EP0589955A1 - Medicinal use of jacaline and fragments thereof, in particular for treating hiv-related diseases - Google Patents

Abstract

Use of jacaline, a major protein component of jackfruit seeds, its fragments or derivatives in the treatment of diseases linked to the HIV virus. Advantageously, a peptide having a biological activity equivalent to that of jacaline is used, comprising the following sequence (II): X V V V R S L T F K T N K K T, in which X represents an uncharged hydrophilic amino acid.

Description

USE OF JACALINE AND ITS FRAGMENTS AS A MEDICAMENT, IN PARTICULAR IN THE TREATMENT OF DISEASES RELATED TO HIV VIRUSES.

The present invention relates to fragments of jacaline.

It is also related to their use and to the use of jacaline as a medicament, in particular in the treatment of diseases linked to HIV viruses.

It also relates to pharmaceutical compositions containing these fragments and this protein.

The CD ^ surface glycoprotein of T cells acts as a cellular receptor for HIV viruses. Its interaction with the glycoprotein of the gp 120 viral envelope is probably one of the first steps in natural infection with the HIV virus.

It seems clear in vivo that the HIV virus escapes the effects of neutralizing antibodies because of the significant variability of the amino acid sequences of gp 120 in the various isolates of the HIV virus. However, despite this heterogeneity, until now, an interaction between the CD ^ and the virus seems mandatory to maintain a cycle of infection for all the characterized variants. Therefore, the inhibition of the fixation of the virus on the target cell by means of a protection of the CD 4 receptor has been one of the major strategies implemented to block the infection by HIV.

Jacaline is a major component protein of jackfruit seeds (Artocarpus heterophyllus). This protein has a quaternary structure combining α chains having an apparent molecular weight of approximately 11-12 kD and β chains composed of 20 to 21 amino acids. (Young et al, Arch. Biochem. Biophys, 270; 596-603, 1989).

The sequencing of these two chains has been carried out and their sequences have been published (Young et al. 1989 previously cited; Young et al., FEBS Lett., 282, 382-384, 1991).

The effect of jacaline has also been experimentally tested on various cell models. The article by HAMED et al. (J. Biosci. 1988, 13 (4),

419-424) concerns the study of the effect of jacaline on tumor cells injected into the peritoneal cavity of the mouse. The authors show that jacaline agglutinates these cells in vitro and inhibits their growth in vivo. No therapeutic application is mentioned.

Jacaline has also been tested for diagnostic purposes on cells of patients affected at various stages by the HIV virus (Pineau et al. Aids, 1989, 3: 659-663). It has been shown in vitro that jacaline specifically activates CD ^ lymphocyte cells. The responses to these activations are very different depending on the stage of the disease of the patients studied, which suggests, according to the authors, the possibility of correlating, for diagnostic purposes, the activation of CD ₄ ⁺ with the subsequent development of AIDS.

This study was followed by an article by PINEAU et al. (Clin. Exp. Immunol., 1990, 80: 420-425) which specifies the in vitro activation of different cell types of blood cells by jacaline. These publications, if they indicate a potential use of jacaline for diagnostic purposes, do not in any way mention the possibility of using jacaline for therapeutic purposes.

The applicant therefore set out to study the effects of jacaline on HIV viruses.

However, the therapeutic use of proteins the size of jacaline can have drawbacks. In fact, the human organism may not perfectly accept such molecules, or it may degrade them, thereby causing a significant reduction in activity. Thus, the applicant has also endeavored to find fragments of jacaline possessing activities against these viruses and presenting no drawbacks in human therapy. The Applicant has surprisingly shown that jacaline and peptide fragments of this protein specifically inhibit the infection of lymphocytes by HIV viruses, and in particular the HIV-1 virus.

The present invention therefore has for first object a peptide having a biological activity equivalent to that of jacaline. Advantageously, this peptide is a fragment or a derivative of a fragment of jacaline.

It can preferably comprise in its sequence at least one phenylalanine residue and at least one lysine residue separated by three or four other amino acids.

This peptide can also comprise a fragment of the jacaline region interacting with the CD ^ receptor.

It can also have structural homology with the gp 120 protein of the HIV1 virus.

Advantageously, said peptide comprises the following sequence (I):

V V V R S L T F K T N K K T (I) or the following sequence (II): X V V R S L T F K T N K K T (II) in which X represents an uncharged hydrophilic amino acid, in particular aspargine, glutamine, threonine, serine or one of their derivatives.

Such a peptide carrying hydrophilic amino acids uncharged at its NH ₂ end has the notable advantage of having better solubility in aqueous media.

The peptides which are the subject of the present invention also have the property of not agglutinating the cells, unlike jacaline. The present invention also includes in its object, the peptides corresponding to the formulas mentioned above and in which a small percentage, of maximum about 15%, of amino acids not essential to the biological activity of the peptide has been substituted. It also includes the biologically active derivatives and fragments of the peptides previously described.

Another subject of the present invention is the use of jacaline, its derivatives or its fragments and in particular one or more of the peptides described above or one or more of their fragments or one or more of their derivatives. biologically active for the manufacture of a medicament for the treatment of diseases linked to HIV viruses. This use has the advantage of being suitable for all strains of the HIV virus and in particular of the HIV1 virus. Indeed, as indicated above, the binding of the virus to the CD _* receptor is an essential step in the infection of the cells. Another advantage of this use lies in the fact that, unlike certain anti-CD ₄ monoclonal antibodies, it does not inhibit normal lymphocyte functions and therefore does not suppress normal immune response of CD ₄ ⁺ cells in HIV-positive patients. not infected with the virus. The action of jacaline therefore does not in any way diminish the normal physiological functions of CD ₄ ⁺ lymphocytes.

The jacaline subunits or their biologically active derivatives or fragments can also be used for this purpose.

These subunits have been described by Young et al. (1991 and 1989, previously cited).

The present invention also relates to a pharmaceutical composition containing an effective amount of jacaline, its derivatives or its fragments and in particular one or more of the peptides described above, or one or more of their fragments or one or more of their derivatives in combination with one or more compatible and pharmaceutically acceptable adjuvants. Such a composition can also comprise or consist of the subunits of jacaline, their fragments or their biologically active derivatives. It is especially intended for the treatment of diseases linked to HIV viruses.

The present invention also relates to a medicament containing jacaline or its fragments and in particular one or more of the peptides described above and / or α or β subunits of jacaline, their fragments or their biologically active derivatives.

The peptides which are the subject of the present invention and the jacaline can be obtained by any method known to a person skilled in the art, and preferably by chemical synthesis from amino acids. However, such peptides can also be obtained by synthetic methods using eukaryotic cells or fractions of these cells or bacteria, or by genetic engineering.

A person skilled in the art will advantageously find the description of such methods in Molecular Cloning, a Laboratory manual (Sambrok et al. 1989, Cold Spring Harbor Laboratory Press, New York). It should be noted that jacaline can also be obtained by the method described by Aucouturier et al. (Molec. Immunol. 24, 503, 1987).

The therapeutic treatment of patients suffering from diseases linked to HIV viruses can be carried out by administration of one or more of the peptides described above, in particular by oral, parenteral or intravenous administration.

The present invention is illustrated by the following examples in which: FIG. 1 is an autoradiogram showing the CD4 antigen precipitated from CEM cells solubilized and detected by an anti-CD4 monoclonal antibody labeled with iodine 125; in well C, CD4 was immunoprecipitated by the anti-CD4 antibody / protein A sepharose system; in well B, CD4 was precipitated by jacaline attached to sepharose beads. Well A represents a negative control made with protein A alone.

Figure 2 illustrates the interaction between jacaline an IgA and an IgG. and the recombinant soluble CD ₄ adsorbed on nitrocellulose.

It also shows, in accordance with data from the literature, the interaction of jacaline with IgA and its non-interaction with IgG. Figure 3 illustrates the effect of different concentrations of jacaline on HIV infection of CD ₄ ⁺ cells.

FIG. 4 is an autoradiogram corresponding to a nitrocellulose sheet on which drops of CD ₄ and gp 120 have been deposited at various concentrations, and which has been brought into contact with a solution containing labeled jacaline.

FIG. 5 represents the influence on the reverse transcriptase activity (on the ordinate) expressed in cpm / ml of different substances including peptides according to the invention, as a function of the number of days after infection of cells (on the abscissa).

FIG. 6 is a diagram illustrating the effect of the peptide of sequence II according to the invention on the response of blood mononuclear cells to a mitogenic agent (OKT3) measured by the incorporation of tritiated thymidine (on the ordinate).

Figure 7A is a photograph of an SDS-PAGE gel of jacaline (a) and purified subunits (b and c). FIG. 7B is a diagram illustrating the effect of the jacaline subunits on the infection of cells by the HIV1 virus (reverse transcriptase activity on the ordinate).

FIG. 7C is a gel illustrating the result of the PCR amplification of DNA from cells infected with the HIV1 virus and treated in particular with the jacaline subunits. EXAMPLE 1

Highlighting the interaction between jacaline and _____ ._.

CEM cells (leukemic T line) are cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% mixture of antibiotics and 1% glutamine. 10 ° CEM cells are dissolved in 1 ml of buffer

TSA (10 mM Tris buffer, pH 8, 140 mM NaCl, 0.025% NaN3) containing 1% NP40 detergent and 1 mM phenyl methyl sulfonyl fluoride (PMSF) for 1 h at 4 ° C. After treatment of the cell lysates with protein A-sepharose alone, fractions of 1 ml of supernatant are treated either with jacaline linked to sepharose beads (Pierce) or with 30 μg / ml of anti-CD4 monoclonal antibodies (1: 1 mixture of F101-69 and F142-63 supplied by C. Carrière, Sanofi, Montpellier, France) and protein A-sepharose. After 12 hours at 4 ^° C with gentle agitation, the samples were centrifuged and the pellets washed successively with TSA buffer, phosphate buffer (PBS) pH 7.2 Tris buffer (50 mM, pH 6.8). After centrifugation, the pellets are resuspended in Laemmli buffer and heated to 100 ° C for 10 minutes. The samples are then electrophoresed and electrotransfered onto a nitrocellulose sheet. The non-specific saturation of the nitrocellulose is carried out in PBS / 0.05% tween 20. The nitrocellulose sheet is then incubated in PBS containing anti-CD4 monoclonal antibodies labeled with iodine 125. Groups radioactive are visualized by autoradiography on Kodak X-Omat AR films.

The autoradiogram of FIG. 1 shows in well A that the protein A-sepharose alone does not precipitate CD4; well B indicates that jacaline-sepharose precipitates, as in the immunoprecipitation of well C, CD4, thereby demonstrating the interaction of jacaline with CD4. EXAMPLE 2-

Fixation of jacaline on the recombinant soluble CD 4.

Different amounts (1; 0.5; 0.25 and 0.1 μg) of sCD4 (supplied by Dr. Klatzmann, Pitié Salpétrière hospital, Paris, France), human IgA and human IgG (Sigma) are adsorbed on a nitrocellulose sheet. . After non-specific saturation, the nitrocellulose is incubated in a solution of jacaline labeled with iodine 125. The molecules interacting with jacaline are visualized by autoradiography.

Figure 2 shows that jacaline binds to CD4; it also shows that, according to the data in the literature, it interacts with IgA and not with IgG, thus highlighting the specific nature of its interaction with CD4. EXAMPLE 3 Inhibition of infection by the HIV virus.

5.10 ⁵ EMFs are incubated for 30 minutes at 4 ^* C with different concentrations of jacaline in a volume of 100 μl in a 96-well plate, then brought into contact for 30 minutes at 4 ^* C with 100 μl of a solution diluted to I / IO3 of a suspension of HIV1-BRU corresponding to 100 TCID- ₀ . The cells are then washed 5 times with 200 μl of culture medium and then recultivated at the concentration of 5.105 cells per ml in a 24-well plate in a volume of 1 ml in the presence of jacaline. Viral production is evaluated twice a week by measuring transcriptase activity reverse in the culture supernatant. Control infections are carried out under the same conditions but in the absence of jacaline.

The different concentrations of jacaline used are 0.01 μg / ml, 0.1 μg / ml, and 1 μg / ml.

FIG. 3 summarizes the results obtained and shows that jacaline delays in a dose-dependent manner the production of virions by CEM cells and exerts a protective effect at a concentration of 1 μg / ml. EXAMPLE 4:

Study of the binding of jacaline on the -CD, recombinant ι and the recombinant σp 120.

5 μl of solutions at 0.2 μg / ml and 0.1 μg / ml of recombinant sCD4 or of recombinant gpl20 are deposited on a sheet of nitrocellulose.

This leaf is then incubated in a jacaline solution marked with "I.

The interaction between jacaline and the two proteins is demonstrated by autoradiography. Figure 4 shows that jacaline does not interact significantly with gpl20, suggesting that inhibition of HIV infection does not result from an interaction between jacaline and gpl20. EXAMPLE 5 Effect of jacaline on the synthesis of interleukin 2

The effect of jacaline at 50 μg / ml in combination with phorbol myristate acetate on the induction of interleukin-2 synthesis in two JURKAT cell lines with CD ₂ ⁺ / CD ₃ ~ / CD phenotypes respectively ₄ ⁺ and CD ₂ ⁺ / CD- ~ / CD ₄ ~ has been studied.

It has been found that despite the absence of CD3 receptors, jacaline is capable of inducing the synthesis of interleukin 2, if the CD ₄ receptor is present.

On the other hand, in CD ₄ ~ cells, there is no production of interleukin 2 during combined stimulation. EXAMPLE 6

Effect of the peptide of sequence (II) (X = Asn) on the infection of CEM cells by the HIV 1 virus.

5.10 ⁵ CEM cells (leukemic T cell line) are incubated for 30 min. at 4 ° C with different molecules in a volume of 100 μl in a 96 microwell plate, then brought into contact for 30 min at 4 ° C with 100 μl of a solution diluted to 1/1000 of a suspension of HIV1-BRU corresponding to 100 TCID50. The cells are then washed five times with 200 μl of culture medium and then recultivated at a concentration of 5.10 cells per ml in a 24-well plate in a volume of 1 ml in the presence of the various substances tested.

Viral production is evaluated twice a week by measuring the reverse transcriptase activity in the culture supernatant.

The different substances tested are:

- an anti-CD ₄ antibody, 1OKT4A (2 μg / ml),

- a fragment of cholecystokinin (CCK) (sequence 26 to 33: DYMGWMDF-NH2), at a concentration of 500 μg / ml,

- the peptide of sequence II in which X is asparagine, at a concentration of 10 μg / ml, 100 μg and 500 μg / ml.

FIG. 5 shows that peptide II inhibits in a dose-dependent manner the infection induced by HIV-1. At 100 μg / ml, the infection is delayed while at 500 μg / ml the infection is completely inhibited.

The reference peptide (CCK fragment) is completely inactive. 0KT ₄ A used as a positive control shows total inhibition at 2 μg / ml. EXAMPLE 7:

Effect of the peptide of sequence II (X = Asn) on the mitoqene response of blood cells induced by OKT-. Peripheral blood mononuclear cells were isolated and cultured as described by FAVERO et al. (Cell. Immunol., 122, 307-318, 1989), in the presence or absence of OKT3 (Orthodiagnostic System) at a concentration of 60 μg / ml, in the absence (A) or in the presence of 100 μg / ml (B) or 500 mg / ml of the peptide of sequence II according to the invention.

After 72 hours of culture, the mitogenic response is evaluated by the incorporation of tritiated thymidine.

The diagram in Figure 6 illustrates the results of these experiments.

These results show that the peptide of sequence II according to the invention does not affect the response of the mononuclear cells of the peripheral blood vis-à-vis the mitogens. EXAMPLE 8 Effect of jacaline subunits on infection with the HIV virus.

The two types of subunits were isolated by SDS-page preparative gels according to AUCOUTURIER et al. (Molec. Immunol. 24, 503-511, 1987) followed by electroelution. The SDS is eliminated by chromatography on a column of Extracti D gel (Pierce) balanced with 0.05 M of Tris buffer (pH 9). The fractions containing the subunits are concentrated by lyophilization and stored at -80 "C.

Figure 7A shows the SDS-PAGE gel. Lanes a, b and c correspond respectively to the native jacaline, the purified high molecular weight subunit and the purified low molecular weight subunit.

The effect of jacaline subunits has been tested on HIV infection of CEM cells. These cells are incubated with OKT ₄ (2 μg / ml), jacaline or its subunits (10 μg / ml) then infected, washed abundantly and cultured without any other additive. Reverse transcriptase activity is measured in the supernatant after six days.

The diagram in FIG. 7B illustrates the results obtained. This figure shows that the two subunits have inhibitory actions against HIV infection, comparable to that of jacaline.

PCR analysis of HIV1 DNA in infected cells in the presence of jacaline subunits was performed.

The production of DNA HIV1 was evaluated as follows. The cells are washed 4 times in 5 ml of PBS buffer. After centrifugation, the pellets are suspended in 50 μl of water and heated to 93 ° C. in the presence of 0.005% of Tween 20, 0.05% of NP40, 0.001% of gelatin, 20 μg of each of the Vpr primers (TGAAACTTATGGGGATACTT- GGG-3 ', and

CTAGGATCTACTGGCTCCATTTC-5 '), and two units of Taq polymerase.

The mixture is subjected to a first denaturation for 5 minutes at 92 C and then ^β to hybridization by the primers for 3 minutes at 55 ^° C and polymerization for 3 minutes at 72 ° C..

30 additional cycles were carried out under the following conditions: denaturation for 7 seconds at 92 "C, hybridization for 30 seconds at 55 ^* C, polymerization for 3 minutes at 72 ^* C (15 minutes for the last cycle).

The amplified product (160 base pairs) is analyzed by electrophoresis on a 1% agarose gel, fixed for two hours on a nylon sheet according to Cuny et al. (Anal. Biochem., 193, 45-48, 1991) and hybrid with an HIVl env probe labeled with 32 P.

The hybridization products were visualized by autoradiography. Figure 7C shows the final image obtained.

Lane 3 corresponds to untreated CEM cells, lane 4 corresponds to cells treated with saturated concentrations of anti-CD ₄ antibody (0KT4 A), lanes 5, 6 and 7 correspond to cells infected with the HIVl virus and treated respectively by jacaline (lane 5), the high molecular weight subunit (lane 6) or the low molecular weight subunit (lane 7) at concentrations of 10 μg / ml.

Uninfected cells (lane 1) and cells chronically infected with the HIV1 BRU virus (lane 2) were used as controls.

Claims

1. Peptide with biological activity equivalent to that of jacaline.

2. Peptide according to claim 1, characterized in that it is a fragment or a derivative of fragment of jacaline.

3. Peptide according to one of claims 1 and 2, characterized in that it comprises in its sequence, at least one phenylalanine residue and at least one lysine residue separated by three or four other amino acids

4. Peptide according to one of claims 1 to 3, characterized in that it comprises a fragment of the jacaline region interacting with the CD ₄ receptor.

5. Peptide according to one of claims 1 to 4, characterized in that it has a sequence homology with the protein gpl20.

6. Peptide according to one of claims 1 to 5, characterized in that it comprises the following sequence (I):

V V V R S L T F K T N K K T (I)

7. Peptide according to one of claims 1 to 5, characterized in that it comprises the following sequence (II):

X V V V R S L T F K T N K K T (II) in which X represents an uncharged hydrophilic amino acid.

8. Peptide according to claim 7, characterized in that said hydrophilic amino acid is asparagine, gluta ine, threonine, serine or one of their derivatives.

9. Biologically active derivative or fragment of the peptides according to one of claims 1 to 8.

10. Use of jacaline, its fragments or its derivatives for the manufacture of a medicament for the treatment of diseases linked to the HIV virus.

11 Use according to claim 10 of a peptide according to one of claims 1 to 9 or one of the jacaline subunits, or one of their derivatives or fragments biologically active for the manufacture of a medicament for the treatment of diseases linked to HIV viruses.

12. Medicinal product containing jacaline, one or more of its fragments or one or more of its derivatives.

13. Medicament according to claim 12 containing a peptide according to one of claims 1 to 9 or one of the subunits of jacaline or one of their biologically active derivatives or fragments.

14. Medicament according to one of claims 12 and 13 for the treatment of diseases linked to HIV viruses.

15. Pharmaceutical composition, characterized in that it contains an effective amount of jacaline, one or more of its fragments or one or more of its derivatives in combination with one or more diluents or compatible and pharmaceutically acceptable adjuvants.

16. Pharmaceutical composition according to claim

15, containing an effective amount of a peptide according to one of claims 1 to 9 or of one of the jacaline subunits or one of their biologically active derivatives or fragments, in combination with one or more compatible diluents or adjuvants and pharmaceutically acceptable.

17. Composition according to one of claims 15 and

16, characterized in that it is intended for the treatment of diseases linked to HIV viruses.