WO1991012271A1 - Synthetic peptides for neutralizing the infectivity of the aids virus (hiv) - Google Patents

Abstract

There is disclosed a new peptide corresponding to the sequence of amino acids FYIFFVEDQKEEDD or to its variants X1YIX2X3VEDQKX4X5DD (X1=T or F, X2=C or F, X3=E or F, X4=E or F, X5=E or F) with activity in vitro against the infection by HIV which allows the micromolar 164 concentration to inhibit up to 95 % the capacity of HIV to form giant cells (syncytes) with Mol-3 cells and to inhibit 75 % of its capacity to synthesize the protein p17 of the core. These peptides have therapeutic potential not only in individuals infected with AIDS but also with AIDS-related diseases. Said peptides could also be useful in the profusion of the disease or to revert it in seropositive asymptomatic individuals. Furthermore, said peptides may be efficient for the prevention of the infection in high risk individuals, with possible exposure to HIV and in infants born from seropositive mothers. Its synthesis, after selecting the most appropriate sequence, is carried out by the method of Marriefield in modified solid phase or by genetic engineering.

Description

Title

SYNTHETIC PEPTIDES TO NEUTRALIZE THE INFECTIVITY OF

AIDS VIRUS (HIV)

Introduction

It is known [GA DalgleisJi, CLP Beverley, RP Clapham, HD Crawford, FM Greves and AR Weiss, Nature 312, 763 (1984)] that the human immunodeficiency virus (HIV) infects T helper lymphocytes through an interaction between the protein gpl20 of the envelope of the virus and the CD4 protein found on the surface of these lymphocytes. When this discovery was made, several groups of researchers [RA Fisher, JM Bertonis, W. Meier, VA Johnson, DS Costopulos, T. Liu, R. Tizard, BB al er, MS Hirsch, RT Schooley, RA Flavell, Nature, 331, 76 (1988); RE Husséy, NE ^* . Richardson, M. Kowalski, NR Brown, HC Chang, RF Siciliano, T. Dorfman, B. Walker, J., Sodroski, El Reinherz, Nature 331, 78 (1988); KC Deen, JS McDougal, R. Inaker, G. Folena-Wasserman, J. Arthos, J. Rosenberg, PJ Madddons, R. Axel and RW Sweet, Nature 331, 82 (1988); A. Traunecker, W. Luke and K. Karjalainen, Natüre 331, 84 (1988)] prepared CD4 using recombinant techniques to use it in blocking the aforementioned interaction, since, at least from a theoretical point of view, it would be foreseeable that in a medium containing T lymphocytes and HIV virus, the addition of free CD4 protein had the effect of blocking viral gpl20. This would prevent the virus from binding to the T lymphocyte and therefore infecting it.

State of the art

The possibility of using free CD4 protein to block viral gpi20 has been tested and proposed by various researchers [J. Kahn, A. Davis, J. Groop an, L. Kaplan, S. Sherwin and PA Volberding. V International AIDS Conference, June 4-9, Montreal, Canada (1989); R. Schooley, D. Ho, P. Gaut, M. Tierney, J. Schindler, S. Henochowicz et al. ibid; V. Chams, T. Jouault, E. Fenouillet, JC Gluck an, D. Klatzmann, ibid .; -N. Letvin, M. Watanabe, KA Reimann, PA DeLong, T. Liu, and RA Fisher, ibid.].

The idea of using peptides representing CD4 protein sequences as a method to neutralize the interaction between the virus gpl20 protein and the CD4 T cell protein has been explored by various authors [J.D. Lifson, K.M. Hwang, P.L. Nara, B. Fraser, M. Padget, N.M. Dunlop and L.E. Eiden Science 241, 712 (1988); P.L. Nara, K.M. Hwang, D.M. Rausch, J.D. Lifson, L.E. Eiden Proc. Nati Acad. Sci. USA. 86, 7139 (1989)]. Indeed, Lifson et al. they synthesized peptides between 12 and 25 amino acids representing the entire sequence of the CD4 protein. In a first experiment, said authors found that a preparation of the peptide corresponding to amino acid sequence 76-94 of said protein inhibited the ability of the HIV-I virus to form syncytia with CEM-SS cells at the 125 micromolar concentration of peptide. In subsequent experiments, Lifson et al. they demonstrated that the purified peptide was actually inactive and that the inhibition was due to the benzylated impurities of the peptide. Realizing this fact, Lifson et al. they performed a synthesis by saving the benzyl group (bzl) in one or more of the amino acids that, in the synthesis of the peptide, had been protected with this group. In this way they came to determine that the purified peptide TYIC- (bzl) -E- (bzl) -VEDQKEE was able to inhibit the formation of syncytia at a concentration of 125 μmol / 1. Recently Nara et al. have published the preparation of various benzylated derivatives of the sequence 81-92 of the CD4 protein. Among them, the amino acid sequence T- (bzl) -YIC- (bzl) -E-

(bzl) -VEDQK- (ac) -EE seems to be the most effective, being able, according to these authors, to inhibit the formation of syncytia at the 31 μmolar concentration of peptide. Brief Description of the Invention

The present invention is situated in an intermediate position between the two lines of investigation described above. On the one hand, the use of the CD4 protein is excluded, while on the other, the proposal is made to replace it with small peptides (12-20 amino acids) that do not necessarily have to be all those that make up the CD4 protein, being enough with its analogy those of that and the complementarity with regions of gpl20 or gp41.

The use of these peptides allows to achieve the same effect as the use of CD4 with the advantage of a lower cost and a lower probability of degradation; on the other hand, since it is a simpler molecule than that of the protein, the formation of antibodies that inactivate the drug is unlikely since it is difficult for the peptide to contain a T determinant (amino acid sequence recognized by the major histocompatibility complex type II) which, as Borras et al. [F. Borras-Cuesta, A. Petit-Camurdan and Y. Fedon. Eur. J. Immunol. 17, 1213 (1987)], would be responsible for providing T-aid to B lymphocytes that synthesize the antiseptic antibodies.

On the other hand, the use of a peptide instead of a complex molecule where some of the amino acids have been benzylated or acetylated leads to products that, due to their similarity to the natural ones, have a lower degree of toxicity for the organism, thus increasing the LD50 of the drug. ; on the other hand, a peptide, formed exclusively by natural amino acids, presents the possibility of being obtained by genetic engineering using recombinant methods that, as is known, allow the production cost to be significantly reduced.

The fundamental basis, therefore of this invention is to use a certain amino acid sequence between those found, together with the CD4 protein component, phenylalanine which, by having the benzyl group in its molecule, avoids the need to benze certain amino acids.

Detailed description of the invention

It is known that the amino acid sequence in the CD4 protein

TYICEVEDQKEE [0] is the determinant of its inhibitory power as long as the amino acids marked in bold (T, C and E) are benzylated.

The present invention is based on synthesizing a peptide (chemically or genetically engineered) based on that sequence but substituting one or more of the amino acids for phenylalanine which, in another case, should be benzylated. In principle, this substitution can be:

TYIFEVEDQKEE [1] TYIFFVEDQKEE [2] substitution that, however, does not have to be limited to these two alternatives but can be extended to other amino acids in the sequence that can also carry the benzyl group: TYIFFVEFQKFE [3]

TYIFFVEFQKFF [4]

TYIFEVFDQKEE [5]

Peptides [3] and [4], due to their low negative charge under the conditions of the antiviral activity assay, showed such low solubility that it was impossible to specify that activity. Peptides [2] and [5], however, by resorting to sonication, sufficient solubility is achieved to perform the positive test.

The hypothesis that the small solubility of the peptide is due to its low net negative charge is confirmed. because, including one or two additional aspartic acids in the C-terminal region (thereby increasing the net negative charge of the peptide), its solubility also increases. The addition must be made in the said C-terminal region and not in the N-terminal where the hydrophobic nature of the peptide resides, presumably involved in the interaction with the gpl20 protein.

In this way, the new peptide, in which the sequence of [2] is maintained, becomes configured as

TYIFFVEDQKEEDD [6] where, since aspartic acid has no other mission than to increase solubility, two molecules are used per peptide so that, without affecting the fundamental sequence or substantially price, the desired effect is achieved.

This same improvement is achieved with the peptide in which the T of [6] was replaced by F, which goes on to present the configuration: FYIFFVED KEEDD [7]

The obtaining of these peptides characterized in that the sequence [0] existing in a part of the CD4 protein is respected, but in which some of the amino acids are substituted by phenylalanine, can be done by chemical or biological synthesis.

In the case of chemical synthesis, this is performed using the Merriefield solid phase method [RB Merrifield, J. Am. Chem. Soc., 85, 2149 (1963)] but using the Fmoc variant [E. Atherton, JC Logan and CR Sheppard, J. Chem. Soc. Perkin Trans., 1, 538 (1981)] in which, unlike the BOC variant, the benzyl protective group is not used with what is allowed to ensure that none of the amino acids of the synthetic peptide contain this group. The synthesis is carried out as follows:

1. Resin preparation

The polyamide gel resin functionalized with sarcosine is treated with ethylenediamine for about 10 hours. It is then washed with abundant dimethylfor amide and the resin is reacted with an activated arm (linkage agent in the Anglo-Saxon literature). The activated arm is preferably the penta-fluorophenyl ester of 4- hydroxy-methyl-phenoxyacetic acid. Once this arm has been added, the Kaiser ninhydrin test is performed which must be negative, thus showing the absence of free amino groups.

2. Union of the first amino acid of the synthesis to the arm attached to the resin.

The symmetric anhydride of the first amino acid is prepared, which has the alpha-amino group protected with the fluoren-methyl-oxycarbonyl group (Fmoc) and any other side chain group that can react, protected with a group compatible with Fmoc technology . To the symmetric anhydride is added dimethyl-amino-pyridine which acts as a catalyst and is allowed to react with the resin between 80 and 100 minutes. At the end of the reaction, it is washed with abundant dimethyl formamide (DMF) and the Fmoc group is removed with 20% piperidine in DMF. This operation exposes the alpha-amino group of the first amino acid to react with the carboxyl group of the second amino acid.

3. Aggregation of the other amino acids in the sequence Once the first amino acid is joined to the arm, the other amino acids of the sequence are added, one by one, using the method of either symmetric anhydrides or active esters .

Symmetric anhydrides are prepared from conveniently protected amino acids, activating them with dicycloexylcarbodium in dichloromethane. Dicycloexil Urea that forms is removed by filtration, dichloromethane is removed on a rotary evaporator and the residue (symmetric anhydride) is dissolved in DMF and added to the resin. It is reacted for 60 to 90 minutes until the ninhydrin assay [E. Kaiser, RL Colescott, CD Bossinger and PI Cook, Anal. Biochem , 34, 595 (1970)] show absence of free amino groups, otherwise this stage is repeated.

In the case of using active esters, the procedure is simpler. The active ester is dissolved directly in DMF containing the same molar amount of 1-hydroxybenzotriazole (HOBT) as the active ester, and added to the reaction mixture.

At the end of each amino acid binding (either by means of symmetric anhydrides or active esters) the ninhydrin test is performed. If this is negative, the Fmoc protective group with 20% piperidine in DMF is removed in order to leave it ready for the next binding to the amino acid that follows in the sequence. This process is repeated for the different amino acids in the sequence, which manages to grow the polypeptide chain in the C-terminal to N-terminal direction.

4. Resin peptide cutting.

The first amino acid of the synthesis (the C-terminal amino acid) is attached to the arm forming a bond that is much weaker than the peptide bond. For this reason it is possible to selectively cut the peptide under conditions of complete stability for the peptide bond. The cutting solvent is such that it allows both the resin peptide to be cut and the protective groups that have been used to protect the side chains from amino acids to be removed. In this sense, 95% trifluoroacetic acid is preferably used as the cutting solvent, containing phenol, ethanedithiol and anisole in variable proportions according to the amino acids used, a composition that affects the time required for the operation.

The added amino acids, in the order N-terminal to C-terminal, are:

1 T or F

2 AND

3 I 4 F

5 E or F

6V

5. Peptide purification. The peptide obtained is purified by high pressure liquid chromatography using a solvent-phase reversed phase C18 column (0.1% [w / v] trifluoroacetic acid in water) and making a linear gradient, up to 70%, of a solution of acetonitrile containing 0.1% (w / v) of trifluoro acetic acid.

Use of the peptide as an inhibitor of HIV virus infectivity.

To test the inhibition power of the various peptides obtained as variants of sequence 76-94 of the CD4 protein (sequence [0]), the degree of infection in the presence or absence of the peptide is compared. For this, the formation of syncytia (giant cells), cell viability and the presence of viral proteins in cells [P.S. Sarin, Sudhir Agrawal, M.P. Civeira, J. Goodchild, T. Ikeuchi and P.C. Zamecnik, Proc. Nati Acad. Sci. USA, 85, 7448 (1988)].

The most active peptide allows, at 164 μmolar concentration, to inhibit in 91% the ability of the HIV virus to form giant cells (syncytia) with Molt-3 cells and in 75% its ability to synthesize the core pl7 protein. These peptides possess therapeutic potential not only in subjects with AIDS but also in AIDS-related conditions. It may also be useful in the profusion of the disease or would reverse in asymptomatic seropositive individuals. On the other hand, they can be effective in preventing infection in high-risk individuals, with possible exposure to the HIV agent and in those born to HIV-positive mothers.

Examples

To understand how to carry out the application of this patent, a series of examples of obtaining peptides and checking their inhibitory effect on virus infectivity are described below.

Example n ^e l

Following the experimental technique described above (sections 1 to 5), a peptide with the amino acid sequence is prepared

TYIFEVFDQKEE being the first amino acid of the synthesis the E (the C-terminal amino acid) followed by: E, K, Q, D, F, V, E, F, IY and T, successively, growing the peptide, as has indicated in the direction C-terminal to N-terminal.

Verifying the test regarding its inhibition ability, according to the techniques of Sarin et al. described above, with Molt-3 cells, and after peptide sonication, the following results were obtained

Concentration (μmol / 1) Percent inhibition 37.5 0

75.0 6 150.0 33

300.0 59 10

Example n ^c 2

Using the same technique described in example 1, the peptide corresponding to the sequence was obtained

TYIFFVEDQKEE

The test regarding its capacity for inhibition was verified, under the same conditions as in example 1 (including sonication) the following results were obtained

Concentration (μmol / 1) Percent inhibition

37.5 26

75.0 62

150.0 95 300.0 100

When a sonication is not done, prior to the inhibition test, it gives a negative result.

Example No ⁸ 3

Using the same technique described in example 1, the peptide corresponding to the sequence was obtained

TYIFFVEDQKEEDD

Verified the test regarding its capacity for inhibition, under the same conditions as in example 1, but without sonication, the following results were obtained

Using the same technique described in example 1, obtained the peptide corresponding to the sequence

FYTFFVEDQKEEDD

Verified the test regarding its inhibition capacity, under the same conditions as in example 1, but without sonication, the following results were obtained

Claims

Claims 1. New peptides corresponding to the amino acid sequence FYIFFVEDQKEEDD or any of the variants corresponding to the general formula X ^ IX ^ VEDQKX ^ DD (X, = T or F, X ₂ = C or F, X ₃ = E or F, X ₄ = E or F, X ₅ = E or F) with in vitro activity against HIV virus infection obtained by chemical synthesis from natural amino acids or by genetic engineering. 2. New peptides with specific antiviral activity according to claim 1, based on the TYICEVEDQKEE amino acid sequence existing in the CD4 protein in which T, C and E are replaced by F and added in the C-terminal region one to three (preferably two) D to increase its net negative charge. 3. New peptides with specific antiviral activity, according to claims 1 and 2, characterized by inhibiting in concentrations below 200 μmol / 1 more than 90% syncytium formation and 100% in concentrations greater than 300 μmolar. 4. Method of obtaining new peptides with specific antiviral activity, according to the claims 1 to 3, in which the amino acid sequence is achieved by chemical synthesis by the solid phase method controlled by the ninhydrin test. 5. Procedure for obtaining new peptides with specific viral activity, according to claims 1 to 3 by genetic engineering. 6. Application of the peptides according to claims 1 to 5 for therapeutic treatment in both subjects with AIDS as in asymptomatic seropositive individuals and in the prevention of infection in high-risk individuals and in those born to HIV-positive mothers.