CONJUGATED MOLECULES COMPRISING A PEPTIDE DERIVED FROM THE CD4 RECEPTOR COUPLED TO AN ANIONIC POLYPEPTIDE FOR THE TREATMENT OF AIDS

Abstract

This invention relates to a conjugated molecule comprising a peptide derived from the CD4 receptor coupled to an organic molecule by means of a linker as well as a process for its preparation. Said organic molecule comprises a 5 to 21 amino acid anionic polypeptide. Such a conjugated molecule can be used in antiviral treatment, namely in the treatment of AIDS.

Claims

1.-17. (canceled)

18. A conjugated molecule comprising a peptide derived from the CD4 receptor, said peptide being coupled to an organic molecule by means of a linker, wherein: the said peptide derived from the CD4 receptor comprises the following general sequence (I):
Xaa.sup.f-P1-Lys-Cys-P2-Cys-P3-Cys-Xaa.sup.g-Xaa.sup.h-Xaa.sup.i-Xaa.sup.j-Cys-Xaa.sup.k-Cys-Xaa.sup.l-Xaa.sup.m,  (I) in which: P1 represents 3 to 6 amino acid residues, P2 represents 2 to 4 amino acid residues, P3 represents 6 to 10 amino acid residues, Xaa.sup.f represents N-acetylcysteine (Ac-Cys) or thiopropionic acid (TPA), Xaa.sup.g represents Ala, Xaa.sup.h represents D-proline, Xaa.sup.i represents Thr, Xaa.sup.j represents biphenylalanine (Bip), phenylalanine, [beta]-naphthylalanine or 4-cyclohexylmethoxy-L-phenylalanine (U.sub.1), Xaa.sup.k represents Thr or Ala, Xaa.sup.l represents Gly, Val or Leu, and Xaa.sup.m represents —NH.sub.2 or —OH, the amino acid residues in P1, P2 and P3 being natural or non-natural, identical or different, said residues of P1, P2 and P3 being all different from the Lys residue and P1, P2 and P3 having a sequence in common or not, and the said organic molecule comprises an anionic polypeptide consisting of 5 to 21 amino acid residues being natural or non-natural, identical or different, wherein at least 3 amino acids are negatively charged, and a molecular group A-Z, wherein: A comprises a group chosen between the groups of formula —CO(CH.sub.2).sub.p—NH—CO—(CH.sub.2).sub.q—, —CO(CH.sub.2—CH.sub.2)—(O—CH.sub.2—CH.sub.2).sub.p—NH—CO—(CH.sub.2).sub.q—, —CO(CH.sub.2).sub.p—NH—CO—(CH.sub.2—CH.sub.2—O).sub.q—(CH.sub.2—CH.sub.2)— and —CO(CH.sub.2—CH.sub.2)—(O—CH.sub.2—CH.sub.2).sub.p—NH—CO—(CH.sub.2—CH.sub.2—O).sub.q—(CH.sub.2—CH.sub.2)—, wherein p represents an integer comprised between 1 and 10 and q represents an integer comprised between 1 and 10, and Z represents a halogen atom, a thiol or a maleimide group, wherein the first carbonyl group of A is coupled to the N-terminal end of the anionic polypeptide, wherein the said linker is covalently bound at one of its extremity to the free amino group (—NH.sub.2) of the amino acid residue Lys present in general sequence (I) of the said peptide derived from the CD4 receptor, and is covalently bound at its other extremity to the Z group of the said organic molecule when Z is a thiol or a maleimide group or to the A group of the said organic molecule when Z is a halogen atom, Z being eliminated in this last case.

19. The conjugated molecule according to claim 18, wherein P1 represents 3 amino acid residues.

20. The conjugated molecule according to claim 18, wherein the anionic polypeptide consists of 10 to 17 amino acid residues being natural or non-natural, identical or different, wherein 3 to 15 amino acids are negatively charged.

21. The conjugated molecule according to claim 18, wherein Xaa.sup.j represents 4-cyclohexylmethoxy-L-phenylalanine (U.sub.1).

22. The conjugated molecule according to claim 18, wherein the sequence of the peptide derived from the CD4 receptor of general sequence (I) is chosen from the group consisting of sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 20 and SEQ ID NO: 21.

23. The conjugated molecule according to claim 18, wherein the linker is chosen from the group consisting of: ##STR00026## with k representing an integer comprised between 2 and 24, ##STR00027## with k1 representing an integer equal to 1, 2, 3, 5 and 10, ##STR00028## when Z represents a thiol group, and among: ##STR00029## when Z represents a maleimide group or a halogen atom.

24. The conjugated molecule according to claim 18, wherein the negatively charged amino acids of the anionic polypeptide are chosen among aspartic acid, sulfotyrosine, tyrosine sulfonate, aminosuberic acid, p-carboxymethyl phenylalanine, and glutamic acid, wherein said sulfotyrosine has the formula: ##STR00030## wherein said tyrosine sulfonate has the formula: ##STR00031## wherein said aminosuberic acid has the formula: ##STR00032## and wherein said p-carboxymethyl phenylalanine has the formula: ##STR00033##

25. The conjugated molecule according to claim 18, wherein the anionic polypeptide consists of 13 amino acids.

26. The conjugated molecule according to claim 18, wherein the anionic polypeptide comprises at least two different amino acids.

27. The conjugated molecule according to claim 26, wherein the anionic polypeptide comprises at least aspartic acid (D) and serine (S).

28. The conjugated molecule according to claim 26, wherein said anionic polypeptide has a sequence of S-(X-D-X-S).sub.n, where n represents an integer comprised between 1 and 5, S represents serine, D represents aspartic acid, X is selected from the group consisting of: tyrosine, sulfotyrosine, tyrosine sulfonate, aminosuberic acid, and p-carboxymethyl phenylalanine and where the various X groups are identical or different.

29. The conjugated molecule according to claim 28, wherein said anionic polypeptide has a sequence of S-X-D-X-S-X-D-X-S-X-D-X-S(SEQ ID NO: 19), where n represents an integer comprised between 1 and 5, S represents serine, D represents aspartic acid, X is selected from the group consisting of: tyrosine, sulfotyrosine, tyrosine sulfonate, aminosuberic acid, and p-carboxymethyl phenylalanine and where the various X groups are identical or different.

30. The conjugated molecule according to claim 28, wherein X groups are identical.

31. The conjugated molecule according to claim 28, wherein said anionic polypeptide has a sequence which is selected in the group consisting of: S-(Y-D-Y-S).sub.n, S-(Y.sub.SO3-D-Y.sub.SO3-S).sub.n, S-(Y.sub.SN-D-Y.sub.SN-S).sub.n, S-(pF-D-pF-S).sub.n, and S-(Asu-D-Asu-S).sub.n, where n represents an integer comprised between 1 and 5, S represents serine, D represents aspartic acid, Y represents tyrosine, Y.sub.SO3 represents sulfotyrosine, Y.sub.SN represents tyrosine sulfonate, pF represents p-carboxymethyl phenylalanine and Asu represents aminosuberic acid.

32. The conjugated molecule according to claim 28, wherein said anionic polypeptide has a sequence which is selected in the group consisting of: S-Y-D-Y-S-Y-D-Y-S-Y-D-Y-S(SEQ ID NO: 8), S-Y.sub.SO3-D-Y.sub.SO3-S-Y.sub.SO3-D-Y.sub.SO3-S-Y.sub.SO3-D-Y.sub.SO3-S (SEQ ID NO: 4), S-Y.sub.SN-D-Y.sub.SN-S-Y.sub.SN-D-Y.sub.SN-S-Y.sub.SN-D-Y.sub.SN-S (SEQ ID NO: 5), S-pF-D-pF-S-pF-D-pF-S-pF-D-pF-S(SEQ ID NO: 6), S-Asu-D-Asu-S-Asu-D-Asu-S-Asu-D-Asu-S (SEQ ID NO: 7), S-Y.sub.SO3-D-Y.sub.SO3-S-Y.sub.SO3-D-Y-S-Y-D-Y-S(SEQ ID NO: 14), and S-Y-D-Y-S-Y-D-Y.sub.SO3-S-Y.sub.SO3-D-Y.sub.SO3-S(SEQ ID NO: 15), where S represents serine, D represents aspartic acid, Y represents tyrosine, Y.sub.SO3 represents sulfotyrosine, Y.sub.SN represents tyrosine sulfonate, pF represents p-carboxymethyl phenylalanine and Asu represents aminosuberic acid.

33. The conjugated molecule according to claim 18, wherein: the peptide derived from the CD4 receptor is chosen from the group consisting of sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 20 and SEQ ID NO: 21, the linker is CO—(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.2NHCO(CH.sub.2).sub.2-pyrrolidinyl-2,5-dione, the organic molecule comprises an anionic polypeptide having the following sequence S-Y.sub.SO3-D-Y.sub.SO3-S-Y.sub.SO3-D-Y.sub.SO3-S-Y.sub.SO3-D-Y.sub.SO3-S as defined in claim 32, which is linked to the linker by a molecular group of formula A-Z, wherein A is —CO(CH.sub.2).sub.3NH—CO(CH.sub.2).sub.2— and Z is a thiol group.

34. A method for treating AIDS comprising the administration to a person in need thereof of an effective amount of a conjugated molecule according to a claim 18.

35. A method for inhibiting the HIV cell-to-cell transmission comprising the administration to a person in need thereof of an effective amount of a conjugated molecule according to claim 18.

36. A pharmaceutical composition comprising a conjugated molecule according to claim 18 and a pharmaceutically acceptable vehicle.

37. A process for the preparation of a conjugated molecule according to claim 18 comprising the following steps: a. contacting the miniCD4 peptide of general sequence (I) as defined in claim 18 with a bifunctional compound carrying two active groups, so that one of the two active groups forms a covalent bond with the free amino group (—NH.sub.2) of the residue of the amino acid Lys present in general sequence (I), in order to obtain an activated peptide carrying the second active group of the bifunctional group, and b. contacting the activated peptide obtained at step (a) with an organic molecule as defined in claim 18, wherein Z can be in a protected form when Z is a thiol group, so that the active group of the activated peptide reacts with the Z group of the organic molecule to form a covalent bond between the organic molecule and the activated peptide, in order to obtain the conjugated molecule.

Description

FIGURES

[0123] FIG. 1 represents the structure of the conjugated molecules of the invention.

[0124] FIG. 2 represents the synthesis scheme of the conjugated molecules of the invention.

[0125] FIG. 3 represents the HPLC profile of reference conjugated molecules (mCD4.1-PS1 and M48U1-PS1).

[0126] FIG. 4 represents the HPLC profile of conjugated molecules according to the invention (mCD4.2-PS1 and mCD4.3-PS1).

[0127] FIG. 5 represent the percentage of intracellular Gag 48h (FIG. 5A) or 96 h (FIG. 5B) after administration of various compounds at various concentrations in a viral cell-to-cell transmission assay using HIV-1 NL4-3.

[0128] FIG. 6 represent the percentage of intracellular Gag 48 h (FIG. 6A) or 96 h (FIG. 6B) after administration of various compounds at various concentrations in a viral cell-to-cell transmission assay using HIV-1 NLAD8.

[0129] FIG. 7 represent the percentage of intracellular Gag 48 h (FIG. 7A) or 72 h (FIG. 7B) after administration of various compounds at various concentrations in a viral cell-to-cell transmission assay using HIV-1 NL4-3.

[0130] FIG. 8 represent the percentage of intracellular Gag 48 h (FIG. 8A) or 72 h (FIG. 8B) after administration of various compounds at various concentrations in a viral cell-to-cell transmission assay using HIV-1 NLAD8.

EXPERIMENTAL EXAMPLES

Material and Methods

Reagents

[0131] All reagents for peptides synthesis were from Applied Biosystems/Life Technolgies, except Fmoc-4,4′-Biphenylalanine (Fmoc-Bip) from Interchim, S-trityl Thiopropionic acid (S-trityl Tpa) and Fmoc-4-cyclohexylmethoxy-L-phenylalanine (Fmoc-U.sub.1) from Iris Biotech GmbH, Fmoc-γAminobutyric acid (Fmoc-γAbu), Fmoc-D-Proline and Fmoc-L-O-sulfo-Tyrosine tetrabutylammonium salt, pseudoproline dipeptides Fmoc-Ser(tBu)-Ser(ψ.sup.Me,.sup.MePro) and Fmoc-Gly-Ser(ψ.sup.Me,.sup.MePro) from Novabiochem. Succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester (NHS-PEG.sub.2-Maleimide) and SATP-NHS (N-Succinimidyl S-Acetylthiopropionate) were from Pierce/Thermo Scientific. PS 2-CT-Ser (tBu) was from Rapp polymere GmbH.

[0132] All peptides and conjugates were controlled by HPLC, mass spectrometry (ESI-MS on Q-Tof micro Waters). Synthesis yields were calculated after quantification by amino acids analysis on a Hitachi L8800 apparatus.

General Protocol for miniCD4 Peptides Synthesis

[0133] MiniCD4 peptides were synthesized on a Rink Amide resin (100 μmoles) using Fmoc chemistry and HATU/DIEA activation for Fmoc amino acids coupling on an ABI433 apparatus. For mCD4.1, Ser11Ser12 and GLy21Ser22 were introduced as pseudoproline dipeptides. After chain elongation, peptides were released from the resin by TFA/H.sub.2O/Thioanisole/Phenol/Ethanedithiol/Triisopropyl silane (82/5/5/5/2/1), 20 ml, 4° C., 2 h30 treatment. Precipitation in cold diethylether afforded the crude peptides that were collected by centrifugation then solubilized in Aqueous 0.08% TFA/acetonitrile and freeze-dried. Crude peptides were purified by C18 Reverse-Phase Medium Pressure Chromatography (C18 RP-MPLC, 30 mm×340 mm column) using a 20-60 linear gradient of acetonitrile in 0.08% aqueous TFA over 60 minutes at 25 ml flow rate. Purified SH peptides (130 mg) were dissolved in 65 ml of water then dilute with 65 ml of 0.1 M pH8.5 TRIS buffer. Folding was performed by sequential addition of 796 mg of GSH and 158 mg of GSSG (20 mM/2 mM final concentration). Folding was followed by analytical RP-HPLC and was complete after 45 minutes. After acidification of the reaction mixture by 1 ml of pure AcOH, folded mini CD4 peptides were isolated by RP-MPLC using a 0-60 linear gradient with the same eluents as above. MiniCD4 peptides were controlled by MS using positive mode.

TABLE-US-00002 Yield MS expected MS found Sequence Peptide (%) Formula [M + H].sup.+ [M + H].sup.+ SEQ IDNO: 16 mCD4.1* 21 C.sub.122H.sub.194N.sub.38O.sub.32S.sub.6 2896.3124 2896.4170 SEQ IDNO: 17 M48U1* 6 C.sub.133H.sub.212N.sub.38O.sub.32S.sub.6 3046.4532 3046.4294 SEQ IDNO: 1 mCD4.2 15 C.sub.127H.sub.208N.sub.38O.sub.33S.sub.6 2986.4169 2986.4021 SEQ IDNO: 2 mCD4.3 21 C.sub.130H.sub.215N.sub.41O.sub.32S.sub.6 3055.4259 3055.4546 *reference peptides
General Protocol for Activated miniCD4 Peptides Synthesis (Lys5/11 (Nε-Maleimide) miniCD4 Peptides)

[0134] To 51 mg of miniCD4 peptide in 26 ml of water was slowly added 3 ml of 0.1 M sodium phosphate buffer pH7.2 under mild agitation to avoid foaming. NHS-PEG2-Maleimide (75 mgs, 10 molar equivalents) in 750 μl of DMSO was dropwise added to the peptide solution. After 10 minutes, the reaction mixture was acidified by 1.2 ml of pure AcOH and purified by C18 RP-MPLC using the same conditions as for miniCD4 peptide. Activated miniCD4 peptides were controlled by MS using positive mode.

TABLE-US-00003 Activated Yield MS expected MS found peptide (%) Formula [M + H].sup.+ [M + H].sup.+ mCD4.1Mal* 77 C.sub.136H.sub.212N.sub.40O.sub.38S.sub.6 3206.4289 3206.6572 M48U1Mal* 80 C.sub.147H.sub.230N.sub.40O.sub.38S.sub.6 3356.5697 3356.4619 mCD4.2Mal 60 C.sub.141H.sub.226N.sub.40O.sub.39S.sub.6 3296.5333 3296.4609 mCD4.3Mal 66 C.sub.144H.sub.233N.sub.43O.sub.38S.sub.6 3365.6024 3365.7483 *reference activated peptides

Protocol for Anionic Polypeptide (γAbu-PS1) Synthesis

[0135] PS1 peptide (SEQ ID NO: 4) was synthesized on a Serine preloaded Chlorotrityl resin, PS 2-CT-Ser (tBu), (100 μmoles). Tyrosine sulfate was incorporated using Fmoc-L-O-sulfo-Tyrosine tetrabutylammonium salt. γAminobutyric acid was introduced at the N-terminus of the peptide chain using standard amino acid coupling protocol. In order to maintain the sulfate group on the tyrosine residues, all the cleavage protocol was performed at 4° C. (ice bath). After 1 h30 in TFA/TIS/H.sub.2O 95/2.5/2.5 (9 ml), precipitation in cold diethylether afforded the crude peptide that was collected by centrifugation, solubilized in 30 ml of 100 mM ammonium hydrogenocarbonate and freeze-dried. Crude peptide was purified by C18 RP-MPLC using a 0-60 linear gradient of acetonitrile in 0.1 M Triethylacetate buffer over 60 minutes. γAbu-PS1 was controlled by MS using negative mode.

TABLE-US-00004 Polyanionic Yield MS expected MS found Sequence peptide (%) Formula [M − H].sup.− [M − H].sup.− SEQ IDNO: 18 γAbu-PS1 37 C.sub.82H.sub.98N.sub.14O.sub.49S.sub.6 2253.3811 2253.3853

Protocol for SATP-γAbu-PS1 Peptide Synthesis

[0136] To 100 mg of γAbu-PS1 peptide in 12 ml of 0.1 M sodium phosphate buffer pH 7.2 was dropwise added 40 mg of SATP-NHS (6 molar equivalents) in 300 μl of DMSO. After 40 minutes, the reaction medium was injected onto C18 RP-MPLC using the same conditions as for γAbu-PS1. SATP-γAbu-PS1 was controlled by MS using negative mode.

TABLE-US-00005 Yield MS expected MS found (%) Formula [M − H].sup.− [M − H].sup.− SATP-γAbu-PS1 76 C.sub.87H.sub.104N.sub.14O.sub.51S.sub.7 2383.3942 2383.3174
General Protocol for miniCD4-PS1 Conjugated Molecule Synthesis

[0137] SATP-γAbu-PS1 peptide (109 mg) was dissolved in 20 ml of 0.1M sodium phosphate buffer pH 7.2. 50 equivalents (2 ml) of a solution containing 0.5 M Hydroxylamine chlorhydrate in 0.1 M sodium phosphate (pH adjusted to 7.2 by 4N NaOH) were added. After 45 minutes, 52 mg of Lys5/11 (NE-maleimide) miniCD4 peptide in 22 ml of water were added. After 35 minutes, miniCD4-PS1 conjugate was purified by C18 RP-MPLC using the same conditions as for γAbu-PS1.

[0138] Final purity was controlled by analytical C18 RP-HPLC (AERIS peptide XB-C18, Phenomenex, 3.6 μm, 100×2.1 mm) using a linear gradient of acetonitrile in 100 mM aqueous Trielthylamine acetate buffer over 20 min at 0.35 ml/min flow rate. HPLC profiles are reported on FIGS. 3 and 4. All peptide conjugates were controlled by MS using negative mode.

TABLE-US-00006 Conjugated Yield MS expected MS found molecule (%) Formula [M − H].sup.− [M − H].sup.− mCD4.1-PS1* 64 C.sub.221H.sub.314N.sub.54O.sub.88S.sub.13 5552.0933 5552.1108 M48U1-PS1* 61 C.sub.232H.sub.332N.sub.54O.sub.88S.sub.13 5702.3572 5702.9180 mCD4.2-PS1 66 C.sub.226H.sub.328N.sub.54O.sub.89S.sub.13 5642.2589 5642.6030 mCD4.3-PS1 66 C.sub.229H.sub.335N.sub.57O.sub.88S.sub.13 5705.9782 5705.9243 *reference conjugated molecules

Antiviral Activity Assays

[0139] The antiviral activity of the tested compounds was determined by pre-incubating 10.sup.4 TZM-bl cells/well in a 96-well plate for 30 min at 37° C. and 5% CO.sub.2 with or without a serial dilution of the compound. Next, 200 TCID.sub.50 of viruses were added to each well and cultures were incubated for 48 h before luciferase activity was quantified. Each compound was tested in triplicate in a single experiment. Antiviral activity was expressed as the percentage of viral inhibition compared to the untreated control and plotted against the compound concentration. Next, non-linear regression analysis was used to calculate the 50% effective concentration (EC.sub.50).

Cytotoxic Activity Assays

[0140] Cytotoxicity was determined using the water-soluble tetrazolium-1 (WST-1) cell proliferation assay, which is based on the cleavage of the tetrazolium salt WST-1 to a formazan dye by cellular dehydrogenases. Because this bioreduction is dependent on the glycolytic production of NAD(P)H in viable cells, the amount of formazan dye formed is correlated directly to the number of viable cells in a culture. Quantification is performed by measuring absorbance at 450 nm in a multiwell plate reader. An amount of 10.sup.4 cells was plated per well in a 96-well plate, and a serial dilution of compound was added. Forty eight hours later, cell proliferation reagent was added and cell viability was measured compared to untreated control cultures. Cell viability was plotted against the compound concentration, and nonlinear regression analysis was performed to evaluate the 50% cytotoxic concentration (CC.sub.50).

Viral Cell-to-Cell Transmission Assays

[0141] Primary CD4+ T cells were purified from human peripheral blood by positive selection (Miltenyi). About 98% of cells were CD4+CD3+. For activation, primary T cells were treated with phytohemagglutinin (PHA) (1 pg/ml) for 24 h and then cultured with interleukin 2 (IL-2) (50 IU/ml) for 3-5 days before use. Primary cells were infected with HIV-1 NL4-3 and NLAD8 as described in Lepelley et al. PLoS Pathog 2011, 7:e1001284 and Malbec et al. J Exp Med 2013, 210:2813-2821. Donor cells were used a few days later, when about 20% of the cells were Gag+. Target cells were labeled with FarRed (2.5 μM; Molecular Probes). Donors were preincubated 1 h with the indicated doses of compounds. Donor and target cells were then mixed at a 1:2 ratio in 96-well plates at a final concentration of 1.5×10.sup.6/ml in 200 μl, in duplicates. After 48, 72 or 96 h, cells were stained for intracellular Gag (KC57 mAb, Coulter) and analyzed by flow cytometry. When stated, Nevirapine (NVP; 12.5 nM) or the NIH45-46 broadly neutralizing antibody (bNAb) (Malbec et al. J Exp Med 2013, 210:2813-2821) was added 1 h before coculture.

Results

Tested Compounds

[0142] The following miniCD4 peptides were synthesized as described in the Material and Methods.

TABLE-US-00007 Desig- SEQ nation Sequence ID NO mCD4.1 Tpa-NLHK.sub.5CQLRCSSLGLLGRCAGS- 16 Bip-CACV-amide M48U1 Tpa-NLHFCQLRCK.sub.11SLGLLGRCAp 17 TU.sub.1CACV-amide mCD4.2 Tpa-NLHK.sub.5CQLRCS.sub.11SLGLLGRCAp  1 TU.sub.1CACV-amide mCD4.3 Tpa-NLHK.sub.5CQLRCR.sub.11SLGLLGRCAp  2 TU.sub.1CACV-amide

[0143] The corresponding miniCD4 conjugates, mCD4.1-PS1, M48U1-PS1, mCD4.2-PS1, and mCD4.3-PS1, were synthesized as described in the Material and Methods.

[0144] mAb VRC01, mAb b12 and Dapivirine (TMC120), used as reference compounds, were obtained from NIH Aids Reagent Program.

Antiviral Activities

[0145] The miniCD4 conjugates were then evaluated for their antiviral activity on TZM-bl cells that express Luciferase under the control of the HIV LTR (collaboration G. Vanham, Anvers) according to the protocol described above. All antiviral results are reported in the table below in the form of EC.sub.50 values (nM).

TABLE-US-00008 Clade- mCD4.1- M48U1- mCD4.2- mCD4.3- mAb mAb Dapivirine Viral strain tropism mCD4.1 PS1 M48U1 PS1 mCD4.2 PS1 mCD4.3 PS1 VRC01 b12 (TMC120) Bal B-R5 236 0.54 0.96 0.13 2 0.034 2.3 0.031 0.63 1.0 1.65 IIIB B-X4 29 0.013 0.08 0.02 0.22 0.007 0.16 0.0025 0.64 0.09 0.84 MN B-X4 20 0.08 0.25 0.12 0.60 0.046 0.37 0.026 2 1.5 1.5 SHIV162p3 B-R5 5372 36 24 3.4 69 2.2 29 1.4 8.2 5.9 >1000 SF162 B-R5 130 0.11 1 0.05 1.24 0.01 0.66 0.008 6.0 0.43 1.3 VI829 C-RS >10000 16 59 1.1 512 4.0 221 3.6 9.2 >100 1.3 VI820 A- 1686 25 3.8 0.1 6.1 0.031 4.2 0.024 11.9 >100 1.95 X4R5 VI1888 AE-R5 >1000 1102 8422 1069 9763 1064 8018 1212 16 60 2.1 (CRF01) MP568 AG-R5 7931 310 129 15 155 4.9 204 4.2 54 >100 1.1 (CRF02) VI824 D-R5 3176 40 29 2.2 56 0.68 39 0.7 >100 >100 1.2 Ca10-3 AE-X4 96 0.57 2.7 0.6 4.73 0.12 3.1 0.076 13 >100 1.3 (CRF01) p246F10 C-R5 >10000 723 718 58 510 33 85 >100 1.4 pREJO.c/2864 B-R5 108 0.66 76 0.27 46 0.28 0.77 >100 1.6 cl2

Cytotoxic Activities

[0146] The miniCD4 conjugates were then evaluated for their cytotoxic activity according to the protocol described above. All cytotoxic results are reported in the table below in the form of CC.sub.50 values (nM).

TABLE-US-00009 mCD4.1- M48U1- mCD4.2- mCD4.3- mCD4.1 PS1 M48U1 PS1 mCD4.2 PS1 mCD4.3 PS1 CC50 45525 >50000 12946 >50000 24036 >50000 15540 >50000 Dapivirine mAb VRC01 mAb b12 (TMC120) CC50 >100 >300 2524

Viral Cell-to-Cell Transmission Inhibition

[0147] mCD4.1, mCD4.2, mCD4.3 and their PS1 conjugates, as well as Nevirapine and NIH45-46, were evaluated for their capacity to inhibit cell-cell HIV transmission. This was performed using primary cells infected with R5 (HIV1-NLAD8) and X4 (HIV1-NL4-3) viruses.

[0148] The results obtained are presented on FIGS. 5A, 5B, 6A, 6B, 7A, 7B, 8A and 8B.

DISCUSSION

[0149] Results from antiviral activities clearly show the great enhancement of miniCD4 peptides activity brought by polyanion (PS1) coupling. The effect of polyanion coupling is still important on miniCD4 peptides that are already very active like M48U1, mCD4.2 and mCD4.3. These results confirm the synergetic effect afforded by polyanion covalent coupling. In particular, the polyanion coupling greatly enhances antiviral activity of MCD4.2-PS1 as compared to mCD4.2 alone (9-190 fold increase).

[0150] The two conjugates according to the invention (mCD4.2-PS1 and mCD4.3-PS1) also show enhanced activity on Circulating Recombinant Form (5CRF) strains that are difficult to inhibit. EC.sub.50 of 4.9 nM and 1.1 μm respectively for CRF02_AG R5 and CRF01_AE R5 were obtained for mCD4.2-PS1.

[0151] It is worth noting that these molecules display much higher antiHIV activity than VRC01, currently considered as one of the most potent broadly neutralizing antibody.

[0152] Importantly, these conjugates display an extraordinary selective index (SI) of 1 to 5.106 when measured on TZM-bl cells (SI=CC50/EC.sub.50). Moreover, polyanion coupling seems to decrease at least by two fold the toxicity of the uncoupled miniCD4.

[0153] The conjugate according to the invention mCD4.2-PS1 displays EC.sub.50 as low as 34 pM (Bal strain, B-R5) and 10 pM (SF162 strain, B-R5).

[0154] When compared to mCD4.1-PS1, a 25 fold increase of HIV antiviral activity has been obtained with mCD4.2-PS1 for R5 strains Ba_L (0.034 nM versus 0.54 nM) and SF162.

[0155] It is worth noting that polyanion coupling in position 5 of miniCD4 peptides rather than in position 11 leads to more active compounds. When compared to M48U1-PS1 (polyanion coupled on Lys11), mCD4.2-PS1 and mCD4.3-PS1 (polyanion coupled on Lys5) show a 1.5 to 5 fold increase in antiviral activity.

[0156] Moreover, on the way to develop a new anti-HIV drug, cell-cell transmission inhibition has also to be evaluated since this form of dissemination appears to be less susceptible to inhibition by antiretroviral drugs than cell-free virus transmission. Even though cell-cell transmission is more difficult to inhibit for R5 viruses than for X4 viruses, mCD4.2-PS1 and mCD4.3-PS1 were able to block cell-cell virus transmission for both viruses (mCD4.1-PS1 also but to a lesser extend). These results are very encouraging when comparing the low molecular conjugates according to the invention (around 5.5 Kd) to the broadly neutralizing antibody NIH45-46 (150 Kd), targeting the CD4binding site of gp120, used as reference at a 15 μg/ml (100 nM). These experiments also confirmed the great importance of PS1 conjugation in enhancing miniCD4 peptides antiviral activity.