RANTES derived peptides with anti-HIV activity

Details RANTES derived peptides with anti-HIV activity RANTES derived peptides with anti-HIV activity

2001-06-22

2004-03-23

Mertz, Prema (Department: 1646)

Drug, bio-affecting and body treating compositions

Lymphokine

C514S002600, C514S008100, C514S013800, C530S326000

Reexamination Certificate

active

06709649

ABSTRACT:

The present invention relates to RANTES-derived peptides having inhibitory activity against the human immunodeficiency virus (HIV).
The peptides of the invention are useful for the treatment of diseases which are connected with the infection of viruses like HIV-1, other primate-lentiretroviruses (HIV-2, SIV) and other viruses which use chemokine receptors to bind the cellular surface and/or to penetrate the target cell, as well as for the treatment of all the diseases, like the allergic or autoimmune diseases, in the pathogenesis of which chemokines play an important role.
BACKGROUND OF THE INVENTION
The term chemokine is used to identify a family of chemotactic cytokines characterized by a high degree of genetic, structural and functional similarity (Immunol. Today 1993, 14:24).
Most known chemokines are grouped in two main families referred to as C-X-C and C-C, depending on the configuration of a conserved motif of two cysteine in their sequence (Ann. Rev. Immunol. 1994, 55:97-179).
Chemokines are important mediators of the inflammatory response which act through the recruitment of specific cellular populations of the immune system in the inflammatory site; the C-X-C chemokines are generally active on neutrophil granulocytes while the C-C chemokines are active on eosinophil and basophil granulocytes, on limphocytes and monocytes.
RANTES, MIP-1&agr; and MIP-1&bgr; are C-C chemokines which have been proposed as possible mediators of autoimmune and allergic diseases.
Recently, a specific antiviral effect against primate lentiretrovirus has been described for those three chemokines (Science, 1995, 270:1811-1815).
RANTES is the most potent among C-C chemokines which inhibit the HIV infection. This chemokine bonds to the CCR5 receptor, which is the main membrane co-receptor for HIV-1, in that it is used by most viral strains present in the population and preferably sexually transmitted. Said receptor is therefore a primary target for possible therapeutical strategies, above all during the asymptomatic phase of HIV disease. However, the therapeutic use of natural chemokines is hampered by their pro-inflammatory activity, in that most chemokines are involved in leucocyte recruitment at the inflammation and infection sites, and in their functional activation.
At the moment a preliminary knowledge exists of the domains involved in the pro-inflammatory activity of some chemokines, but not of the domains involved in the antiviral activity. A number of recent studies have suggested that an element crucial for the chemokineinduced receptor activation is located at the molecule's NH
2
-terminus (J. Biol. Chem. 1991; 266:23128-23134; Biochem. Biophys. Comm. 1995, 211:100-105). Actually, a preliminary study (Nature, 1996, 383:400) and a more detailed study (Science; 1997, 276-282), both recently published, have shown that RANTES-chemokine analogues modified at the NH
2
terminus (through the deletion of 8 amino acids, or through the covalent bond of a complex chemical radical [amino-oxy-pentane or AOP], respectively) maintain the anti-HIV activity even though they do not induce chemotaxis in vitro or they induce it to a very low extent.
Peptides corresponding to the sequences 7-68 to 10-68 of RANTES are disclosed in WO97/44462. RANTES mutants such as Leu-RANTES and Met-RANTES are disclosed in WO96/17935 and WO98/13495. However, in therapeutical applications, the use of small molecules or peptides is preferred, compared with the whole protein, although in the recombinant form, for a number of reasons, such as easiness of synthesis and possibility of minimizing any side-effects caused by the molecule regions which are not useful or even harmful. A number of examples of peptides in the pre-clinical phase for anti-HIV therapy can, in fact, be found in literature: 1) Judice et al., PNAS 94:13426,1997, disclose structurally rigid peptides deriving from the gp41 sub-unit of HIV envelope, which inhibit the fusion of the cell membraneo 2) Prieto et al., AIDS Res Hum Retroviruses 12:1023,1996, disclosed modified peptides (benzyl-conjugated) deriving from CD4; or 3) Robinson et al., J Leuk Biol 63:94, 1998, disclose the activity of Indolicin 13-mer, a natural peptide of bovine origin, neutrophil and capable of inhibiting HIV virus at doses comprised from 60 to 100 &mgr;M; 4) Heveker et al., Curr Biol 8:369, 1998: describe peptides deriving from the N-terminus of SDF-1, capable of inhibiting HIV and which also lack the pro-inflammatory activity when the first two amino acids are deleted.
DISCLOSURE OF THE INVENTION
It has now been found that RANTES-derived peptides are particularly active in inhibiting HIV viral infection.
The peptides of the invention have 12 to 30 amino acids with sequence identical to a portion of at least 5 consecutive aminoacids of the sequence 9-38 of RANTES.
The peptides of the invention consist of either natural amino acids of the D or L series or of modified or “non-protein ” amino acids.
Preferably, the peptides of the invention have from 15 to 35 amino acids, more preferably from 18 to 25 amino acids.
Preferably, the peptides of the invention have a sequence derived from or identical to at least 10 consecutive amino acids of the sequence 9-38 of RANTES, more preferably derived from or identical to at least 12 amino acids and even more preferably derived from or identical to at least 15 amino acid, the other amino acids in the peptide sequence deriving from conservative substitutions of the natural amino acids in the native sequence of RANTES.
The sequence identity of the peptides of the invention with the above mentioned RANTES regions, for example the sequence comprised between the amino acid in position 9 and the amino acid in position 38 of the RANTES native sequence, is preferably of at least 50%, preferably at least 80% and more preferably of at least 90%.
The substitutions of the natural amino acids of the native sequence are preferably of conservative type, both with other natural amino acids and with non-proteic amino acids. “Conservative substitution ” herein means, for example, the substitution of a hydrophobic amino acid with another hydrophobic amino acid, of a basic amino acid with another basic amino acid and so on.
“Derived from ” means that the native sequence may be further modified by substituting the natural amino acid with the corresponding amino acid of the D series or with non-protein amino acid and/or by inverting the sequence from the carboxy-terminus to the N-terminus and/or by forming dimers through cysteine disulfide bonds and/or by single deletion of amino acids of the native sequence. The invention also refers to peptides having two or more of the above characteristics sequences or domains linked by a suitable linker, e.g. an hydrophilic linker or a metabolic resistant linker. Said derivatives are obtainable according to known methods and criteria.
The invention also provides derivatives of said peptides, chemically modified in order to increase their in vivo stability.
According to a further aspect, the invention provides chimeric proteins which are obtained through conventional techniques by inserting the antiviral domains described above into proteins showing the desired biological characteristics.
Finally, the invention provides antiviral, antiinflammatory and antiallergic pharmaceutical compositions containing the above defined peptides or proteins as the active ingredient.
Preferred peptides of the invention have the following general formula (l):
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17 -X18-X19-X20
wherein:
X1=H, Ace, or any amino acid both N-acylated and non-acylated, or a dipeptide of sequence Y1-Y2 both N-acylated and non-acylated in which Y1 and Y2, are the same or different and represent any amino acid;
X2=any hydrophobic amino acid;
X3=Ala, Pro, Val, Thr, lie, thioproline, hydroxyproline;
X4=any hydrophobic amino acid;
X5=any hydrophobic amino acid;
X6=Ala, Pro, Val, Thr, Ile, thioproline, hydroxyproline;
X7=any basic amino acid;
X8=Pro, Val, Th

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