of pH follows a bell-shaped curve, with an optimum between pH 6.0 and 7.0 (Figure 17).

This profile is very similar to the one described for EcPDF by Pei et al. [16, 22] using either the FDH-coupled assay or the APN-coupled assay but differs from the EcPDF profile established by the same group when they used the assay monitoring the release of a thiol [44].

HsPDF inhibition An important application of this newly developed assay is the screening and study of HsPDF inhibitors. Actinonin is a naturally occurring inhibitor of bacteria peptide deformylase with an IC. 50 value of 0.8 to 90 nM depending on the metal cation form of the enzyme [23]. Using the HsPDF- beads assay we found an ICso of 60 nM (data not shown) in agreement with the IC50 of 45 nM found with HsPDF using the FDH coupled assay (in press). We found that actinonin inhibits HsPDF in a competitive way (Figure 18).

Using HsPDF immobilized on a solid support together with the natural substrate fMAS and fluorescamine, we have developed a highly sensitive assay for this less active form of the peptide deformylase. It can be used with any PDF substrate and therefore is very useful for studying the substrate specificity of the enzyme. The assay is also particularly useful for the screening of PDF inhibitors. The concept described here could be applied to the development of assays for other proteases.

REFERENCES Al. Chang, S. Y. , McGary, E. C., and Chang, S. 1989.

Methionine aminopeptidase gene of Escherichia coli is essential for cell growth. J Bacteriol 171: 4071-4072.

A2. Solbiati, J. , Chapman-Smith, A. , Miller, J. L. , Miller, C. G. , and Cronan, J. E. , Jr. 1999. Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal. J Mol Biol 290: 607-614.

A3. Varshavsky, A. 1996. The N-end rule: functions, mysteries, uses. Proc Natl Acad Sci U S A 93: 12142- 12149.

A4. Ragusa, S. , Blanquet, S. , and Meinnel, T. 1998. Control of peptide deformylase activity by metal cations. J Mol Biol 280: 515-523.

A5. Margolis, P. S. , Hackbarth, C. J., Young, D. C. , Wang, W., Chen, D. , Yuan, Z. , White, R. , and Trias, J. 2000.

Peptide deformylase in Staphylococcus aureus: resistance to inhibition is mediated by mutations in the formyltransferase gene. Antimicrob Agents Chemother 44 : 1825-1831.

A6. Margolis, P. , Hackbarth, C. , Lopez, S. , Maniar, M., Wang, W. , Yuan, Z. , White, R. , and Trias, J. 2001.

Resistance of Streptococcus pneumoniae to deformylase inhibitors is due to mutations in defB. Antimicrob Agents Chemother 45: 2432-2435.

A7. Li, Y. , Chen, Z. , and Gong, W. 2002. Enzymatic properties of a new peptide deformylase from pathogenic bacterium Leptospira interrogans. Biochem Biophys Res Commun 295: 884-889.

A8. Cynamon, M. H., Alvirez-Freites, E. , and Yeo, A. E. 2004.

BB-3497, a peptide deformylase inhibitor, is active against Mycobacterium tuberculosis. J Antimicrob Chemother 53: 403-405.

A9. Meinnel, T. 2000. Peptide deformylase of eukaryotic protists: a target for new antiparasitic agents? Parasitol Today 16: 165-168.

A10. Giglione, C. , Serero, A. , Pierre, M. , Boisson, B. , and Meinnel, T. 2000. Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms. Embo J 19: 5916-5929.

All. Bracchi-Ricard, V. , Nguyen, K. T. , Zhou, Y., Rajagopalan, P. T. , Chakrabarti, D. , and Pei, D. 2001.

Characterization of an eukaryotic peptide deformylase from Plasmodium falciparum. Arch Biochem Biophys 396: 162-170.

A12. Dirk, L. M. , Williams, M. A. , and Houtz, R. L. 2001.

Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis. Plant Physiol 127: 97-107.

A13. Lee, M. D. , Antczak, C. , Li, Y. , Sirotnak, F. M. , Bornmann, W. G. , and Scheinberg, D. A. 2003. A new human peptide deformylase inhibitable by actinonin. Biochem Biophys Res Commun 312: 309-315.

A14. Nguyen, K. T. , Hu, X. , Colton, C. , Chakrabarti, R., Zhu, M. X. , and Pei, D. 2003. Characterization of a Human Peptide Deformylase: Implications for Antibacterial Drug Design. Biochemistry 42: 9952-9958.

A15. Serero, A. , Giglione, C. , Sardini, A. , Martinez-Sanz, J. , and Meinnel, T. 2003. An Unusual Peptide Deformylase Features in the Human Mitochondrial N- terminal Methionine Excision Pathway. J Biol Chem 278: 52953-52963.

A16. Guilloteau, J. P. , Mathieu, M. , Giglione, C. , Blanc, V., Dupuy, A. , Chevrier, M. , Gil, P. , Famechon, A. , Meinnel, T. , and Mikol, V. 2002. The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the

structure-based design of antibacterial agents. J Mol Biol 320: 951-962.

A17. Giglione, C. , Pierre, M. , and Meinnel, T. 2000. Peptide deformylase as a target for new generation, broad spectrum antimicrobial agents. Mol Microbiol 36: 1197- 1205.

A18. Kumar, A. , Nguyen, K. T. , Srivathsan, S. , Ornstein, B., Turley, S. , Hirsh, I., Pei, D. , and Hol, W. G. 2002.

Crystals of peptide deformylase from Plasmodium falciparum reveal critical characteristics of the active site for drug design. Structure (Camb) 10: 357- 367.

A19. Gordon, J. J. , Kelly, B. K. , and Miller, G. A. 1962.

Actinonin: an antibiotic substance produced by an actinomycete. Nature 195: 701-702.

A20. Xu, Y. , Lai, L. T. , Gabrilove, J. L. , and Scheinberg, D. A.

1998. Antitumor activity of actinonin in vitro and in vivo. Clin Cancer Res 4: 171-176.

A21. Xu, Y. , and Scheinberg, D. A. 1995. Elimination of human leukemia by monoclonal antibodies in an athymic nude mouse leukemia model. Clin Cancer Res 1: 1179-1187.

A22. Chen, D. Z. , Patel, D. V. , Hackbarth, C. J. , Wang, W., Dreyer, G. , Young, D. C. , Margolis, P. S. , Wu, C. , Ni, Z. J. , Trias, J. , et al. 2000. Actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor. Biochemistry 39: 1256-1262.

A23. Thompson, J. D. , Higgins, D. G. , and Gibson, T. J. 1994.

CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.

Nucleic Acids Res 22: 4673-4680.

A24. Meinnel, T., Blanquet, S. , and Dardel, F. 1996. A new subclass of the zinc metalloproteases superfamily revealed by the solution structure of peptide deformylase. J Mol Biol 262: 375-386.

A25. Chan, M. K. , Gong, W. , Rajagopalan, P. T. , Hao, B. , Tsai, C. M. , and Pei, D. 1997. Crystal structure of the Escherichia coli peptide deformylase. Biochemistry 36: 13904-13909.

A26. Velculescu, V. E. , Zhang, L. , Vogelstein, B. , and Kinzler, K. W. 1995. Serial analysis of gene expression. Science 270: 484-487.

A27. Jackson, A. L. , Bartz, S. R. , Schelter, J. , Kobayashi, S. V. , Burchard, J. , Mao, M. , Li, B. , Cavet, G. , and Linsley, P. S. 2003. Expression profiling reveals off- target gene regulation by RNAi. Nat Biotechnol 21: 635- 637.

A28. Persengiev, S. P. , Zhu, X. , and Green, M. R. 2004.

Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). Rna 10: 12-18.

A29. Semizarov, D. , Frost, L. , Sarthy, A. , Kroeger, P., Halbert, D. N. , and Fesik, S. W. 2003. Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci U S A 100: 6347-6352.

A30. Elmore, S. P. , Nishimura, Y. , Qian, T. , Herman, B. , and Lemasters, J. J. 2004. Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer. Arch Biochem Biophys 422: 145- 152.

A31. Nieminen, A. L. , Dawson, T. L. , Gores, G. J. , Kawanishi, T., Herman, B. , and Lemasters, J. J. 1990. Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals. Biochem Biophys Res Commun 167: 600-606.

A32. Mazel, D. , Pochet, S., and Marliere, P. 1994. Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation. Embo J 13: 914-923.

A33. Hao, B. , Gong, W. , Rajagopalan, P. T. , Zhou, Y., Pei, D. , and Chan, M. K. 1999. Structural basis for the design of antibiotics targeting peptide deformylase. Biochemistry 38: 4712-4719.

A34. Durand, D. J. , Gordon Green, B. , O'Connell, J. F. , and Grant, S. K. 1999. Peptide aldehyde inhibitors of bacterial peptide deformylases. Arch Biochem Biophys 367: 297-302.

A35. Clements, J. M. , Beckett, R. P. , Brown, A. , Catlin, G., Lobell, M. , Palan, S. , Thomas, W. , Whittaker, M. , Wood, S. , Salama, S. , et al. 2001. Antibiotic activity and characterization of BB-3497, a novel peptide deformylase inhibitor. Antimicrob Agents Chemother 45: 563-570.

A36. Hackbarth, C. J. , Chen, D. Z. , Lewis, J. G. , Clark, K., Mangold, J. B. , Cramer, J. A. , Margolis, P. S. , Wang, W., Koehn, J. , Wu, C. , et al. 2002. N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity. Antimicrob Agents Chemother 46: 2752-2764.

A37. Gupta, M. K. , Mishra, P. , Prathipati, P. , and Saxena, A. K.

2002.2D-QSAR in hydroxamic acid derivatives as peptide deformylase inhibitors and antibacterial agents. Bioorg Med Chem 10: 3713-3716.

A38. Jones, R. N. , and Rhomberg, P. R. 2003. Comparative spectrum and activity of NVP-PDF386 (VRC4887), a new peptide deformylase inhibitor. J Antimicrob Chemother 51: 157-161.

A39. Umezawa, H. , Aoyagi, T. , Tanaka, T. , Suda, H. , Okuyama, A. , Naganawa, H. , Hamada, M. , and Takeuchi, T. 1985.

Production of actinonin, an inhibitor of aminopeptidase M,. by actinomycetes. J Antibiot (Tokyo) 38: 1629-1630.

A40. Tieku, S. , and Hooper, N. M. 1992. Inhibition of aminopeptidases N, A and W. A re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme. Biochem Pharmacol 44: 1725-1730.

A41. Giglione, C. , and Meinnel, T. 2001. Organellar peptide deformylases: universality of the N-terminal methionine cleavage mechanism. Trends Plant Sci 6: 566-572.

A42. Giglione, C. , Vallon, O., and Meinnel, T. 2003. Control of protein life-span by N-terminal methionine excision.

Embo J 22: 13-23.

A43. Emanuelsson, O., and von Heijne, G. 2001. Prediction of organellar targeting signals. Biochim Biophys Acta 1541 : 114-119.

A44. Penta, J. S. , Johnson, F. M. , Wachsman, J. T. , and Copeland, W. C. 2001. Mitochondrial DNA in human malignancy. Mutat Res 488: 119-133., A45. Taanman, J. W. 1999. The mitochondrial genome: structure, transcription, translation and replication. Biochim Biophys Acta 1410: 103-123.

A46. Sawafuji, K. , Miyakawa, Y. , Weisberg, E. , Griffin, J. D., Ikeda, Y. , and Kizaki, M. 2003. Aminopeptidase inhibitors inhibit proliferation and induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta pathways. Leuk Lymphoma 44: 1987-1996.

A47. Zhao, Q. , Wang, J. , Levichkin, I. V. , Stasinopoulos, S., Ryan, M. T. , and Hoogenraad, N. J. 2002. A mitochondrial specific stress response in mammalian cells. Embo J 21: 4411-4419.

A48. Zinszner, H. , Kuroda, M. , Wang, X. , Batchvarova, N., Lightfoot, R. T. , Remotti, H., Stevens, J. L. , and Ron, D.

1998. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev 12: 982-995.

A49. Costantini, P. , Jacotot, E. , Decaudin, D. , and Kroemer, G. 2000. Mitochondrion as a novel target of anticancer chemotherapy. J Natl Cancer Inst 92: 1042-1053.

A50. Desagher, S. , and Martinou, J. C. 2000. Mitochondria as the central control point of apoptosis. Trends Cell Biol 10 : 369-377.

A51. Nieminen, A. L. 2003. Apoptosis and necrosis in health and disease: role of mitochondria. Int Rev Cytol 224: 29- 55.

A52. Bernard, P. 1996. The permeability transition pore.

Control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death. Biochim Biophys Acta 1275: 5-9.

A53. Zoratti, M., and Szabo, I. 1995. The mitochondrial permeability transition. Biochim Biophys Acta 1241: 139- 176.

A54. Cavalli, L. R. , and Liang, B. C. 1998. Mutagenesis, tumorigenicity, and apoptosis: are the mitochondria involved? Mutat Res 398: 19-26.

A55. Modica-Napolitano, J. S. , Koya, K. , Weisberg, E., Brunelli, B. T. , Li, Y. , and Chen, L. B. 1996. Selective damage to carcinoma mitochondria by the rhodacyanine MKT-077. Cancer Res 56: 544-550.

A56. Fantin, V. R. , Berardi, M. J. , Scorrano, L. , Korsmeyer, S. J. , and Leder, P. 2002. A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth. Cancer Cell 2: 29-42.

A57. Lazennec, C. , and Meinnel, T. 1997. Formate dehydrogenase-coupled spectrophotometric assay of peptide deformylase. Anal Biochem 244: 180-182.

1. C. Flinta, B. Persson, H. Jornvall and G. von Heijne, Sequence determinants of cytosolic N-terminal protein processing, Eur J Biochem 154 (1986) 193-6.

2. S. Y. Chang, E. C. McGary and S. Chang, Methionine aminopeptidase gene of Escherichia coli is essential for cell growth, J Bacteriol 171 (1989) 4071-2.

3. J. Solbiati, A. Chapman-Smith, J. L. Miller, C. G. Miller and J. E. Cronan, Jr. , Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal, J Mol Biol 290 (1999) 607-14.

4. D. Mazel, S. Pochet and P. Marliere, Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation, Embo J 13 (1994) 914-23.

5. B. Hao, W. Gong, P. T. Rajagopalan, Y. Zhou, D. Pei and M. K. Chan, Structural basis for the design of antibiotics targeting peptide deformylase, Biochemistry 38 (1999) 4712-9.

6. C. Giglione, M. Pierre and T. Meinnel, Peptide deformylase as a target for new generation, broad spectrum antimicrobial agents, Mol Microbiol 36 (2000) 1197-205.

7. J. P. Guilloteau, M. Mathieu, C. Giglione, V. Blanc, A.

Dupuy, M. Chevrier, P. Gil, A. Famechon, T. Meinnel and V. Mikol, The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the structure-based design of antibacterial agents, J Mol Biol 320 (2002) 951-62.

8. C. Giglione, A. Serero, M. Pierre, B. Boisson and T.

Meinnel, Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms, Embo J 19 (2000) 5916-29.

9. L. M. Dirk, M. A. Williams and R. L. Houtz, Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast- targeted enzymes in Arabidopsis, Plant Physiol 127 (2001) 97-107.

10. A. Serero, C. Giglione and T. Meinnel, Distinctive features of the two classes of eukaryotic peptide deformylases, J Mol Biol 314 (2001) 695-708.

11. V. Bracchi-Ricard, K. T. Nguyen, Y. Zhou, P. T.

Rajagopalan, D. Chakrabarti and D. Pei, Characterization of an eukaryotic peptide deformylase from Plasmodium falciparum, Arch Biochem Biophys 396 (2001) 162-70.

12. A. Kumar, K. T. Nguyen, S. Srivathsan, B. Ornstein, S.

Turley, I. Hirsh, D. Pei and W. G. Hol, Crystals of peptide deformylase from Plasmodium falciparum reveal critical characteristics of the active site for drug design, Structure (Camb) 10 (2002) 357-67.

13. Y. Xu and D. A. Scheinberg, Elimination of human leukemia by monoclonal antibodies in an athymic nude mouse leukemia model, Clin Cancer Res 1 (1995) 1179-87.

14. Y. Xu, L. T. Lai, J. L. Gabrilove and D. A. Scheinberg, Antitumor activity of actinonin in vitro and in vivo, Clin Cancer Res 4 (1998) 171-6.

15. C. Lazennec and T. Meinnel, Formate dehydrogenase- coupled spectrophotometric assay of peptide deformylase, Anal Biochem 244 (1997) 180-2.

16. Y. Wei and D. Pei, Continuous spectrophotometric assay of peptide deformylase, Anal Biochem 250 (1997) 29-34.

17. C. Apfel, D. W. Banner, D. Bur, M. Dietz, T. Hirata, C.

Hubschwerlen, H. Locher, M. G. Page, W. Pirson, G. Rosse and J. L. Specklin, Hydroxamic acid derivatives as potent peptide deformylase inhibitors and antibacterial agents, J Med Chem 43 (2000) 2324-31.

18. D. Groche, A. Becker, I. Schlichting, W. Kabsch, S.

Schultz and A. F. Wagner, Isolation and crystallization of functionally competent Escherichia coli peptide deformylase forms containing either iron or nickel in the active site, Biochem Biophys Res Commun 246 (1998) 342-6.

19. P. T. Rajagopalan and D. Pei, Oxygen-mediated inactivation of peptide deformylase, J Biol Chem 273 (1998) 22305-10.

20. T. Meinnel and S. Blanquet, Enzymatic properties of Escherichia coli peptide deformylase, J Bacteriol 177 (1995) 1883-7.

21. S. Ragusa, S. Blanquet and T. Meinnel, Control of peptide deformylase activity by metal cations, J Mol Biol 280 (1998) 515-23.

22. P. T. Rajagopalan, A. Datta and D. Pei, Purification, characterization, and inhibition of peptide deformylase from Escherichia coli, Biochemistry 36 (1997) 13910-8.

23. D. Z. Chen, D. V. Patel, C. J. Hackbarth, W. Wang, G.

Dreyer, D. C. Young, P. S. Margolis, C. Wu, Z. J. Ni, J.

Trias, R. J. White and Z. Yuan, Actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor, Biochemistry 39 (2000) 1256-62.

24. H. Umezawa, T. Aoyagi, T. Tanaka, H. Suda, A. Okuyama, H. Naganawa, M. Hamada and T. Takeuchi, Production of actinonin, an inhibitor of aminopeptidase M, by actinomycetes, J Antibiot (Tokyo) 38 (1985) 1629-30.

25. S. Tieku and N. M. Hooper, Inhibition of aminopeptidases N, A and W. A re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme, Biochem Pharmacol 44 (1992) 1725-30.

26. D. J. Durand, B. Gordon Green, J. F. O'Connell and S. K.

Grant, Peptide aldehyde inhibitors of bacterial peptide deformylases, Arch Biochem Biophys 367 (1999) 297-302.

27. LI. M. Clements, R. P. Beckett, A. Brown, G. Catlin, M.

Iiobell, S. Palan, W. Thomas, M. Whittaker, S. Wood, S.

Salama, P. J. Baker, H. F. Rodgers, V. Barynin, D. W. Rice and M. G. Hunter, Antibiotic activity and characterization of BB-3497, a novel peptide deformylase inhibitor, Antimicrob Agents Chemother 45 (2001) 563-70.

28. C. J. Hackbarth, D. Z. Chen, J. G. Lewis, K. Clark, J. B.

Mangold, J. A. Cramer, P. S. Margolis, W. Wang, J. Koehn, C. Wu, S. Lopez, G. Withers, 3rd, H. Gu, E. Dunn, R.

Kulathila, S. H. Pan, W. L. Porter, J. Jacobs, J. Trias, D. V. Patel, B. Weidmann, R. J. White and Z. Yuan, N- alkyl urea hydroxamic acids as a new class of peptide

deformylase inhibitors with antibacterial activity, Antimicrob Agents Chemother 46 (2002) 2752-64.

29. M. K. Gupta, P. Mishra, P. Prathipati and A. K. Saxena, 2D-QSAR in hydroxamic acid derivatives as peptide deformylase inhibitors and antibacterial agents, Bioorg Med Chem 10 (2002) 3713-6.

30. R. N. Jones and P. R. Rhomberg, Comparative spectrum and activity of NVP-PDF386 (VRC4887), a new peptide deformylase inhibitor, J Antimicrob Chemother 51 (2003) 157-61.

31. P. S. Margolis, C. J. Hackbarth, D. C. Young, W. Wang, D.

Chen, Z. Yuan, R. White and J. Trias, Peptide deformylase in Staphylococcus aureus: resistance to inhibition is mediated by mutations in the formyltransferase gene, Antimicrob Agents Chemother 44 (2000) 1825-31.

32. P. Margolis, C. Hackbarth, S. Lopez, M. Maniar, W. Wang, Z. Yuan, R. White and J. Trias, Resistance of Streptococcus pneumoniae to deformylase inhibitors is clue to mutations in defB, Antimicrob Agents Chemother 45 (2001) 2432-5.

33. J. D. Thompson, D. G. Higgins and T. J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res 22 (1994) 4673-80.

34. T. Meinnel, S. Blanquet and F. Dardel, A new subclass of the zinc metalloproteases superfamily revealed by the solution structure of peptide deformylase, J Mol Biol 262 (1996) 375-86.

35. M. K. Chan, W. Gong, P. T. Rajagopalan, B. Hao, C. M. Tsai and D. Pei, Crystal structure of the Escherichia coli peptide deformylase, Biochemistry 36 (1997) 13904-9.

36. P. Anton, J. O'Connell, D. O'Connell, L. Whitaker, G. C.

O'Sullivan, J. K. Collins and F. Shanahan, Mucosal

subepithelial binding sites for the bacterial chemotactic peptide, formyl-methionyl-leucyl- phenylalanine (FMLP), Gut 42 (1998) 374-9.

37. C. Giglione and T. Meinnel, Organellar peptide deformylases: universality of the N-terminal methionine cleavage mechanism, Trends Plant Sci 6 (2001) 566-72.

38. C. Giglione, O. Vallon and T. Meinnel, Control of protein life-span by N-terminal methionine excision, Embo J 22 (2003) 13-23.

39. O. Emanuelsson and G. von Heijne, Prediction of organellar targeting signals, Biochim Biophys Acta 1541 (2001) 114-9.

40. J. S. Penta, F. M. Johnson, J. T. Wachsman and W. C.

Copeland, Mitochondrial DNA in human malignancy, Mutat Res 488 (2001) 119-33.

41. Mazel, D. , Pochet, S. , and Marliere, Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation. Embo J, 1994.13 (4): p. 914-23.

42. Meinnel, T. and Blanquet, S. , Characterization of the Thermus thermophilus locus encoding peptide deformylase and methionyl-tRNA (fMet) formyltransferase. J Bacteriol, 1994. 176 (23): p. 7387-90.

43. Nguyen, K. T. , Hu, X. , Colton, C. , Chakrabarti, R. , Zhu, M. X. , and Pei, D. , Characterization of a human peptide deformylase : implications for antibacterial drug design.

Biochemistry, 2003.42 (33): p. 9952-8.

44. Guo, X. C. , Ravi Rajagopalan, P. T. , and Pei, D. , A direct spectrophotometric assay for peptide deformylase.

Anal Biochem, 1999.273 (2): p. 298-304.

45. Bantan-Polak, T., Kassai, M. , and Grant, K. B. , A comparison of fluorescamine and naphthalene-2, 3- dicarboxaldehyde fluorogenic reagents for microplate-

based detection of amino acids. Anal Biochem, 2001.

297 (2): p. 128-36.

46. M. D. Lee, C. Antczak, Y. Li, F. M. Sirotnak, W. G.

Bornmann, and D. A. Scheinberg. A new human peptide deformylase inhibitable by actinonin. Biochem.

Biophys. Res. Comm. (2003) Article in press.

47. M. F. Fromm. Genetically determined differences in P-glycoprotein function: implications for disease risk. Toxicology (2002) Dec 27; 181-182: 299-303.

48. A. Serero, C. Giglione, A. Sardini, J. Martinez-Sanz, and T. Meinnel. An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway. J. Biol. Chem. (2003) Oct 7. Epub ahead of print.

49. Gordon, J. J. , Kelly, B. K. , Miller, G. A. , Actinonin: an antibiotic substance produced by an actinomycete.

Nature, London, 1962.195 : p. 701.

50. Tuschl, T. , Elbashir S. , Harborth, J. and Weber Klaus.

The siRNA user guide (revised May 6, 2004). http ://www. rockefeller. edu/labheads/tuschl/sirna. html.

51. Opalinska, J. B. and Gewirtz, A. M. Nucleic-Acid Therapeutics : Basic Principles and Recent Applications.

Nature, July 2002. Vol. 1. p. 503.

52. Branch A. D. A Good Antisense Molecule is Hard to Find.

TIBS, Feburary 1998. Vol. 23. p. 45.