WO1993003055A1

WO1993003055A1 - Peptide compounds inhibiting hiv adsorption on target cells

Info

Publication number: WO1993003055A1
Application number: PCT/FR1992/000772
Authority: WO
Inventors: Edmond De Mil; Robert Michelot
Original assignee: Edmond De Mil; Robert Michelot
Priority date: 1991-08-05
Filing date: 1992-08-05
Publication date: 1993-02-18
Also published as: FR2680174A1

Abstract

An activity inhibiting fixing of the HIV-1 virus on cells is achieved according to the invention by providing analogue derivatives of the N-terminal portion of Substance P: Arg-Pro-Lys-Pro (see diagram). These novel molecules could constitute a new therapeutic strategy for HIV infections. Furthermore, said molecules may correspond to new immune system cell surface markers (lymphocytes CD4+, macrophages, etc...) which might be used in the diagnosis and biological monitoring of certain immunological diseases.

Description

PEPTIDE COMPOUNDS, INHIBITORS OF HIV ADSORPTION ON ITS TARGET CELLS

The present invention relates to a new class of chemicals having antiviral activity and, in particular, activity inhibiting the binding of the HIV virus.

These peptide compounds which inhibit the binding of the HTV-1 virus to cells of a human lymphoblastoid line are described below. The therapeutic applications of said inhibitor products form part of the present invention. HTV viruses have been described by F. Barré-Sinoussi et al. (Science, 1983, 2Ω. 868: 871) and by Clavel et al. (Science, 1986, 233, 343: 346). In this document, the terms "HIV" or "HIV" will be used interchangeably to designate these viruses.

In the spread of retroviral infection, the addition of the virus to the cell membrane represents a preliminary step, which depends on the binding of the viral glycoprotein GP120 to the CD4 cell receptor present in certain groups of T lymphocytes and cells of the system. immune. The inhibition of this phenomenon can therefore constitute an interesting target for antiviral chemotherapy. Several substances with inhibitory properties against HIV infection (heparin, pentosan polysulfate, dextran sulfate, PVAS and PAVAS) and certain short and hydrophobic peptides (Pro-Phe) have recently been described for similar properties (Finberg et al, Science, 1990, 249. 287: 291).

We have found that substance "P" or C-terminal peptides, acylpeptides, ester peptides and amide peptides, analogues of the N-terminal and hydrophilic part of Substance P: Arg-Pro-Lys-Pro-OH, have a inhibitory activity on the binding of HIV-1 virus with the surface of human lymphoblastoid cells. By way of example, substance P has been described in J. Physiology, 1931, 2 ”74:87, by Von Euler and Gaddum, then characterized by Chang et al. (J. Biol. Chem., 1970, 245. 4784).

The preferred peptides according to the invention correspond to the general formula:

A-Arg-Pro-Lys-Pro-R A ≈Boc, H, CH3-CO-, CH ₃ - (CH ₂ ) m-CO-; m = 2, 4, 6, 10

Boc = terbutyloxycarbonyl

R = -OH, -OCH3, -NH- (CH ₂ ) _n -CH ₃ ; n = 3, 5, 7, 11

B. These are, in particular, the following PI, P2, P3, P4 and P5 products:

P1 = H-Arg-Pro-Lys-Pro-OH P2 = H-Arg-Pro-Lys-Pro-OCH3 P3 = CH ₃ - (CH2) ιo-CO-Arg-Pro-Lys-Pro-OCH3 P4 = H -Arg-Pro-Lys-Pro-NH- (CH ₂ ) ιι-CH3 ^' P5 = CH3- (CH ₂ ) ιo-CO-Arg-Pro-Lys-Pro-OH

products whose inhibitory activities have been evaluated using in vitro screening tests for substances with antiviral activity.

Figure 1 shows the structural formula of the derivatives described.

I - SUMMARY OF PEPTIDE DERIVATIVES

Summary diagram

* Boc Proline methylester, or Boc Proline alkylamide, is deprotected with trifluoroacetic acid at 40% in dichloromethane, at 25 ° C. Deprotection is followed by chromatography, on a thin layer (Merck silica plates, solvent: methanol / chloroform 1/9 volume). The coupling with Nα Boc-L- (ω carbobenzoxy) lysine is carried out at -15 ° C, in the presence of N-methylmorpholine, the proline derivative being in slight excess (M. Bodanszky and A. Bodanszky - la: "The

Practice of Peptide Synthesis "; Springer Verlag Berlin, 1984, p. 109: 110).

The coupling agent is isobutyl chloroformate. The dipeptide formed: Boc Lys (Z) -Pro-R is then deprotected on the N-terminal part with trifluoroacetic acid at 40%. This process is repeated until the tetrapeptide A-Arg-Pro-Lys-Pro-R is obtained, which is ultimately deprotected on the side chains by anhydrous hydrofluoric acid (see Bodanszky p. 174). * The purification is carried out by gel chromatography and reverse phase chromatography, high performance (HPLC), on grafted silica.

The absence of racemization is verified by coupling the hydrolyzate with the Marfey reagent and HPLC. The purity criteria are PHLC, mass spectometry (FAB), NMR and amino acid analysis.

* The pseudopeptides have been synthesized in order to avoid degradation by proteases and to modify the structure of the reference peptide sequence (Arg-Pro-Lys-Pro).

A-Arg-Pro-y (CH2-NH) -Lys-Pro-R A = Boc-, H-, CH ₃ -CO-, CH ₃ - (CH ₂ ) m-CO-; m = 2, 4, 6, 10

R = -OH, -OCH3, -NH- (CH ₂ ) _n -CH ₃ ; n = 3, 5, 7, 11

These pseudopeptides have been synthesized by chain extension, like the corresponding homologous peptides.

The methylene amino-y bond (CH ₂ -NH) -, reduced peptide bond, is obtained by condensation, according to the method of Sasaki and Coy (Peptides, 1989, &, 119: 121).

Thus, a Boc-aminoaldehyde is condensed with the amino function of an amino acid or a peptide. The intermediate Schiff base is reduced in situ by sodium cyanoborohydride (NaBH ₃ CN).

The purity criteria are, again, defined by HPLC, mass spectrometry (FAB), NMR and amino acid analysis. The structural formula of the derivatives described is presented in diagram No. 1.

The inhibitory activity of these peptide compounds on the binding of the HIV-1 virus to these target cells was evaluated according to the following method

H - STUDY OF THE INHIBITION OF HIV ADSORPTION ON THE SURFACE OF TARGET CELLS BY AN ANALYSIS

FLOW CYTOMETRY

A - MATERIAL USED FOR THIS STUDY

1) The molecules tested The peptides PI, P2, P3, P4 and P5 were synthesized according to the synthesis method described above. These products were dissolved in water, stored and frozen at -20 ° C.

2 Levinιs HIV-l

The HIV virus (LAV-1 strain) was produced on infected T lymphoblastoid cells (CEM-LAV-1 line). Every 3 or 4 days, the cells are centrifuged, resuspended in new medium and mixed at 50% with uninfected EMFs at the final concentration of 0.5.10 ⁶ cells per milliliter.

The virus present in the supernatant was concentrated 150 times by precipitation with 10% polyethylene glycol for 24 hours at 4 ° C. After centrifugation at 5000 rpm for 30 min, the pellet was resuspended in an NTE buffer (pH 8) (0.1M NaCl, 0.01M Tris, 0.0001M EDTA), placed on a 30-40% sucrose cushion and centrifuged for one hour at 26,000 rpm (Rotor Kontron TST 28-38).

The purified virus was recovered, diluted in NTE buffer (pH 8), then concentrated by centrifugation at 26,000 rpm for 1 hour. The pellet was resuspended in the NTE buffer, aliquoted and stored at -80 ° C.

The HIV-1 virus used for this study has a titer greater than 10 ⁸ IU / ml (IU: Infectious Unit) for MT4 cells and a titer of 10 ⁹ cpm ml in reverse transcriptase assay.

3) SupTl cells (Smith et al. Cancer Research, 1984,, 5657: 5660)

D. is a continuous line of human lymphoblastoid cells. These cells are cultured in RMPI 1640 medium 10% FCS (Flow), 1% PSN (penicillin, streptomycin, neomycin (GIBCO), 1% glutamine (GIBCO)).

4) The anticor s

a) Anή-GP 110 (anti-HIV glycoprotein envelope 1):

This antibody was produced by Genetic System, it is used to the 100th in a solution of PBS, BSA 1%, Azide 0.1% (PBA). b) Anti-mouse anti-mouse:

This antibody is marketed by the Amersham Company (Ref. RN 1001). π is used and diluted to 25th in PB solution A. The revelation of this antibody is obtained by streptavidin phycoer thrine (Becton Dickson).

B) METHOD OF STUDYING THE FIXATION OF THE HIV-1 VIRUS TO SA

TARGET CELL

This method has been described by Mac Dougal et al, Journal of Immunology, 1986, 137. 2937: 2944).

The SupTl cells (5.10 ⁵ cells) were washed twice in the PB A solution and incubated individually with 10 " ⁶ M of each product PI, P2, P3, P4 and P5 respectively (samples 11, 12, 13, 9a and 10a ), for 30 min at 37 ° C, then 1 μg of concentrated HTV-1 virus was added and the whole was maintained at 37 ° C for 30 min.

The cells were incubated with the anti-GPHO antibody, then with the biotinylated anti-mouse antibody and, finally, with streptavidin phycoerythrin. Each incubation is carried out at 4 ° C. for 30 min and the cells are washed twice with the PB A solution, after each of these steps. Prior to cytofluorograph (Ortho) analysis, the cells were resuspended in the PBA solution supplemented with 1% formalin.

For these experiments, several controls were carried out:

* Positive controls:

- Control of the fixation of the HTV-1 virus and of the revelation system: the cells were incubated with the HTV-1 virus, then, respectively, with the anti-GPHO, the biotinylated anti-mouse and the streptavidin phycoerythrin ( samples 1 and la).

- Control of the blocking of the fixation of the HTV-1 virus with the OKT4a antibody (Ortho Diagnostics): the cells were incubated with the OKT4a antibody, then, respectively, with the HIV-1 virus, the anti-GPHO, biotinylated anti-mouse and streptavidin phycoerythrin (samples 2 and 2a). * Control, negatives:

The cells were incubated with medium, then, respectively, with anti-GPHO, biotinylated anti-mouse and streptavidin phycoerythrin (samples 3 and 3a).

The cells were incubated with each of the products individually, then successively with medium, anti-GPHO, biotinylated anti-mouse and streptavidin phycoerythrin (samples 4, 5, 6 and 4a, 5a).

- Control of the presence of the CD4 molecule: the cells were incubated with the OKT4a antibody, then a fluorescent anti-mouse (samples 6a and 7).

- Control of the effect of the substances on the binding of the OKT4a antibody on the CD4 molecule (samples 8, 9, 10 and 7a, 8a).

C) RESULTS

Table I presents the results of the cytofluorograph analysis of the samples PI, P2 and P3. Under our experimental conditions, 53% of the cells binding the HIV-1 viras are detected (sample 1).

When the binding of the HIV-1 virus is blocked by preincubation of the cells with the anti-CD4 antibody (OKT4a), only 1% of fluorescent cells are detectable (sample 2). The products PI, P2 and P3 do not modify the binding of the antibody OKT4a to the SupT1 cells.

Pretreatment of the cells with the products allows a significant reduction in the binding of the HIV-1 virus with the products PI and P3 (respectively samples 11 and 13) and a total blocking of the fixation with the product P2 (sample 12).

Table II shows the results obtained with products P4 and P5. The various witnesses present the same observations as those previously described for the products PI, P2 and P3.

The pretreatments of the cells with the products P4 and P5 show a reduction in the binding of the HTV-1 virus (samples 9 and 10), compared to the binding of the HIV-1 virus on these untreated cells (sample 1a). in - CONCLUSIONS

Under these experimental conditions, five products studied modify the binding of the HIV-1 virus to SupT1 cells.

At a concentration of 10-6M, the following products lead to the blocking of virus fixation in the following reports:

% Inhibition formula

Pl = H-Arg-Pro-Lys-Pro-OH 89

P2 = H-Arg-Pro-Lys-Pro-OCH ₃ 96

P3 = CH3- (CH ₂ ) _n -CO-Arg-Pro-Lys-Pro-OCH3 89

P4 = H-Arg-Pro-Lys-Pro-NH- (CH ₂ ) n-CH ₃ 85

P5 = O CH ^ n-CO-Arg-Pro-Lys-Pro-OH 80

The present invention also covers the derivatives of these substances and, in particular, derivatives which cannot be hydrolyzed by modification, in particular, of peptide bonds, if these derivatives retain an inhibitory activity equivalent to the original products.

TABLE I

Cytofluograph analysis of PI, P2 and P3 samples

Percentage nomenclature

SAMPLES used of fluorescent cells

* Positive witnesses

= Cells then HTV-1 virus then anti-GPHO then anti¬ 53% biotinylated mouse then streptavidin phycoerythrin

= Cells then OKT4 then HIV-1 virus then 1% anti-GPHO then biotinylated anti-mouse then streptavidin phycoerythrin

* Negative controls:

= Cells then medium then anti-GPHO then anti-mouse 2% biotinylated then streptavidin phycoerythrin

= Cells then product then medium then anti-GPHO then anti-biotinylated mouse then streptavidin phycoerythrin

PI 4 product 1%

Product P2 5 1%

Product P3 6 1%

* Control of the çp4 molecule:

= Cells then OKT4a antibody then 99% fluorescent anti-mouse

= Cells then product then OKT4a antibody then fluorescent anti¬ mouse

TABLE II

Cytofluograph analysis of samples P4 and P5

Nomenclature Percentag SAMPLES used of fluorescent cells

* Positive witnesses:

= Cells then HTV-1 virus then anti-GPUO then anti-53.41% biotinylated mouse then streptavidin phycoerythrin

= Cells then OKT4 then HIV-1 virus then anti-GPl 10 2a 8.57% then biotinylated anti-mouse then streptavidin phycoerythrin

* Negative controls:

= Cells then medium then anti-GPUO then anti-mouse 3a 10.47% biotinylated then streptavidin phycoerythrin

= Cells then product then medium then anti-GPUO then biotinylated anti-mouse then streptavidin phycoerythrin

Product P4 4a NT *

Product P5 5a NT *

* Control of the CD4 molecule:

= Cells then OKT4a antibody then anti-mouse 6a 95.5% fluorescent

= Cells then product then OKT4a antibody then fluorescent anti¬ mouse

Product P4 7a 89.28%

Product P5 8a 92.20%

* Experience:

= Cells then product then HIV-1 virus then anti-GPl 10 then biotinylated anti-mouse then streptavidin phycoerythrin

Product P4 9a 19.43%

Product P5 10a 15.15%

NT *: not tested

Claims

1. New use of substances of general formula

A-Arg-Pro-Lys-Pro-R

- in which A denotes a group chosen from:

Boc-, H-, CH ₃ CO-,

C ^ CH ^ m-CO- with m = 2, 4, 6, 10.

- in which R represents a group chosen from:

-OH, -OCH3,

-NH-.CH ^ n-CHs with n = 3, 5, 7, 11.

for the inhibition of the binding of the HIV-1 virus on human target cells.

2. The invention is characterized by substances according to claim 1, intended to be used in the manufacture of a medicament inhibiting the binding of the HIV-1 virus. These substances are characterized by the peptide and / or pseudo-peptide sequence: Arg-Pro-Lys-Pro.