FIELD:

The present invention relates to peptides that antagonise CXCR4 and their use in the treatment of CXCR4-mediated diseases.

BACKGROUND:

The human immunodeficiency virus (HIV) is responsible for the acquired immunodeficiency syndrome (AIDS), a condition characterized by CD4+ T cell depletion and consequent susceptibility to opportunistic infections (Moir, Chun et al. 2011). In 2017 there were 36.9 million people globally living with HIV, with nearly one-half of this population being treated with a combined antiretroviral therapy (cART) (UNAIDS 2018). The cART, which consists in a triple drug regimen directed towards at least two distinct molecular targets, is broadly accepted as the most efficient way to control viraemia (Gunthard, Aberg et al. 2014). However, this therapy does not lead to complete virus elimination and the potential acquisition and transmission of HIV drug resistant strains poses a challenge in drug discovery.

The steady development of peptide-based drugs over the last few years is one of the most promising fields in drug discovery (Vlieghe, Lisowski et al. 2010) (Kaspar and Reichert 2013). The high specificity and affinity of peptides towards their molecular targets is one of their main advantages, as well as low toxicity (Castel, Chteoui et al. 2011). There are several peptides known for their anti-HIV activity, targeting different steps of the HIV life cycle, such as fusion with the host cell (Rausch 1990) (Jin BS et al. 2000), reverse transcription (Gleenberg, Herschhorn et al. 2007) (Agopian, Gros et al. 2009), integration (Oz Gleenberg, Avidan et al. 2005) (Li HY 2006), and maturation (Schramm HJ 1996) (John M. Louis 1998). T20 (enfuvirtide, fuzeon) is emblematic of fusion inhibitor peptides used in the clinical management of HIV infection, preventing the merging of viral and cellular membranes, thus precluding virus entry. HIV entry is a multistep process mediated by the viral envelope glycoprotein complex (Env), which consist of a turner of heterodimers, each formed by a gpl20 surface glycoprotein and a gp41 transmembrane protein. Binding of gpl20 to CD4 and to one of the chemokine receptors that serve as HIV co-receptor (CCR5 or CXCR4) triggers conformational changes, leading to the exposure of a hydrophobic peptide at the N-terminus of gp41, which inserts into the cellular membrane. Then, two alpha helices (HRl and HR2) on gp41 fold onto each other, forming a six-helix bundle for each Env trimer. This rearrangement brings together the viral and cellular membranes, leading to their fusion. T20, the only clinically approved fusion inhibitor, is a 36 amino acid peptide whose sequence derives from the HR2 that, by binding to HR1, prevents the formation of the six-helix bundle.

T20 has been shown to suppress replication of HIV variants with multidrug resistance to reverse transcriptase and protease inhibitors (Thakur, Qureshi et al. 2012) (Qureshi et al. 2013) (Kilby JM 1998) (Lalezari JP 2003) (Ashkenazi, Wexler-Cohen et al. 2011). However, its administration by high-dosage injection, plus short in vivo half-life (Naider and Anglister 2009), results in low efficacy and has limited its clinical application to “salvage” treatment. As with any antiretroviral, the emergence of T20 resistant viral variants was observed both in vitro and in 3 patients (Wei X 2002) (Matthews, Salgo et al. 2004) (Rimsky L T et al. 1998) (Naider and Anglister 2009) (Ashkenazi, Wexler-Cohen et al. 2011) (Welch, Francis et al. 2010). Antimicrobial peptides (AMPs) are a group of molecules known for their broad-spectrum activity against bacteria, fungi and viruses. Despite the considerable number of AMPs described, and the fact that some have been shown to directly inhibit one or more steps in the HIV replication cycle, (Thakur, Qureshi et al. 2012) (Guangshun Wang 2012), in-depth studies of their anti-HIV potential are scarce.

PepR, a multifunctional peptide derived from the Dengue virus capsid protein (Freire, Veiga et al. 2013) (Freire, Veiga et al. 2014), has been reported to display antibacterial activity against both Gram-positive and -negative bacteria (Freire, Almeida Dias et al. 2015).

SUMMARY:

The present inventors have discovered a protease-resistant peptide fragment (termed pepRFl) of the Dengue virus capsid protein that is a potent antagonist of C-X-C motif chemokine receptor 4 (CXCR4). This peptide fragment may be useful, for example, in the treatment of CXCR4 mediated conditions, such as HIV and cancer.

A first aspect of the invention provides a pepRFl peptide consisting of the amino acid sequence of SEQ ID NO: 3. The pepRFl peptide may consist of residues 66 to 82 of the capsid protein of Dengue virus 2, 1 or 3; or residues 65 to 81 of the Dengue virus 2 capsid protein; or the corresponding residues in another Dengue virus capsid protein. For example, the pepRFl peptide may consist of any one of SEQ ID NOs: 10 to 14.

A second aspect of the invention provides a pharmaceutical composition comprising a pepRFl peptide of the first aspect and a pharmaceutically acceptable excipient. A third aspect of the invention provides a method of treating a CXCR4-mediated disease comprising administering a therapeutically effective amount of a pepRFl peptide of the first aspect to an individual in need thereof.

A fourth aspect of the invention provides a method of mobilising haematopoietic stem cells (HSCs) in an individual comprising administering a therapeutically effective amount of a pepRFl peptide of the first aspect to an individual.

A fifth aspect of the invention provides a method of stem cell transplantation comprising mobilising haematopoietic stem cells (HSCs) in a donor individual using a method of the fourth aspect, obtaining a sample of peripheral blood from the donor individual, isolating HSCs from the sample of peripheral blood and administering the isolated HSCs to a recipient individual.

A sixth aspect of the invention provides a pepRF 1 peptide of the first aspect for use in a method of treating a CXCR4-mediated disease, for example a method according to the third aspect; a method of mobilising HSCs, for example a method according to the fourth aspect; or a method of stem cell transplantation, for example a method according to the fifth aspect.

A seventh aspect of the invention provides the use of a pepRF 1 peptide of the first aspect in the preparation of a medicament for use in a method of treating a CXCR4-mediated disease, for example a method according to the third aspect; a method of mobilising HSCs, for example a method according to the fourth aspect; or a method of stem cell transplantation, for example a method according to the fifth aspect.

Other aspects and embodiments of the invention are described in more detail below.

DETAILED DESCRIPTION:

This invention relates to the finding that a peptide fragment of the Dengue virus capsid protein (termed herein a pepRFl peptide) is an antagonist of C-X-C motif chemokine receptor 4 (CXCR4). PepRFl was also found to display potent anti-HIV activity as well as being highly stable in serum and exhibiting low toxicity. PepRFl peptides as described herein may be useful for the antagonism CXCR4 in vitro, for example in the treatment of CXCR4 mediated conditions, such as HIV and cancer.

A pepRFl peptide described herein may consist of residues 66 to 82 of a Dengue virus 2 capsid protein, Dengue virus 1 capsid protein, or Dengue virus 3 capsid protein; residues 65 to 81 of Dengue virus 4 capsid protein; or the corresponding residues in another Dengue virus capsid protein. An exemplary Dengue virus 2 capsid protein sequence may has the database accession number NP 739591.2 (C4PK10) and is shown in SEQ ID NO: 1. An exemplary Dengue virus 1 capsid protein sequence may has the database accession number NP 722457.2 and is shown in SEQ ID NO: 2. An exemplary Dengue virus 3 capsid protein sequence may has the database accession number YP 001531165 and is shown in SEQ ID NO: 3. An exemplary Dengue virus 4 capsid protein sequence may has the database accession number NP 740314.1 and is shown in SEQ ID NO: 4. The structure of Dengue virus capsid protein has been characterised in the art (Ma et al PNAS (2004) 101 10 3414-3419; PDB 1R6R).

A pepRFl peptide described herein may consist of residues 1 to 17 of a pepR peptide. Exemplary pepR peptide sequences are shown in SEQ ID NOs: 5 to 8.

A pepRFl peptide described herein may consist of the amino acid sequence of: LX1RWGX2X3KKX4X5AIX6X7LX8 wherein Xi is K or A; X ₂ is T, S or Q; X ₃ is I, F or L; X ₄ is S or N; X ₆ is K or G; X ₆ is N or K; X ₇ is V or I, and X ₈ is R, K or I (SEQ ID NO: 9).

For example, the pepRFl peptide may consist of the amino acid sequence of SEQ NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

A pepRFl peptide described herein may be modified. Suitable peptide modifications are well known in the art (see for example, R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2004).

In some embodiments, one or more amino acid residues may be non-naturally occurring. For example, one or more of the amino acid residues of the peptide may be replaced by the corresponding D-amino acid, N-methyl amino acid, homo amino acid, alpha-methyl amino acid, beta (homo) amino acid, gamma amino acid, or backbone modification peptoid. One or more of the peptide bonds in the pepRFl peptide may be replaced by other linkages, such as N- alkyl (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46,47), retro-inverse amide (Chorev, M and Goodman, M., Acc. Chem. Res, 1993, 26, 266), thioamide (Sherman D. B. and Spatola, A. F. J. Am. Chem. Soc., 1990, 112, 433), thioester, phosphonate, ketomethylene (Hoffman, R. V. and Kim, H. O. J. Org. Chem., 1995, 60, 5107), hydroxymethylene, fluorovinyl (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297), vinyl, methyleneamino (Sasaki, Y and Abe, J. Chem. Pharm. Bull. 199745, 13), methylenethio (Spatola, A. F., Methods Neurosci, 1993, 13, 19), alkane (Lavielle, S. et. al., Int. J. Peptide Protein Res., 1993, 42, 270) or sulfonamido bonds (Luisi, G. et al. Tetrahedron Lett. 1993, 34, 2391). Aspects of the invention provide peptidomimetics of the pepRFl peptide of SEQ ID NO: 9 in which all amino acid residues and/or peptide bonds are non-naturally occurring. Peptidomimetics may include retro inverso peptides, peptoids and depsipeptides. The term “pepRFl peptide” may refer to a peptide or a peptidomimetic and the term amino acid residue ^' may refer to the equivalent sub-unit of a peptidomimetic. In other embodiments, the pepRFl peptide may comprise a chemical modification. Examples of such modifications include, alkylation, acylation, such as acetylation, amidation, carboxyl ation, ester formation, glycosylation, lipidation, such as myristoylation, palmitoylation or prenylation, phosphorylation, hydroxylation, citrullination, methylation, succinyl ation, ubiquitinylation, PEGylation, labelling with a label compound, β-elimination, deimidation, and deamidation.

The pepRFl peptide may further comprise a C terminal modification. For example, a chemical group such as a hydroxyl group, amino group, azide group, carboxamide group, ester group, thioester group, lipid moiety, carbohydrate moiety, polyethylene glycol moiety, or small chemical group, such as lysine or cysteine, may be linked covalently or non-covalently to the C terminus of the pepRFl peptide. In some preferred embodiments, the pepRFl peptide may further comprise a carboxamide group at the C terminus.

The pepRFl peptide may further comprise an N terminal modification. For example, a chemical group, such as an acetyl group, biotinyl group, fluorenyl group, methoxy group, carbonyl group, formyl group, acyl group, such as a palmitoyl, myristyl, or stearyl group, polyethylene glycol, fluorescein, t-butyloxycarbonyl group, dansyl group, 2, 4-dinitrophenyl group, carbobenzoxyl group, or 7-methoxycoumarin acetyl (Mca) group, may be linked covalently or non-covalently to the N terminus of the pepRFl peptide.

A heterologous element is an element which is not associated or linked to the subject feature in its natural environment i.e. association with a heterologous element is artificial and the element is only associated or linked to the subject feature through human intervention.

One, two, three, four, five or more heterologous amino acids, for example a heterologous peptide or heterologous polypeptide sequence, may be joined, linked or fused to a pepRFl peptide set out herein. The one or more heterologous amino acids may include amino acid sequences from a non-Dengue virus source. For example, a pepRFl peptide as described herein may be part of a fusion protein which contains one or more heterologous amino acid sequences additional to the pepRFl peptide sequence set out above. The one or more heterologous amino acid sequences may be attached to the N or C terminal of the pepRFl peptide sequence and may not be the corresponding amino acid sequences from pepR peptide or Dengue virus capsid protein (i.e. the pepRFl peptide sequence is the only sequence in the conjugate or fusion derived from pepR or the Dengue virus capsid protein). For example, the fusion protein comprising the pepRFl peptide may further comprise one or more additional domains which improve the stability, pharmacokinetic, targeting, affinity, purification and production properties of the pepRFl peptide. A pepRF 1 peptide as described herein may be part of a conjugate or fusion protein which contains one or more additional chemical moieties, in addition to the pepRF 1 peptide sequence set out above. The one or more chemical moieties may be attached to the N or C terminal of the pepRF 1 peptide sequence. The chemical moieties may include lipid groups or glycosyl groups. Lipidation of the pepRF 1 peptide may, for example, increase membrane affinity and improve the interaction of the pepRF 1 peptide with membrane receptors, such as CXCR4. Suitable techniques for the conjugation of peptides to chemical moieties, for example peptide lipidation and glycosylation, are well established in the art.

A pepRF 1 peptide as described herein may be isolated, in the sense of being free from contaminants, such as other pepR peptide fragments, other polypeptides and/or cellular components.

The pepRF 1 peptide may be in the free form, or any pharmacologically acceptable salt form, for example, a form of acid salt, metal salt, alkaline earth metal salt, or amine salt.

A pepRF 1 peptide which consists of a defined sequence from a Dengue virus capsid protein, or a fusion protein or conjugate comprising such a pepRF 1 peptide, may be devoid of additional residues that are contiguous with the defined sequence of the pepRF 1 peptide in the Dengue virus capsid protein sequence i.e. the pepRF 1 peptide may be devoid of the amino acid sequence that is naturally located N or C terminal of the pepRF 1 peptide sequence in the Dengue virus capsid protein.

A pepRF 1 peptide as described herein may be provided using synthetic or recombinant techniques which are standard in the art. Conveniently, a pepRF 1 peptide as described herein may be produced by solid phase synthesis. Peptides are typically synthesized by solid phase synthesis in a stepwise fashion from the C terminus to the N terminus. In an initial step, an N protected amino acid is covalently attached to an insoluble solid support via its carbonyl group. Suitable groups for N protecting the amino acid include 9-fluorenylmethyloxycarbonyl group (Fmoc) and t-butyloxycarbonyl (Boc). Following covalent attachment of the N protected amino acid, the N protecting group is removed and the deprotected NH2 group of the attached amino acid is reacted with the carboxylic acid group of the next N protected amino acid to generate a nascent peptide comprising 2 amino acids that is covalently attached to the solid phase. This process is repeated until the complete peptide sequence is built up on the solid phase. In some embodiments, protecting groups may be employed to prevent functional groups in the side chains of amino acids from reacting with an incoming N protected amino acids. These side chain protecting groups may be present throughout the synthesis of the peptide and may be removed in a final deprotection step. A method of producing a pepRFl peptide may comprise synthesising a peptide consisting of SEQ ID NO: 3 by solid or liquid phase peptide synthesis.

Methods of solid phase peptide synthesis are well-established in the art (see for example Coin et al Nature Protocols 2, 3247-3256 (2007) Stawikowski (2002) Curr Protoc Protein Sci. 2002 Unit-18.1 oi: 10.1002/0471140864. psl801s26; Chan and White; Fmoc Solid Phase Peptide Synthesis - A Practical Approach. Oxford University Press, 2000; Stewart, J. M.; Young, J. D. Solid-Phase Peptide Synthesis (2nd ed.), Pierce Chemical Co., Rockford, IL, 1984; Atherton, E.; Sheppard, R. C., Solid-Phase Peptide Synthesis: A Practical Approach. Oxford University Press: New York City, 1989; M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); J. H. Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford 1991; in Applied Biosystems 430A User’s Manual, ABI Inc., Foster City, California; G. A. Grant, (Ed.) Synthetic Peptides, A User’s Guide. W. H. Freeman & Co., New York 1992, and G.B. Fields, (Ed.) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol. 289). Academic Press, New York and London 1997); Merrifield, J. Amer. Chem. Soc. 85:2149-54(1963)). Methods of liquid phase peptide synthesis are also well-established in the art (U.S. Pat. No. 5,516,891).

An example of a suitable method of peptide synthesis is described in detail below.

A pepRFl peptide described herein antagonises C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 (Gene ID: 7852) is a G-protein coupled receptor specific for CXCL12 (also called SDF1; Gene ID 6387) and is involved in a number of cellular processes, including embryogenesis, immune surveillance, inflammation response, tissue homeostasis, and tumor growth and metastasis. Human CXCR4 has been well-characterised in the art and may, for example have the amino acid sequence of database accession number NP OO 1008540.1, NP_001334985.1, NP_001334988.1, NP_001334989.1, CAA12166.1 or NP_003458.1.

A pepRFl peptide binds to CXCR4 and inhibits CXCR4-mediated signalling. The pepRF 1 peptide may bind to CXCR4 on the surface of a mammalian cell without triggering any CXCR4-mediated response. For example, the pepRFl peptide may bind to CXCR4 without simulating intracellular Ca2+ signalling or the internalisation of CXCR4.

A pepRFl peptide may prevent or inhibit the binding and/or activation of CXCR4 by its natural ligand CXCL12. For example, the pepRFl peptide may inhibit the intracellular Ca2+ signalling elicited by CXCL12. Suitable methods of measuring intracellular Ca2+ signalling are described in more detail below. A pepRFl peptide may display anti- viral activity, preferably anti-HIV activity. For example, a pepRFl peptide may specifically inhibit the CXCR4-mediated entry of a virus into mammalian cells by binding to CXCR4.

Preferably, a pepRFl peptide displays anti-HIV activity. HIV may include HIV1 and HIV2. The pepRFl peptide may display activity against CXCR4 tropic or dual tropic HIV.

A pepRFl peptide may inhibit the infection of mammalian cells by a virus, such as HIV. A suitable pepRFl peptide may inhibit the infectivity of HIV in mammalian cells, such as TZM- bl cells, with an IC50 of less than 20nM, less than 10nM, less than 5 nM or less than 2nM, as determined in a single-cycle infectivity assay. Suitable methods of measuring viral inhibition, including single-cycle infectivity assays, are described in more detail below.

A pepRFl peptide described herein is protease-resistant and is stable in serum. For example, a suitable pepRFl peptide may display a half-life of 90 mins or more in 25% (v/v) human serum at 37°C. Suitable methods of measuring protein stability are described in more detail below.

A pepRFl peptide described herein may display low toxicity. Toxicity may be determined by measuring the effect of the peptide on mammalian cell viability. For example, a suitable pepRFl peptide may display no cytotoxic effects on mammalian cells, such as TZM- bl cells at concentrations of 80 μM or less. Suitable methods of determining cell viability are described in more detail below.

A pepRFl peptide as described above may be formulated into a pharmaceutical composition. A pharmaceutical composition is a formulation comprising one or more active agents and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be capable of eliciting a therapeutic effect.

A pharmaceutical composition may comprise a pepRFl peptide and a pharmaceutically acceptable excipient.

A method of making a pharmaceutical composition may comprise; admixing a pepRFl peptide as described above with a pharmaceutically acceptable excipient.

The term “pharmaceutically acceptable” relates to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound veterinary or medical judgement, suitable for use in contact with the tissues of a subject (e.g. human or other mammal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable excipients and carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants, and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH-adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers. The pharmaceutical compositions described herein are not limited by the selection of the carrier. The preparation of these pharmaceutically-acceptable compositions, from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art.

Suitable carriers, excipients, etc. may be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences [71] and The Handbook of Pharmaceutical Excipients, 4th edit., eds. R. C. Rowe et al, APhA Publications, 2003.

A pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the one or more isolated immunogenic polypeptides into association with a carrier or excipient as described above which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both.

Pharmaceutical compositions described herein may be produced in various forms, depending upon the route of administration. The pharmaceutical compositions may be prepared for administration to subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories. Pharmaceutical compositions may also be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze- drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant. Pharmaceutical compositions can also be made in the form of suspensions or emulsions.

Pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use.

The pharmaceutical composition may be administered to a subject by any convenient route of administration. In some embodiments, administration is by systemic routes, including oral, or more preferably parenteral routes. For example, the pharmaceutical composition may be administered by intravenous, intraperitoneal or subcutaneous injection.

A pepRFl peptide or pharmaceutical composition as described herein may be for use in a method of treatment of the animal or human body, for example a CXCR4-mediated disease in an individual.

A method of treating a CXCR4-mediated disease may comprise administering a therapeutically effective amount of a pepRFl peptide or pharmaceutical composition as described herein to an individual in need thereof.

An individual with a CXCR4-mediated disease may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of a CXCR4- mediated disorder in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine such as Harrison’s Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001. In some embodiments, the individual may have been previously identified or diagnosed with a CXCR4- mediated disorder or a method of the invention may comprise identifying or diagnosing the presence of a CXCR4-mediated disorder in the individual, prognosing a CXCR4-mediated disorder or assessing the risk of onset of a CXCR4-mediated disorder in the individual.

Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the CXCR4-mediated disease, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the CXCR4-mediated disease, cure or remission (whether partial or total) of the CXCR4-mediated disease, preventing, delaying, abating or arresting one or more symptoms and/or signs of the CXCR4-mediated disease or prolonging survival of a subject or patient beyond that expected in the absence of treatment.

Treatment as a prophylactic measure (i.e. prophylaxis) is also included (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset). For example, an individual susceptible to or at risk of the occurrence or re-occurrence of a CXCR4-mediated disease, such as cancer may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of CXCR4-mediated disease or one or more symptoms thereof in the individual.

An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human.

In some preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.

A CXCR4-mediated disease is a disorder or condition in which CXCR4/CXCL12 signalling plays an active role in the onset, development, maintenance or parthenogenesis of the disorder or condition.

CXCR4 mediated diseases include HIV infection, for example HIV1 or HIV2 infection.

HIV uses CXCR4 as a receptor to facilitate its entry into host CD4+ T cells. Antagonism of CXCR4 using a pepRFl peptide as described herein inhibits the entry of HIV into a cell and thus inhibits viral infection.

HIV infection may include CXCR4-tropic HIV infection or dual-tropic HIV infection.

HIV may also use CCR5 as a co-receptor to facilitate its entry into host CD4+ T cells. In some embodiments, a pepRFl peptide may be administered in combination with a CCR5 inhibitor. For example, a method of treating HIV infection may comprise administering therapeutically effective amounts of a pepRFl peptide in combination with a CCR5 inhibitor to an individual in need thereof. Suitable CCR5 inhibitors are known in the art and include maraviroc, aplaviroc, vicriviroc, leronlimab (PRO 140), AOP-RANTES, NNY-RANTES, Cl, C5-RANTES, TAK- 799 TAK-652 TAK-220 and INCB9471.

A pepRFl peptide may be administered in combination with one or more anti-viral agents, for example, anti-retroviral agents. For example, a method of treating HIV infection may comprise administering therapeutically effective amounts of a pepRFl peptide in combination with an anti-retroviral agent to an individual in need thereof.

Anti-retroviral agents may include nucleoside reverse transcriptase inhibitors (NRTIs), such as abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir and zidovudine; non- nucleoside reverse transcriptase inhibitors (NNRTIs), such as delavirdine, doravirine, efavirenz, etravirine, nevirapine, elsulfavirine and rilpivirine; protease inhibitors, such as atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfmavir, ritonavir, saquinavir, and tipranavir, and integrase inhibitors, such as bictegravir, cabotegravir, dolutegravir, elvitegravir, or raltegravir.

Anti-retroviral agents may also include fusion inhibitors, such as enfuvirtide, tifuvirtide, TRI-999 and TRI-1144; CD4-gpl20 binding inhibitors, such as Ibalizumab (TNX-355), Fostemsavir, PRO-542, BMS-378806 (l-[(2R)-4-benzoyl-2-methylpiperazin-l-yl]-2-(4- methoxy-lH-pyrrolo[2,3-b]pyridin-3-yl)ethane-l,2-dione), BMS-488043 (l-(4- benzoylpiperazin-l-yl)-2-{4,7-dimethoxy-lH-pyrrolo[2,3-c]pyr idin-3-yl}ethane-l,2-dione), and zintevir; entry inhibitors, such as griffithsin, and DCM205 (5-[(E)-2-[[(E)-2-(3,4,5- trihydroxyphenyl)ethenyl]sulfonylmethylsulfonyl]ethenyl]benz ene-l,2,3-triol); and attachment inhibitors, such as cyanovirin-N, PRO 2000, glycyrrhizin, suramin and aurintricarboxylic acid (ATA).

CXCR4-mediated diseases also include cancer. The CXCR4-CXCL12 axis is involved in tumour biology and antagonism of CXCR4 using a pepRFl peptide may exert an anti-cancer effect.

Cancer is characterised by the abnormal proliferation of malignant cancer cells and may include leukaemias, such as AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP). Preferred CXCR4-mediated cancers include breast, pancreatic, cervical, prostate, lung, and colorectal cancer; primary brain cancers, such as glioblastoma; melanoma, leukaemia, multiple myeloma; and non-Hodgkin’s lymphoma.

In some embodiments, a pepRFl peptide may be useful in inhibiting or reducing the metastasis of a cancer. For example, a method of reducing or inhibiting metastasis in an individual with cancer may comprise administering therapeutically effective amounts of a pepRFl peptide to the individual.

In some embodiments, the individual may have minimal residual disease (MRD) after an initial cancer treatment.

Cancer treatment may include inhibiting cancer growth, including complete cancer remission, and/or inhibiting cancer metastasis. Cancer growth generally refers to any one of a number of indices that indicate change within the cancer to a more developed form. Thus, indices for measuring an inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumour volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumour growth, a destruction of tumour vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of T cells, and a decrease in levels of tumour-specific antigens.

In some embodiments, a pepRFl peptide described herein may be useful eliciting an immune response against cancer cells in an individual. For example, inhibition of CXCR4 using a pepRFl peptide may promote immune cell activation and/or reduce or inhibit immune suppression within the tumour microenvironment. Inhibition of CXCR4 using a pepRFl peptide may for example alleviate desmoplasia and increase T-lymphocyte infiltration.

In some embodiments, a pepRFl peptide may be administered in combination with another cancer therapy, such as immuno-, or chemo- or radio-therapy. For example, a method of treating cancer may comprise administering therapeutically effective amounts of a pepRFl peptide in combination with an anti-cancer agent to an individual in need thereof.

CXCR4 mediated diseases also include autoimmune diseases, such as rheumatoid arthritis; inflammatory diseases; neurodegenerative diseases; non-alcoholic fatty liver diseases (NAFLD), such as non-alcoholic steatohepatitis (NASH); and cardiovascular diseases, such as atherosclerosis.

A pepRFl peptide or pharmaceutical composition as described herein may be useful in the mobilization haematopoietic stem cells (HSCs) in an individual. For example, the pepRFl peptide may increase or promote the mobilisation of HSCs from the bone marrow to the peripheral blood. A method of HSC mobilisation may comprise administering a therapeutically effective amount of a pepRFl peptide or pharmaceutical composition described herein to an individual in need thereof, thereby increasing the amount or proportion of haematopoietic stem cells (HSCs) in the peripheral blood.

HSCs may include CD34+ HSCs.

HSC mobilisation may be useful, for example in a method of stem cell transplantation (SCT). A method of stem cell transplantation may comprise administering a therapeutically effective amount of a pepRFl peptide or pharmaceutical composition as described herein to a donor individual, obtaining a sample of peripheral blood from the donor individual, isolating HSCs from the sample of peripheral blood and administering the HSCs to a recipient individual.

The donor individual and the recipient individual may be the same (i.e. autologous stem cell transplantation) or different (i.e. allogeneic stem cell transplantation).

In some embodiments, the donor and recipient may be human leukocyte antigen (HLA) matched.

The recipient individual may have a damaged or defective bone marrow or immune system. For example, the recipient individual may have a disorder that damages the immune system, for example anaemia, such as sickle cell anaemia or severe aplastic anaemia, a cancer, such as leukaemia, lymphoma, or myeloma, multiple sclerosis, or a metabolic disorder, such as thalassemia.

The recipient individual may have undergone a therapy that damages the immune system, such as high dose chemo- or radio-therapy, for example for the treatment of cancer.

In some embodiments, the pepRFl peptide or pharmaceutical composition as described herein may be administered in combination with another HSC mobilizing agent, such as G-CSF or plerixafor.

It will be appreciated that appropriate dosages of the therapeutic agent, and compositions comprising the therapeutic agent, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular cells, the route of administration, the time of administration, the rate of loss or inactivation of the cells, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of cells and the route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of therapeutic polypeptides are well known in the art (Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of a pepRFl peptide described herein may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the pepRFl peptide described herein is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the pepRFl peptide described herein and the nature of any detectable label or other molecule attached to the pepRFl peptide described herein.

A typical dose of a pepRFl peptide will be in the range of 0.1 mg/kg to 10Omg/kg. For example, a dose in the range 100 μg to 1 g may be used for systemic applications. An initial higher loading dose, followed by one or more lower doses, may be administered. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated. For example, a composition may be administered in combination with vaccination, immune checkpoint inhibition, other immunotherapies and potentially chemotherapy and radiotherapy.

Treatment may comprise the administration of a therapeutically effective amount of a pepRFl peptide or pharmaceutical composition to the individual. “Therapeutically effective amount" relates to the amount of a pepRFl peptide or pharmaceutical composition that is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio. For example, a suitable amount of a pepRFl peptide or pharmaceutical composition for administration to an individual may be an amount that generates a therapeutic effect in the individual. A therapeutic effect may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). In some embodiments, pharmaceutical compositions may be administered more than once to the same individual with sufficient time interval to obtain a boosting effect in the individual, e.g., at least 1 week, 2 weeks, 3 weeks or 4 weeks, between administrations, preferably about 2 weeks. A prime dose of the pharmaceutical composition may be administered to the individual followed by a booster dose. For example, a prime dose may be administered to a let at 1-4 weeks old and a booster dose at 3-6 weeks old. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation and the subject being treated. For example, in some embodiments, the prime dose of the pharmaceutical composition may be administered when the levels of maternal derived antibodies in the let have declined (e.g. after 2-4 weeks) followed by a booster dose two weeks later. Single or multiple administrations may be carried out with the dose level and pattern being selected by the veterinary surgeon or physician.

The treatment schedule for an individual may be dependent on the pharmocokinetic and pharmacodynamic properties of the pepRFl peptide, the route of administration and the nature of the condition being treated.

Treatment may be periodic, and the period between administrations may be about 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 96 hours or more, or one week or more. Suitable formulations and routes of administration are described above and may be readily determined by a physician for any individual patient.

In other embodiments, a pepRFl peptide as described herein may be administered in combination with one or more other therapies. For example, a pepRFl peptide as described herein may be administered in combination with cytotoxic chemotherapy or radiotherapy for the treatment of cancer or in combination with a CCR5 inhibitor for the treatment of HIV infection.

When the therapeutic agents are used in combination with additional therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route. When a therapeutic agent is used in combination with an additional therapeutic agent active against the same disease, the dose of each agent in the combination may differ from that when the therapeutic agents are used alone. Appropriate doses will be readily appreciated by those skilled in the art.

Another aspect of the invention provides an in vitro method of detecting CXCR4-tropic HIV or dual-tropic HIV, the method comprising; determining the infectivity of an HIV sample in the presence and absence of a pepRFl peptide, wherein a decrease in infectivity of HIV sample in the presence relative to the absence of pepRFl peptide is indicative that the HIV sample is CXCR4-tropic HIV or dual-tropic HIV.

Another aspect of the invention provides a method of determining the responsiveness of an HIV infection in an individual to treatment comprising; contacting a sample of HIV from the individual with mammalian cells in the presence and absence of a pepRFl peptide, wherein a reduction in infection of the cells by the HIV in the presence relative to the absence of pepRFl peptide is indicative that the HIV infection is responsive to treatment with a CXCR4 antagonist, such as a pepRFl peptide.

The method may further comprise contacting a sample of HIV from the individual with mammalian cells in the presence and absence of a CCR5 inhibitor, wherein a reduction in infection of the cells by the HIV in the presence relative to the absence of the pepRFl peptide or the CCR6 is indicative that the HIV infection is responsive to treatment with a combination of an CXCR4 antagonist and a CCR5 inhibitor.

Infectivity may be determined using conventional techniques as described herein. For example, the HIV sample may be contacted with CD4+ cells, such as CD4+ T cells, in the presence and absence of pepRFl peptide and the infection of the CD4+ cells determined.

The HIV sample may be a sample from an individual with an HIV infection or suspected of having an HIV infection. Inhibition of infection of the CD4+ cells may be indicative that the individual has a CXCR4-tropic or dual-tropic HIV infection. This may be useful for example in the diagnosis, prognosis or treatment of the individual.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of’ and the aspects and embodiments described above with the term “comprising” replaced by the term ” consisting essentially of’.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

FIGURES:

Figure 1 shows the in vitro evaluation of pepR activity against HIV-1NL4.3 infection of TZM-bl cells. Inhibition of viral infection was assessed against the lab-adapted strain HIV- 1NL4.3 (100 TCID50) in the presence of increasing concentrations of pepR. Viral infectivity was quantified 48 h post-infection through luciferase reporter-enzyme activity and converted to percentage of viral infection inhibition. Data points represent the average of results obtained from three independent experiments.

Figure 2 shows pepR stability in human serum. (A) Time course of pepR decay in human serum (green circles, determined as % of starting HPLC peak) and of emerging main by-product, fragment I (white circles). The first curve was fitted to an exponential decay for half-life (tl/2) estimation. (B) RP-HPLC chromatograms of pepR, human serum and pepR human serum digests at various time points (0 to 90 min). Peaks labeled I to VI in the red trace correspond to pepR degradation fragments, with the primary structures outlined in Table 1.

Figure 3 shows an in vitro evaluation of pepRFl activity against HIV-1NL4.3 infection of TZM-bl cells. Inhibition of viral infection was assessed against the lab-adapted strain HIV- 1NL4.3 (100 TCID50) in the presence of increasing concentrations of pepRFl. Viral infectivity was quantified 48 h post-infection through luciferase reporter-enzyme activity and converted to percentage of viral infection inhibition. Data points represent the average of results obtained from three independent experiments.

Figure 4 shows sequence-activity relationship analysis of pepR-derivatives. Dose- response curves (A), and corresponding mean IC50 values (B) for inhibition of viral infection assessed against the lab-adapted strain HIV- 1NL4.3 (100 TCID50) in the presence of increasing concentrations of pepR-derivatives. PepR and pepRFl were included for comparative analysis. Viral infectivity was quantified 48 h post-infection through luciferase reporter-enzyme activity and converted to percentage of viral infection inhibition. Data points represent the average of results obtained from three independent experiments.

Figure 5 shows in vitro evaluation of pepRFl time-of-action and efficacy against HIV- 1NL4.3 entry into TZM-bl cells. (A, B) IC50 values were determined for pepR and pepRFl by treating cells with increasing concentrations of each peptide at -1 h, 0 h, +1 h, +2 h, +3h, or +4 h relatively to the moment of HIV-1NL4.3 addition (100 TCID50). IC50 values represent mean values obtained from three independent experiments. (C) Anti-HIV- 1 activity of pepRFl over time relative to reference anti-HIV inhibitors. HIV-1NL4.3 (100 TCID50) was incubated with cells for 1 h and unbound virus was subsequently removed by repeated washing to synchronize infection. Cells were treated with 1 μM of pepRFl and reference inhibitors (Dextran Sulphate, AMD3 100, T20, and AZT) at 0 h, + 1 h, + 2 h, + 3 h, or + 4 h relatively to the moment of virus addition. The percentage of infection was taken relatively to the control (viruses in the absence of inhibitors). Data points represent the average of results obtained from three independent experiments.

Figure 6 shows in vitro evaluation of pepRFl activity against HIV-1NL4.3-VSV-G and HIV-1NL4.3. The antiviral activity of pepRFl against HIV-1NL4.3-VSV-G was determined by infecting TZM-bl cells in the presence of increasing concentrations of the peptide. T20, which targets HIV-1 gp41, was tested against HIV-1NL4.3-VSV-G as a control. The effect of pepRFl on HIV-1NL4.3-VSV-G infection was compared to HIV-1NL4.3. Viral infectivity was quantified 48 h post-infection through luciferase reporter-enzyme activity and converted to percentage of viral infection inhibition: HIV-1NL4.3 wt + pepRFl (IC50= 1.5 ± 0.4 nM); HIV- 1NL4.3-VSV-G + pepRFl (IC50= n.a.); HIV-lNL4.3-VSV-G + T20 (IC50= n.a.). Data points represent the average of results obtained from three independent experiments.

Figure 7 shows the effect of pepRFl on the target cell. Cells were pre-incubated for 1 h with increasing concentrations of pepRFl or T20 (control). After incubation, (A) HIV- 1NL4.3 (100 TCID50) was added to cells and infection was allowed for proceed for 3 h in the presence of peptides: pepRFl IC50= 1.5 ± 0.4 nM; T20 IC50= 33 ± 9.1 nM; or (B) cells were washed to remove peptides, HIV-1NL4.3 (100 TCID50) was added to cells, and infection was allowed for proceed for 3 h in the absence of peptides: pepRFl IC50= 22 ± 12 nM; T20 IC50= n.a. Viral infectivity was quantified 48 h post-infection through luciferase reporter-enzyme activity and converted to percentage of viral infection inhibition. Data points represent the average of results obtained from three independent experiments. Figure 8 shows a virus-cell fusion assay. An assay based on the use of virions containing the β-lactamase-Vpr pepRFl peptide was performed using the CXCR4-tropic HIV-1 strains: Blam NL4.3 (A) and Blam NL4.3DIM (T20 resistant) (B); and the CCR5-tropic HIV-1 strain: Blam NLAD8 (C). MT4R5 cells treated with increasing concentrations of pepRFl, pepR or T20 (control) were exposed to the different HIV-1 strains. Viral fusion and its inhibition were quantified by measuring the percentage of cells in which a fluorescent substrate of β-lactamase (CCF2) was cleaved, as compared to untreated controls. Data points represent the average of results obtained from three independent experiments.

Figure 9 shows the antiviral effect of pepRFl on HIV-1 viruses carrying primary CXCR4 and CCR5 HIV-1 envelopes. Dose-response curves (A), and corresponding mean IC50 values (B) for inhibition of viral infection of pepRF 1 against viruses carrying primary CXCR4- tropic envelopes (clones X4-1 and X4-2) and CCR5-tropic envelopes (clones R5-1 and R5-2) isolated from a patient, and against the reference strains NL4.3 (CXCR4-tropic) and NLAD8 (CCR5 -tropic). Infection inhibition was evaluated by infecting TZM-bl cells in the presence of increasing concentrations of pepRFl. Viral infectivity was measured 40 h post-infection through β-galactosidase reporter-gene expression and converted to percentage of viral infection inhibition. Data points represent the average of results obtained from two independent experiments.

Figure 10 shows the effect of pepRFl and pepR on CXCR4 expression on cell surface. Primary CD4+ T-lymphocytes were pre-incubated for 90 min with different concentrations of pepRFl and pepR at 37°C. The cells were then stained with the mAbs 12G5 (A) or 1D9 (B), and were analyzed by flow cytometry. T20, AMD3100 and CXCL12 were used as controls. The results are expressed as percent of cells positive for the surface expression of CXCR4, as compared to untreated controls. Data points represent the average of results obtained from three independent experiments.

Figure 11 shows the impact of pepRFl and pepR on CXCR4-associated Ca2+ intracellular mobilization. Time-resolved fluorescence emission intensity profiles of Fluo-4 within THP-1 cells upon treatment with pepRFl (1.25 μM), pepR (1.25 μM), AMD3100 (50 μM), and CXCL12 (50 nM), alone (A) or in combination (B, C). Fluo-4 basal fluorescence emission intensity was collected for 30 s prior to addition of individual compounds and combinations, and followed for an additional 270 s. A mark was added to the t horizontal axis, at 30 s, for clarity. Data was normalized to the basal Fluo-4 fluorescence emission intensity values, measured at t = 0 s. Each data point represents the average value of the fluorescence emission intensity measured from three independent experiments. Figure 12 show cytotoxicity of pepR and pepRFl. TZM-bl cells were incubated for 3 h with the indicated concentrations of pepR (light bars) and pepRFl (dark bars). After 45 h, the effect of the peptides on the metabolic activity of cells was measured by quantifying the amount of resazurin, a non-fluorescent indicator dye, converted to red-fluorescent resofurin via the reduction reactions of metabolically active cells. The effect of peptides on was also studied 3 h after incubation of cells with peptides and similar results were obtained. Data points represent the average of results obtained from three independent experiments.

EXAMPLE:

Material & Methods

Peptide synthesis

Peptides were synthesized in C-terminal carboxamide form on a Liberty BlueTM automated microwave peptide synthesizer (CEM Corporation, Matthews, NC) using Fmoc protocols. Peptides were assembled at 0.05 mmol scale on a H-Rink Amide-ChemMatrix resin of 0.50 mmol/g substitution (PC AS BioMatrix, Montreal, Canada). Couplings were performed at 90 °C with five-fold excess of Fmoc-amino acid/N,N’-diisopropylcarbodiimide (DIC)/Oxyma (1:2.5:5 molar ratio) in N,N-dimethylformamide (DMF), and deprotection with piperidine (20 % v/v in DMF), followed by extensive DMF washes. After chain assembly, peptides were fully deprotected and cleaved from the resin by acidolysis with trifluoroacetic acid (TFA)/H20/3,6-dioxa-l,8-octanedithiol (DODT)/ triisopropylsilane (TIS) (94:2.5:2.5:1 v/v) for 90 min. Peptides were precipitated by cold diethyl ether followed by 3x5 min centrifugation at 4800 rpm, 4°C; the pellet was taken up in water and lyophilized. Crude peptides were inspected by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS), and purified by preparative RP-HPLC as described below. Fractions with the expected mass and with HPLC purity > 95% were pooled and lyophilized.

Cell culture

Human embryonic kidney 293 T (HEK293T) and HeLa-derived TZM-bl cell lines, were purchased through the American Tissue Cell Culture Collection (ATCC) (Manassas, VA, USA). Cells were cultured in complete medium composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Pen-Strep). THP-1 cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (Bethesda, MD, USA). Cells were cultured in complete medium composed of Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% (v/v) FBS and 100 U/mL Pen-Strep. MT4R5 cells (PMID: 12551993) were grown in complete RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS and 100 U/mL Pen-Strep. Primary CD4+ T were isolated from peripheral blood mononuclear cell (PBMC) of a donor and were activated with 1 μg/mL of phytohemagglutinin (PHA). One day after activation, they were grown in complete RPMI- 1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 100 U/mL Pen-Strep and IL-2 (100 IU/mL). DMEM, RPMI-1640, FBS, and Pen-Strep were purchased from Gibco (Thermo-Fisher, Waltham, MA, USA).

All cell cultures were maintained at 37°C in a humidified atmosphere with 5% C02.

Virus production

The full-length molecular clone pNL4.3, the env-defective molecular clone pNL4.3Δenv, and the pHEF-VSVG vector were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Infectious HIV-1NL4.3 viruses and env-defective HIV-1NL4.3 viruses pseudotyped with VSVG (HIV-1NL4.3-VSV-G) were produced by transfecting HEK293T cells with pNL4.3 and pNL4 3Δenv clones, respectively, through the calcium phosphate co-precipitation method (Rato, Maia et al. 2010) (Kwon and Firestein 2013). Briefly, a total of 3.5 μg/well of plasmid DNA was used to transfect HEK293T cells (5x10 ⁵ cells/well) in tissue culture-treated 6-well microplates from TPP (Trasadingen, Switzerland). Calcium-phosphate-DNA transfection mixtures were prepared by diluting plasmid DNA in 1 mM Tris-HCl, 0.1 mM EDTA, 250 mM CaC12, pH 7.6. The DNA mixture was then added to an equal volume of 50 mM HEPES (Merck, Darmstadt, Germany), 280 mM NaCl (Merck, Darmstadt, Germany), 1.5 mM Na2HP04 (Merck, Darmstadt, Germany), pH 7.05, under gentle agitation. Transfection mixtures were allowed to incubate at room temperature, for 20 min, before addition to cells. After 18 h, transfection mixtures were replaced with fresh complete medium. Viral supernatants were collected 48 h post-transfection, centrifuged at 315 g for 5 min to remove cell debris, and stored at -80°C until used.

Viral supernatants were titered through the Reed-Muench method (Reed LJ Muench 1938) in a single-cycle viral infectivity assay on TZM-bl cells. TZM-bl cells contain a HIV long terminal repeat (LTR)-driven β-galactosidase and luciferase reporter cassettes that are activated by HIV-1 Tat expression. Briefly, TZM-bl cells were seeded at 2x10 ⁴ cells/well in tissue culture-treated 96-well flat-bottomed polystyrene microplates from Corning (Corning, NY, USA), and incubated for 24 h. Cells were then incubated with two-fold serial dilutions of viral supernatants for 3 h, after which the supernatant was replaced with fresh complete medium. After 45 h, infection of TZM-bl cells was quantified through luciferase reporter-gene expression levels (Wood KV 1990) using the Luc-Screen® luciferase chemiluminescence detection system from Applied Biosystems (Thermo-Fisher), according to manufacturer’s instructions. Luminescence intensity, L, was measured in an Infinite M200 microplate reader from Tecan (Mannedorf, Switzerland). Cells were considered infected if the respective luminescence intensity was five-fold higher compared to the intensity from control cells (in the absence of viruses). Titration was performed with at least four replicates to allow accurate estimation of HIV-1NL4-3 and HIV- 1 NL4 3Δenv 50% tissue culture infectious dose (TCID50) of viral supernatants.

To measure the inhibition potency on clinically relevant viruses, we used four previously described full-length HIV-1 molecular clones carrying envelope sequences with different coreceptor tropism isolated from one patient plasma (patient T28) (PMID: 16140761). Isolates X4-1 and X4-2 are CXCR4-tropic, while isolates R5-1 and R5-2 are CCR5-tropic. Virus particles were produced by transfecting subconfluent HEK293T cells in T75 with 20 μg of each plasmid. DNA was diluted in 588μL of H20 with 195μL of calcium chloride, and added to an equal volume of HEPES (Sigma Aldrich, XX) under agitation. Transfection mixtures were incubated at room temperature for 30 min, before addition to cells. After 16 h, transfection mixtures were replaced with fresh complete medium. Viral supernatants were collected 40 h post-transfection, centrifuged at 600 g for 5 min to remove cell debris, filtered with 0.45μm filter and stored at -80°C until used. Viral supernatants were titred in a viral infection assay on TZM-bl cells. 24 h before infection, 1x10 ⁴ TZM-bl cells/well were seeded in tissue culture- treated 96-well flat-bottom microplates. Cells were then incubated with several dilutions of viral supernatants. After 48 h, infection of TZM-bl cells was quantified using a chemiluminescent β-galactosidase reporter-gene expression assay (Roche, XX), according to manufacturer’s instructions. Luminescence intensity, L, was measured in a Varioskan Flash device (Thermo Fisher Scientific).

The efficiency of viral entry into target cells was evaluated by the β-lactamase-Vpr assay (PMID: 12355096). Virus stocks for this assay were produced by transfection of sub-confluent HEK293T cells in T75 flasks by Jet Pei (Polyplus Inc. Illkrich, France) following the manufacturer’s instructions. 12 μg of pNL4.3 or pNLAd8 (PMID: 7745752) or pNL4.3DIM (PMID: 15078945) and 4 μg of a plasmid coding the Vpr gene fused to the β-lactamase gene (a kind gift from Michael D. Miller) were co-transfected. Medium was changed 16 h later, and the virus-containing supernatant was collected 40 h post-transfection, filtered with 0.45 pm filter and overlaid on a 20% (X/X) sucrose cushion in a Beckman SW32 tube, after which particles were pelleted by centrifugation (98,000 g, 4 °C) for 90 min. Viral pellets were re-suspended in RPMI medium to obtain a 10-fold concentration as compared with the initial culture supernatant, separated into several aliquots, and frozen at -80°C.

Viral inhibition assays

The anti-HIV activity of the peptides was evaluated through single-cycle viral infectivity assays using TZM-bl reporter cells, as previously described (Montefiori DC 2009) (Borrego, Calado et al. 2012). TZM-bl cells were seeded at 2x10 ⁴ cells/well in tissue culture- treated 96-well flat-bottomed polystyrene microplates (Corning) and incubated for 24 h at 37°C with 5% C02. Cells were then incubated for 3 h with 100 TCID50/well ofHIV-lNL4.3 orHIV- 1NL4.3-VSV-G in the presence of two-fold serial dilutions of pepR, covering the 0.004 - 5 μM range, and four- fold serial dilutions of pepRFl and pepR-derivatives, covering the 0.000005 - 5 μM range. For the time-of-addition experiments, serial dilutions of pepR (0.004 - 5 μM) and pepRFl (0.000005 - 5 μM) were instead added to cells at -1 h, 0 h, +1 h, +2 h, +3 h or +4 h relatively to the moment of infection with HIV-1NL4-3. After a 3 h incubation period, cells were washed with PBS and fresh complete medium was added. To compare the antiviral activity of pepRFl with known HIV-1 inhibitors, cells were incubated with HIV-1NL4.3 alone or in combination to lμM of pepRFl, dextran sulfate, AMD3100, T20, and AZT. After 1 h, cells were washed with PBS to remove all unbound viruses and synchronize the infection, as described elsewhere (Daelemans, Pauwels et al. 2011). lμM of pepRFl, dextran sulfate, AMD3100, T20, and AZT were then added to cells at +1 h, +2 h, +3 h, +4 h, relatively to the moment of the virus addition to cells. The concentration used correspond to at least 100-fold the IC50 of each inhibitor, as established in a TZM-bl single-cycle infectivity assay (data not shown). Untreated cells were used as a control. Infection of TZM-bl cells was quantified 45 h later through luciferase reporter-gene expression levels as described above. L measurements were performed in an Infinite M200 microplate reader. L values were analyzed through non linear regression with the classical dose-response relationship (median-effects model based on mass action) (Chou T-C 1976); fa is the fraction of inhibited viruses, IC50 is the concentration that inhibits 50% of viral infection, m is a slope parameter equivalent to the Hill slope, [P] is the peptide concentration, and Lo is the luminescence intensity in the absence of peptides.

Dextran sulfate and AMD3100 were purchased from Sigma- Aldrich (St. Louis, MO, USA). T20 and AZT were obtained through the NIH AIDS Reagent Program. At least three independent experiments were performed for each assay.

Susceptibility of primary viruses to inhibition

24 h before infection, lxl 0 ⁴ TZM-bl cells/well were seeded in tissue culture-treated 96- well flat-bottom microplates. TZM-bl cells were treated prior to infection with four-fold serial dilutions of pepR and pepRFl, covering the 0.0012 - 1.25 μM range. Cells were then incubated with the viral supernatants of the primary virus (R5-1, R5-2, X4-1 and X4-2) and the laboratory strains NL4.3 (CXCR4-tropic) or NLAd8 (CCR5 -tropic). After 48 h, infection of TZM-bl cells was quantified through β-galactosidase reporter-gene assay (Roche), according to manufacturer’s instructions. Luminescence intensity, L, was measured in a Varioskan Flash instrument (Thermo Fisher Scientific).

RP-HPLC and LC-MS analysis

Analytical RP-HPLC was performed on an LC-20AD instrument (Shimadzu, Kyoto, Japan) equipped with a Luna C18 column (4.6 mm x 50 mm, 3 pm; Phenomenex, Torrance, CA) using linear gradients of solvent B (0.036% TFA in acetonitrile (ACN)) into A (0.045% TFA in H20) over 15 min, with 1 mL/min flow rate and UV detection at 220 nm. Preparative RP-HPLC separations were performed on an LC-8 A instrument (Shimadzu) fitted with a Luna Cl 8 column (21.2 mm x 250 mm, 10 pm; Phenomenex), using linear gradients of solvent D (0.1% TFA in ACN) into C (0.1% TFA in H20) over 30 min, at a 25 mL/min flow rate. Mass spectrometry analysis was performed on an LC-MS 2010EV instrument (Shimadzu) fitted with an XBridge C18 column (4.6 mm x 150 mm, 3.5 pm, Waters, Cerdanyola del Valles, Spain), eluting with linear gradients of F (0.08% formic acid in ACN) into E (0.1% formic acid in H20) over 15 min at a 1 mL/min flow rate. pepR stability assay

To evaluate pepR stability in vitro, 10OμL aliquots containing 25 % (v/v) pre-warmed human serum (Sigma) and 250μM pepR were incubated at 37°C with gentle agitation. At different time points (0.1, 5, 20, 30, 60, 90, 120, 180 and 360 min), the incubation was stopped by 20μL trichloroacetic acid (15 % v/v in H20). Samples were cooled in ice for 15 min, centrifuged at 13000 rpm for 10 min and the supernatant was collected and analyzed by analytical RP-HPLC and LC-MS as described above. Percentage of peptide was calculated by peak integration of chromatograms, and kinetic data were fitted to an exponential decay model to determine the half-life (tl/2).

Cell viability studies

The cytotoxic effect of pepR and pepRFl on TZM-bl cells was studied using a resazurin reduction fluorometric assay (alamarBlue® cell viability reagent, Invitrogen, Thermo-Fisher). Resazurin, the active compound in alamarBlue®, is a blue dye that can be reduced to a pink fluorescent intermediate, resorufin, as a result of cell metabolic activity. TZM-bl cells (2x10 ⁴ cells/well) were seeded in tissue culture-treated 96-well black flat-bottomed polystyrene plates (Corning) and incubated for 24 h at 37 °C with 5% C02. Cells were then incubated for 3 h with four-fold serial dilutions of pepR and pepRFl covering the 0.07 - 80 μM range. After incubation, cells were washed with PBS, alamarBlue® was added at a final concentration of 10% (v/v) and incubated for 3 h. In a complementary assay performed at the same time, after washing the cells with PBS, fresh complete DMEM medium was added and after 45 h, alamarBlue® (10% v/v) was added to cells and incubated for 3 h. Untreated cells were used as a control. After the 3 h incubation of cells with alamarBlue®, resorufin production was monitored by measuring the fluorescent emission intensity (excitation 560 nm, emission 590 nm) in an Infinite M200 microplate reader. The percentage of metabolic active cells was expressed as the percentage of resazurin reduction relative to the reduction measured for the untreated sample after correcting the data with background fluorescence emission intensity from resazurin in cell-free medium, according to the following formula:

I corresponds to the resorufin fluorescence emission intensity in the presence of peptide, Icontrol to the fluorescence emission intensity in the absence of peptides and Ibackground to the background fluorescence emission from the non-reduced alamarBlue® reagent. At least three independent experiments were performed for each assay.

Viral particle integrity studies

The effect of the peptides on the integrity of the viral particles was first evaluated using a Western blot-based assay. 100 TCID50 of HIV-1NL4.3 were incubated with 5 μM of pepR or pepRFl, for 1 h at 37°C. Untreated HIV-1NL4.3 virions (in the absence of peptides), which will remain intact and be able to cross a 20% (w/v) sucrose cushion, were used as positive control for viral integrity. HIV-1NL4.3 virions treated with 0.5% (v/v) Triton X-100 for 1 h, were used as negative controls for viral integrity (positive controls for viral disruption). Samples were then centrifuged through a 20% (w/v) sucrose cushion for 2 h at 40,000 rpm and 4 °C. The viral pellet was ressuspended and lysed directly in Laemmli buffer composed of sodium dodecyl sulfate (SDS, 4% w/v), 125 mM Tris-HCl (pH 6.8), 2-mercaptoethanol (10% v/v), glycerol (20% v/v), and bromophenol blue (0.02% w/v). Viral proteins were denatured by boiling for 10 min at 95 °C. Proteins were then separated by 12% (w/v) SDS-PAGE (National Diagnostics, Atlanta, GA, USA), transferred to nylon membranes (Hybond, Amersham Biosciences, GE Healthcare Life Sciences, Chicago, IL, USA), and reacted with anti-HIV p24 mAh (NIH AIDS Reagent Program, Division of AIDS, NIAID). Membranes were then incubated with goat anti-mouse IgG (H+L)-HRP conjugate (Bio-Rad, Milan, Italy) and visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences, GE Healthcare, BM, UK) using the Chemidoc XRS+ System (Bio-Rad, Hercules, CA, USA).

A complementary assay was also performed to investigate the integrity of viral particles using the QuickTiterTMLentivirus Titer Kit from Cell Biolabs (San Diego, CA, USA), which is an enzyme immunoassay developed for the specific detection and quantification of HIV- 1 capsid (CA) protein (p24) associated to intact viral particles only. This can be achieved through the use of viral bind lentivirus reagents (ViraBindTM, patented technology) that form complexes with the intact virions while free p24 remain in the supernatant. Briefly, HIV- 1NL4.3 virions (100 TCID50) were incubated with 5μM of pepR or pepRFl for 1 h at 37 °C. After incubation, a virus pulldown (using the ViraBindTM lentivirus reagents provided with the QuickTiterTM Lentivirus Titer Kit) was performed to recover intact viral particles from viral supernatants. Virions were disrupted using sample diluent (provided by the QuickTiterTM Lentivirus Titer Kit) and were then added to an anti-p24 antibody coated plate, and incubated for 4 hours at 37 °C. Detection of viral-associated p24 protein was performed using FITC- conjugated anti-p24 mAh followed by HRP-conjugated anti-FITC mAh (provided by the QuickTiterTMLentivirus Titer Kit) following manufacturer’s instructions. Quantification of HIV-1NL4.3 p24 protein was determined by adding HRP-substrate solution to each sample followed by measuring absorbance at 450 nm. Untreated HIV-1NL4.3 virions (in the absence of peptides) were used as positive control for viral integrity. HIV-1NL4.3 virions treated with 70% ethanol or 0.5% (v/v) Triton X-100 for 1 h, were used as negative controls for viral integrity (positive controls for viral disruption). At least three independent experiments were performed for each assay. β-lactamase viral fusion assay

To measure the efficiency of virus entry into target cells, and the impact of peptides on this process, we used a previously described Vpr-BlaM assay (Cavrois, De Noronha et al. 2002). In this assay, the β-lactamase enzyme fused to the viral protein Vpr is incorporated into virions and delivered into the cytosol of target cells, where β-lactamase activity can be quantified by the cleavage of a fluorescent substrate (CCF2). To this end, 2.0x10 ⁵ MT4R5 cells treated with a four-fold serial dilution of pepR, pepRFl and T20 covering the 0.0003 - 1.25 μM were exposed to the virus preparation of Blam NL4.3, Blam NLAD8 or Blam NL4-3DIM for 4 h at 37 °C, using a virus concentration that leads to on approximately 30% of positive cells in the absence of treatment.

Cells were then washed and loaded with the CCF2 substrate (CCF2-AM loading kit, Invitrogen) in the presence of 1.8 mM probenecid (Sigma- Aldrich). Cells were incubated overnight at 15 °C with C02 independent medium (Invitrogen), washed with PBS containing 1% (X/X) BSA and 0.05% (X/X) Saponin (Sigma-Aldrich) and fixed with paraformaldehyde (PFA). The cleaved CCF2 fluorescence was measured by flow cytometry on a FacsCanto II system with FACSDiva software (BD Bioscience). FlowJo, version 10 (Tree Star), was used to analyze and quantify the data.

Expression of HIV coreceptors

The level of expression of CXCR4 on the cell surface was studied by flow cytometry using two different anti-CXCR4 monoclonal antibodies (mAbs), targeting two distinct epitopes: 12G5 (PE mouse anti-human CD184 clone 12G5 (RUO), BDBiosciences) and 1D9 (PE rat anti human CD184 clone 1D9 (RUO), BDBiosciences). Activated primary CD4+ T-lymphocytes were incubated for 90 min at 37 °C with a four-fold serial dilution of pepR, pepRFl and T20 covering the 0.0012 - 1.25 μM range, or a four-fold serial dilution of the CXCR4-antagonist AMD3100 covering the 0.049 - 50 μM range, or a four-fold serial dilution of CXCL12 (the natural ligand of CXCR4) covering the 0.098 - 100 nM range. Then, each sample was split into two aliquots and the cells were separately stained with the two antibodies in the presence of PBS-BSA 1% (X/X) 45 min at room temperature in the dark.

The expression of CCR5 on the cell surface was studied using human CCR5 fluorescein mAh (clone 45502) (R&D Systems). Activated primary CD4+ T lymphocytes were incubated 10 min at room temperature with three dilutions (1250 nM, 78 nM, 5 nM) of pepR, pepRFl, T20 and AMD3100. Then, the cells were stained with the mAh in presence of PBS-BSA 1% (X/X) 45 min at 4°C in the dark. Fluorescence was measured by flow cytometry on a FacsCanto II system with FACSDiva software (BD Bioscience). FlowJo, version 10 (Tree Star), was used to analyze and quantify the data.

Intracellular Ca2+ mobilization assay

CXCR4-mediated intracellular Ca2+ mobilization was assessed using a Fluo-4-based fluorescence assay (Princen, Hatse et al. 2003). Fluo-4 is a fluorescently labelled probe that exhibits an increase in fluorescence emission intensity upon binding to Ca2+ (Gee, Brown et al. 2000). Fluo-4 AM ester (Invitrogen, Thermo-Fisher) was dissolved in pure DMSO (Merck, Darmstadt, Germany) to a final concentration of 4 mM. Fluo-4 stock solutions were diluted to final working concentrations in Hank’s Balanced Salt Solution (HBSS, Gibco, Thermo-Fisher). DMSO content was kept below 0.25 % (v/v) to prevent cell death.

THP-1 monocytes were cultured in complete RPMI medium. Cells were washed with HBBS, resuspended in HBBS with 5 μM Fluo-4, at a density of 2.5x10 ⁶ cells/mL, and incubated for 1 h at 37 °C, to allow Fluo-4 probe internalization into the cytosol. To remove the extracellular probe, cells were washed and resuspended in fresh HBSS and allowed to stabilize for 10 min at 37 °C, before starting fluorescence measurements.

Variations in cytosolic Ca2+ levels were followed by time-resolved Fluo-4 fluorescence emission intensity measurements. Measurements were carried out in a FLS920 spectrofluorometer (Edinburgh Instruments, Livingston, UK) at 37 °C. The excitation and emission wavelengths were 491 nm and 519 nm, respectively. Excitation and emission slits were 3 nm and 8 nm, respectively. Fluorescence emission intensity of labelled cells (1.25x10 ⁶ cells/mL) was collected for 30 s, before addition of CXCL12 (Sigma, 50 nM), pepR (1.25 μM), pepR-F (1.25 μM) or AMD3100 (50 μM), individually or in combination, after which the signal was collected for an additional 270 s. Data was corrected for dilution and background noise. CXCL12 and inhibitors solutions were prepared in HBSS. At least three independent experiments were performed for each assay.

Statistical analysis

Statistical analysis was performed using GraphPad Prism® version 7.0 for Macintosh (GraphPad Software, San Diego, California USA, www.graphpad.com). Error bars on presented data represent the standard deviation (SD).

Results pepR anti-HIV-1 activity and serum stability

The antiviral activity of pepR against the HIV-1 laboratory-adapted strain NL4.3 (HIV- 1NL4.3) was evaluated using a single-cycle infectivity assay. As shown in Figure 1, pepR inhibited infection of TZM-bl cells with an IC50 of 37 ± 3.8 nM. The susceptibility of pepR to human serum was investigated by HPLC-MS (Figure 2). PepR underwent relatively fast decay (Figure 2A), with >90 % of the initial product consumed after 90 min. A tl/2 of 3.7 min was determined by fitting of an exponential function to experimental data. The HPLC profile of the digest (Figure 2B) shows a main peak (Fragment I) reaching maximum intensity at ~16 min and remaining constant henceforth.

Based on MS data, a primary structure (LKRWGTIKKSKAINVLR, Table 1, SEQ ID NO: 10) could be unequivocally proposed for fragment I, which could plausibly result from trypsin-like cleavage of pepR at Argl7-Glyl8. An alternative, more complex pathway would entail chymotrypsin-like cleavage at Phel9-Arg20 to give intermediate LKRWGTIKKSKAINVLRGF (one among possible fragment V candidates, Table 1, SEQ ID NO:21), which would then undergo stepwise carboxypeptidase clipping of C-terminal Phe and Gly.

Aside from fragment I, the other five peaks (II- VI) observed by HPLC (Figure 2B) are of much lower intensity. For fragment III, an unequivocal sequence can be assigned from MS data while, for fragments II and IV- VI, two or more quasi-isobaric sequences can be proposed (Table 1, SEQ ID NO: 15 to SEQ ID NO:25), suggesting degradation pathways of minor importance compared to those leading to fragment I accumulation. It was obvious at this point that, given its superior serum stability and predictable reduction in cost of production, as a result of pepR’ s >50% size reduction, fragment I (herein pepRFl) should be explored as an alternative antiviral candidate over pepR. Accordingly, a synthetic replica (in C-terminal carboxamide version) was also prepared and evaluated (Table 2).

The antiviral activity of pepRFl against HIV-1NL4.3 was assessed, and the IC50 value obtained, 1.5 ± 0.4nM, revealed it as even more potent than its parent pepR, on the inhibition of infection of TZM-bl cells (Figure 3). Importantly, pepRFl has no cytotoxic effects on TZM- bl cells for concentrations up to 80 μM, the highest concentration tested (Figure 12). This corresponds to a therapeutic window > 53000.

The N-terminal domain of pepR is responsible for its anti-HIV-1 activity

A sequence-activity relationship analysis was carried out to unveil the domain of pepR responsible for its anti-HIV- 1 activity. pepR analogs were obtained by stepwise residue deletions at both N- and C-termini (Table 3, SEQ ID NO:26 to SEQ ID NO:32). As shown in Figure 4, assays against HIV-1NL4.3 revealed that the domain responsible for the antiviral activity of pepR is located in the N-terminal region. The results clearly show that the sequential deletion of amino acid residues towards the N-terminal region of the peptide resulted in an increase of the anti-HIV- 1 activity: IC50= 29 nM for ΔC4-pepR, IC50= 27 nM for ΔC8-pepR, and IC50= 6 nM for ΔC12-pepR (Figure 4A).

These results are in agreement with the IC50 value obtained for the peptide fragment pepRFl, 1.5 nM, which corresponds to ΔC 18-pepR (Figure 4B). On the other hand, the peptides generated by the sequential deletion of amino acid residues of the peptide N-terminal region (DN4-, DN8-, DN 12-pepR) resulted in peptides that are not active against HIV-1NL4.3. pepRFl targets an early step in HIV-1 entry

The HIV replication cycle consist of different stages that occur in a well-established chronological order (Frankel, A.D. 1998). Time-of-addition (TOA) experiments are a commonly used approach to study the mode of action of HIV inhibitors (Daelemans, Pauwels et al. 2011), namely which replication step is being targeted.

First, we determined if pepRFl targets HIV-1 before, during, or after viral entry into cells. pepRFl was incubated with HIV-1NL4.3 before addition to cells (t= -1 h), added to cells at different time-points during infection (t= 0, 1, 2 h), or added to cells after infection (t= 3, 4 h). The inhibitory effect of pepRFl was only observed when the peptide was added before or during, but not after the virus infection period, indicating that it acts during viral entry into cells (Figure 5A). This effect was more pronounced when the peptide was pre-incubated with virions (t= -1 h) (IC50= 1.2 ± 0.6 nM), or added to the cells together with virions (t= 0 h) (IC50= 2.5 ± 1.4 nM), suggesting that the peptide targets an early event during viral entry. The results also showed that pepRFl was more potent than pepR at inhibiting HIV-1 infection at all the time- points tested.

To better discriminate which step(s) of HIV-1 replication are inhibited by pepRFl, we studied its activity against HIV-1NL4.3 over time relative to reference drugs (Daelemans, Pauwels et al. 2011). HIV-1NL4.3 virions were added to cells and incubated for 1 h at 37°C. pepRFl and the well-characterized inhibitors dextran sulphate, AMD3100, T20, and Zidovudine (AZT) were added to cells together with HIV-1NL4.3 virions (t= 0 h), or at different time-points during the early phase of the virus replication cycle (t= 1, 2, 3, 4 h). As expected, dextran sulphate, an inhibitor of viral adsorption to the host cell (Daelemans, Pauwels et al. 2011) (Ito M 1987), became ineffective if added 1 h after infection. In this case the calculated time-of-drug-addition that maintains 50% inhibition (t50) was 0.8 h (t50 = 0.8 h) (Figure 5B). The bicyclam AMD3100, a chemokine receptor 4 (CXCR4) antagonist that prevents HIV-1 attachment to the coreceptor (De Clerq E 1994), became ineffective soon after infection (t50 = 1.4 h for AMD3100). The viral fusion inhibitor T20 remained effective up to a slightly latter time (t50 = 1.8 h), which is consistent with its inhibition of the fusion event, occurring after receptor binding (Kilby JM 1998) (Lalezari JP 2003). Finally, AZT blocked HIV-1 replication even if added with significant delay (t50 = 3.1 h), which is in accordance with its intracellular targeting of the reverse transcription process (Daelemans, Pauwels et al. 2011) (Gonzalez- Ortega, Mena et al. 2010). PepRFl inhibited infection with a t50 of 1.3 h, similar to AMD3100 (Figure 5C). This suggests that pepRFl blocked HIV entry into cells by interfering with a process in between virus adsorption and fusion, possibly by blocking attachment of viral glycoproteins to cell receptors.

To test this hypothesis, a viral inhibition assay was performed using HIV-1 virions pseudotyped with the envelope glycoprotein G from the vesicular stomatitis virus (VSV-G), HIV-1NL4.3-VSV-G. This modified virus enters the cells via endocytosis and later fusion within acidified endosomes, without the requirement for HIV glycoproteins (gpl20 and gp41) and cell membrane HIV receptor (CD4) and co-receptors (CCR5 and CXCR4). As shown in Figure 6, pepRFl was not able to inhibit infection of HIV- 1NIA3 -VSV-G. The same was observed for pepR and T20. This clearly shows that viral inhibition by pepRFl targets the HIV Env-mediated entry process.

PepRFl inhibits HIV-1 entry by targeting the host cell

HIV-1 entry is a multistep process that progresses through viral gpl20 binding to CD4 cell receptor, coreceptor engagement (CCR5 or CXCR4), and fusion with the host cell membrane (Miyauchi, Kim et al. 2009). Several peptides have been shown to block HIV infection by interfering with any of these steps in the viral entry process (Qureshi, Thakur et al. 2013). The above results showed that pepRFl blocks HIV-1 entry into the host cell but the specific molecular target of this drug remained elusive. To investigate if pepRFl targets the virion, the cell, or both, TZM-bl cells were pre-incubated with increasing concentrations of the peptide. T20 was used as a control since it is known to target the viral gp41. After incubation, HIV-1NL4.3 was added to cells and infection was allowed to proceed for 3 h in the presence of pepRFl or T20. In a different set of experiments, after pre-incubation with the peptides, cells were washed to remove the peptides, and HIV-1NL4.3 was added to cells for 3 h. As shown in Figure 7A, both pepRFl and T20 were able to inhibit the cells infection by HIV-1NL4.3 when present at the time that viruses were added to the cells. However, when the viruses were added after washing the cells for peptide removal, the ability of T20 to inhibit infection was completely abrogated (Figure 7B). This was expected because exposure of T20 target (the HRl domain in gp41) only takes place after the virus has interacted with its receptors. On the other hand, pepRFl was able to inhibit infection in spite of cell washing. This shows that during the incubation period with cells, the peptide was able to establish a durable interaction with a cellular component.

To further exclude a possible direct action of pepRFl at the level of the viral membrane, the impact of pepRFl on HIV-1 structural integrity was studied and the result showed that the peptide does not cause viral particle disruption.

PepRFl inhibits HIV-1 cell entry in a coreceptor specific manner The above reported results show that pepRFl specifically inhibits HIV entry (Figure 6) by interacting with a cellular factor (Figure 7B). To study the target used by pepRFl to block HIV-1 entry, a direct virus-cell fusion assay was performed based on the incorporation of Vpr- β-lactamase pepRFl peptides (Vpr-BlaM) into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a result of virion fusion. This transfer was then detected by enzymatic cleavage of the CCF2 dye, a fluorescent substrate of β-lactamase (BlaM), loaded into the target cells. This is the validated assay to assess the extent and the kinetics of HIV- 1 fusion with target cells (Cavrois, De Noronha et al. 2002) (Miyauchi, Kim et al. 2009) (Daecke, Fackler et al. 2005). HIV-1 pseudoviruses bearing the nrG-β-lactamase chimera (Vpr-BlaM) with a CXCR4-tropic envelope (NL4-3) (PMID: 3016298) were produced and used to infect MT4R5 cells in the absence and in the presence of increasing concentrations of pepRFl. The ability of pepRFl to inhibit viral fusion was compared to pepR and T20. The results showed that pepRFl was the most efficient (IC50= 2.8 nM) when compared to pepR (IC50= 30.8 nM) and T20 (IC50= 45.1 nM) (Figure 8A). In addition, pepRFl was able to inhibit the HIV-1 strain resistant to T20 (HIV-1NL4.3 DIM) with an IC50 of 5.3 nM (Figure 8B). Interestingly, in contrast to T20, neither pepRFl nor pepR were able to inhibit infection by the CCR5 -tropic virus, Blam NLAd8 (Figure 8C), showing specificity towards CXCR4-tropic viral strains. PepRFl inhibition of CXCR4-tropic HIV-1 patient-derived isolates We also investigated the ability of pepRFl to inhibit infection of four different HIV-1 strains carrying primary CXCR4 and CCR5-specific HIV-1 envelope glycoproteins. These strains carry gpl20 and the extracellular part of gp41 issued from variants present in one patient, as described elsewhere (Skrabal, Saragosti et al. 2005). As shown in Figure 9 (A, B), pepRFl potently inhibited the two CXCR4-tropic viruses, X4-1 and X4-2, in addition to the CXCR4 reference strain used in the previous assays (HIV-1 NL4.3). In contrast, it was not able to inhibit the two CCR5-tropic viruses, R5-1 and R5-2, and the CCR5 reference strain (NLAD8). The same was observed for pepR. These results clearly reinforce the specificity of pepRFl towards CXCR4-tropic HIV strains. PepRFl prevents viral fusion without inducing CXCR4 internalization

Based on the above observations, the most likely target of pepRFl appeared to be the coreceptor CXCR4. To unravel the mechanism underlying the peptide action, the effect of pepRFl on CXCR4 expression at the surface of the cell membrane was studied by flow cytometry on primary CD4+ T-lymphocytes. AMD3100, T20, and the chemokine CXCL12 (C- X-C motif ligand 12), the natural ligand of CXCR4, also known as SDF-1 (stromal cell-derived factor 1) (Schols D 1997) (Mishra, Shum et al. 2016), were used as controls.

To determine whether pepRFl and pepR interact directly with CXCR4, we first used the monoclonal antibody (mAh), 12G5, which is directed against a bridging epitope spanning the first and second extracellular loops of CXCR4 (Zirafi, Kim et al. 2015). As shown in Figure 10, CXCR4 detection at the surface of the cell was reduced in a dose dependent fashion, to undetectable levels, by both pepRFl and pepR. The same was observed for AMD3100, in accordance with previous studies (Schols D 1997) (Zirafi, Kim et al. 2015). As expected, T20 had no effect on CXCR4 detection. These results suggest that pepRFl and pepR interact directly with CXCR4, inhibiting CXCR4-epitope recognition by 12G5 mAh, which could ensue by competitive binding, by changing the conformation of the co-receptor, or by inducing its internalization.

We next explored if the loss of CXCR4 detection from the surface of the cell was due to coreceptor internalization rather than competitive binding or conformational change. A different antibody, 1D9, that targets the N-terminal domain of CXCR4 (Zirafi, Kim et al. 2015), was used. The binding of this mAh will not be perturbed by changes in the conformation of the coreceptor at the ligand binding site (Camec X, 2005). In this way, loss of detection of 1D9 from the surface of the cells would be strongly indicative of internalization of CXCR4. As shown in Figure 10, 1D9 binds equally well to CXCR4 in the presence and absence of pepRFl and pepR, showing that the coreceptor is still on the cell surface, but is occupied/modified by the peptides, which prevented binding of the 12G5 antibody. The same was observed for AMD3100, which does not induce CXCR4 internalization (S. Hatse 2002). The same experiments were performed using CXCL12, which upon interaction with the coreceptor induces its internalization and activates a complex cascade of intracellular signaling pathways (Schols D 1997) (Mishra, Shum et al. 2016) (Zirafi, Kim et al. 2015) (S. Hatse 2002) (Oberlin 1996). At the surface level, pepRFl, pepR, AMD3100, and CXCL12, reduced the recognition of CXCR4 by 12G5 mAh (Figure 10A). In contrast, only CXCL12 decreased recognition of CXCR4 by 1D9 mAh (Figure 10B), as described in previous studies (Mishra, Shum et al. 2016) (Oberlin 1996). To exclude a possible effect of pepRFl and pepR on CCR5 expression on the surface of cells, primary cells were also labelled with an anti-CCR5 antibody. Results show that the surface expression level was unaffected by pepRFl, pepR, AMD3100, and T20, in agreement with the infection data showing an effect only on CXCR4-mediated HIV entry. Overall the data show that pepRF 1 binds to CXCR4 co-receptor, preventing its use by HIV, but without inducing its internalization.

PepRFl is an antagonist of CXCR4

The transient increase in cytosolic Ca2+ concentration is an essential signalling pathway that is activated upon CXCR4 stimulation (Schols D 1997) (Mishra, Shum et al. 2016) (Zirafi, Kim et al. 2015) (S. Hatse 2002) (Oberlin 1996) (Donzella GA 1998). The binding of the natural ligand, CXCL12, to CXCR4 elicits a transient increase in intracellular Ca2+ concentration, classifying the ligand as a CXCR4 agonist (Schols D 1997) (Mishra, Shum et al. 2016) (Zirafi, Kim et al. 2015) (S. Hatse 2002) (Oberlin 1996) (Donzella GA 1998). On the other hand, the compound AMD3100 by itself does not induce intracellular Ca2+ signaling and when both are present, AMD3100 antagonizes the action of CXCL12 (Schols D 1997) (Mishra, Shum et al. 2016) (Zirafi, Kim et al. 2015) (S. Hatse 2002) (Oberlin 1996) (Donzella GA 1998). For this reason, AMD3100 has been defined as a pure and specific CXCR4 antagonist (Schols D 1997) (Mishra, Shum et al. 2016) (Zirafi, Kim et al. 2015) (S. Hatse 2002) (Oberlin 1996) (Donzella GA 1998).

In this study, we used a cell-based fluorescence assay to measure intracellular Ca2+ levels and determined the agonist or antagonist nature of the pepRFl and pepR. CXCL12 and AMD3 100 were used as controls. THP-1 monocytes were first loaded with the fluorescent Ca2+ indicator Fluo-4 AM and incubated for 1 h. The agonist or antagonist effect of the compounds on the intracellular Ca2+ influx was then determined by adding the compounds to cells (alone or in combination), followed by immediate detection of variations in cytosolic Ca2+ levels. Treatment of cells with CXCL12 alone elicited a transient increase in Ca2+ influx, which is in accordance with its expected agonist function (Schols D 1997) (Mishra, Shum et al. 2016) (Zirafi, Kim et al. 2015) (S. Hatse 2002) (Oberlin 1996) (Donzella GA 1998) (Figure 11A). In contrast, as AMD3100, pepRFl had no effect on cytosolic Ca2+ levels (Figure 11A). Surprisingly, pepR showed strong agonistic activity that lasted for the entire duration of the assay (Figure 11A). Like AMD3100, pepRFl was able to antagonize CXCL12 action (Figure 11B), showing that it acts as CXCR4 antagonist. Interestingly, when compared to AMD3100, pepRFl was more efficient at counteracting the strong agonist activity of pepR (Figure 11C).

The above study demonstrates a new powerful CXCR4-targeted inhibitor of HIV- 1 entry, pepRFl, derived from the Dengue virus capsid protein. This highly stable peptide was the resistant fraction of the proteolytic degradation in serum of pepR, a peptide previously reported to have antibacterial activity and cell-penetrating properties (Freire, Veiga et al. 2013) (Freire, Veiga et al. 2014) (Freire, Dias et al. 2015). Remarkably, pepRFl showed an impressive anti-HIV- 1 activity when compared to T20, the only peptide-based HIV-1 fusion inhibitor approved by FDA for clinical use. In addition, pepRFl was able to inhibit a T20-resistant strain, HIV-1NL4.3 DIM, at a low nanomolar concentration range.

Our detailed studies on the mechanism of action revealed that pepRFl inhibitory activity was specific for CXCR4-tropic HIV-1 strains, preventing viral entry into target cells by binding to CXCR4 coreceptor without inducing its internalization. Like AMD3100, a small molecule inhibitor that strongly restricts CXCR4 HIV-1 infection (E De Clercq 1994) (Oberlin 1996) (Schols D 1997) (Donzella GA 1998) (S. Hatse 2002) (Zirafi, Kim et al. 2015) (Mishra, Shum et al. 2016), pepRFl did not elicit intracellular Ca2+ influx indicating that the peptide does not act as a CXCR4 agonist. Moreover, pepRFl, as opposed to pepR, potently inhibited the intracellular Ca2+ signalling elicited by the CXCR4 natural ligand CXCL12, acting as an antagonist. Despite these differences both peptides were strong inhibitors of HIV- 1 infection.

Inhibition of CXCR4 as a strategy to fight HIV-1 infection is important, not only because CXCR4-tropic HIV strains are considered to be more pathogenic than CCR5-tropic strains, but also because their appearance during an extended time course of infection correlates with a decline in the CD4+ T-cell count leading to a more rapid progression to AIDS symptoms (Connor RI 1997). Furthermore, drug resistance is also more often linked to CXCR4-tropic strains (Wagner TA 2008), posing an urgent need for the development of effective drugs that block CXCR4-mediated HIV infection. In this perspective, the use of CXCR4 antagonists may be useful in delaying the onset of disease. In combination with CCR5 inhibitors, such as Maraviroc (Selzentry), the only coreceptor-targeted drug in clinical use, pepRFl would be a potent complement to improve the options available for patients predominantly infected with X4 or dual-tropic HIV-1 strains. Indeed, in more than 50% of HIV-1 infected individuals, CCR5 viruses are usually present as mixtures together with CXCR4 viruses (Timothy J. Wilkin 2012) (Melby T 2006). Moreover, some reports have shown that HIV gpl20 can trigger signaling and chemotactic events specifically through CCR5 and CXCR4, and in a CD4-independent manner (Misse D 1999) (Balabanian, Harriague et al. 2004). Such effects induced by the viral envelope, could also be effectively blocked by CCR5/CXCR4 antagonists, being a further advantage of pepRF 1.

In spite of the growing interest in CXCR4 as a druggable target, the development of CXCR4 inhibitors has been challenging. In fact, to date no CXCR4 inhibitor has been approved for clinical use as an anti-HIV-1 agent. AMD3100 (Plerixafor or Mozobil), one of the most potent CXCR4 antagonist described, was initially developed to be used as an anti-HIV- 1 drug (Donzella GA 1998) but clinical trials were interrupted due to undesired effects (Hendrix CW 2004) (Henrich TJ 2013). This molecule is now being used to mobilize hematopoietic stem cells from the bone marrow to the bloodstream (Hendrix CW 2000) in patients with non-Hodgkin's lymphoma and multiple myeloma (Zhang, Kang et al. 2016). Thus, the quest for druggable CXCR4-targeting compounds for the treatment of HIV- 1 infection continues.

Like AMD3100, the selective antiviral activity of pepRFl against CXCR4-tropic HIV strains is based on the specific blockage of CXCR4-mediated virus entry via direct interaction with CXCR4, counteracting the effects of CXCL12, without triggering any response by itself upon binding to CXCR4 (Schols D 1997) (Zirafi, Kim et al. 2015) (Mishra, Shum et al. 2016) (S. Hatse 2002) (Oberlin 1996) (Donzella GA 1998). In addition, the fact that pepRFl is resistant to proteolytic degradation, is also indicative of its potential use at low doses by oral administration and favorable pharmacokinetics.

Taken together, our findings point towards a promising role of pepRFl as an anti-HIV- 1 lead that could be used in the future, alone or in combination with other entry inhibitors in order to block infection of CXCR4- and dual tropic (CXCR4 and CCR5) - HIV strains, and achieve measurable declines in overall plasma viremia delaying the progression of the disease. It is also worth stressing that pepRFl could also be used in the future as a novel therapeutic strategy for CXCR4-related diseases. CXCR4 is a G-protein-coupled receptor (GPCR) that upon interaction with its cognate ligand, the chemokine CXCL12, activates a complex cascade of intracellular signaling pathways, which regulate a large number of physiological processes including HIV-1 infectivity (Schols D 1997) (Zirafi, Kim et al. 2015) (Mishra, Shum et al. 2016) (S. Hatse 2002), tumorigenesis (Xu, Zhao et al. 2015) (Burger, Glodek et al. 2003) (Kijima T 2002) (Geminder, Sagi-Assif et al. 2001) (Hwang, Hwang et al. 2003) (Peled, Klein et al. 2018) (Schimanski, Bahre et al. 2006) (Darash-Yahana, Pikarsky et al. 2004), stem cell migration (Vagima, Lapid et al. 2011), autoimmune diseases (Debnath, Xu et al. 2013) (Launay, Paul et al. 2013), and inflammation (Kircher, Herhaus et al. 2018). CXCR4 expression has been implicated in a variety of other diseases, such as in rheumatoid arthritis (Debnath, Xu et al. 2013) (Nagafuchi, Shoda et al. 2016), artherosclerosis (Galkina and Ley 2009), neurodegenerative diseases (Bonham, Karch et al. 2018), and in various types of cancers, playing a pivotal role in tumor development and metastasis (Xu, Zhao et al. 2015) (Burger, Glodek et al. 2003) (Kijima T 2002) (Geminder H 2001) (Hwang JH 2003) (Amnon Peled 2018) (Schimanski CC 2006) (Darash-Yahana M 2004). This has been demonstrated for a variety of cancer entities, including breast (Chao Xu 2015), prostate (Arya M 2004), lung (Burger M 2003) (Kijima T 2002) and colorectal cancer (Kim, Takeuchi et al. 2005), as well as primary brain tumors such as glioblastoma (Ehtesham, Mapara et al. 2009). Hence, the disruption of the CXCR4-CXCL12 axis by using a CXCR4-antagonist provides a promising molecular target for future specific therapies in areas beyond HIV-1 infection.

Table 1. Fragments resulting from pepR digestion by serum. Primary structures for peaks I to VI on figure IB, proposed on the basis of MS data. Table 2. F ragment I (pepRF 1 )

Table 3. Peptide derivatives obtained with sequential truncation of pepR

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