Platelet-derived growth factor analogues

Details Platelet-derived growth factor analogues Platelet-derived growth factor analogues

1999-11-02

2002-02-26

Celsa, Bennett (Department: 1627)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S002600, C514S013800, C514S014800, C514S015800, C530S327000, C530S328000, C530S399000, C424S185100

Reexamination Certificate

active

06350731

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to platelet-derived growth factor (PDGF) analogues and their use as cell antiproliferative agents.
DESCRIPTION OF THE RELATED ART
Relevant background material is incorporated herein by reference in the text to the listed references in the appended bibliography.
Platelet-derived growth factor (PDGF) is a potent mitogen for connective tissue cells and promotes the proliferation of fibroblasts and smooth muscle cells (SMC) [333]. The growth factor is a 28-31 KD dimeric, highly basic (Pi=9.8-10) glycoprotein consisting of two highly homologous (up to 60% sequence homology) polypeptide chains which are the products of distinct genes. The gene products designated A (on chromosome 7) and B (on chromosome 22) are assembled to form either a disulphide-linked heterodimer (PDGF-AB) or a homodimer (PDGF-AA or PDGF-BB). Analysis of the PDGF present in human platelets reveals that it is a mixture of all three dimeric forms with AB being the predominant form (up to 70%) [10;12]. The human prot-oncogene, c-sis, which codes for the PDGF-B chain [21] has been identified as the human homologue of the v-sis oncogene of the transforming retrovirus, simian sarcoma virus. This oncogene codes for the protein p28 v-sis which has been identified as PDGF-BB [5].
The cloning and amino acid sequencing of the A and B chains of human PDGF have shown that both chains are synthesised as precursor molecules with hydrophobic leader sequences and both chains undergo proteolytic cleavage at the N-termini during maturation. The B chain is also processed at the C-terminal end [21;20].
The three isoforms of PDGF exert their biological effects by binding with different affinities to two different receptor types, denoted &agr; and &bgr;. Ligand binding induces dimerization of receptors; the A-subunit of PDGF binds to a-receptors whereas the B-subunit binds to both &agr;- and &bgr;-receptors [2].
When PDGF diner is treated with reducing agents, the protein loses its biological activity irreversibly, suggesting that the protein conformation is disturbed by reduction of critical disulphide bonds [16]. PDGF has 8 cysteine residues which are highly conserved between the two chains. Six residues are involved in 3 intra-molecular disulphide bonds: Cys-16—Cys-60, Cys-49—Cys-97 and Cys-53—Cys-99. The other two cysteine residues are involved in asymmetrical inter-molecular disulphide bonds, Cys-43—Cys-52 [11].
A systematic analysis of the abilities of different peptides, derived from the PDGF-B chain sequence, to compete with
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I-PDGF-BB for binding to PDGF &bgr;-receptors, has led to the identification of two regions in the B-chain corresponding to amino acid residues 35-40 and 78-83 that seem important for receptor binding. A peptide corresponding to the two sequences (ANFLVW—EIVRKKP) (SEQ ID NOS: 12 and 13, respectively) has been found to be effective as an antagonist for PDGF, although detailed analysis has shown the pure peptide to be less active [6].
Site-directed mutagenesis studies, using deletion and substitution mutants of PDGF-BB or of the homologous v-sis gene as well as PDGF-A/B chimeras, have also identified a number of amino acid residues which are important for the biological activity of PDGF. The region Ile-25—Phe-38 has been identified as a binding domain by site directed mutagenesis of the v-sis gene [9]. Amino acid residue Asn-34 has been found to be essential for the PDGF-B-like transforming efficiency of PDGF-A/B chimera [27]. Using a different functional assay, which selects for mutants with reduced binding to both receptor types, Ile-30 and, to a lesser extent, Arg-27 have been shown to be important [3]. Basic polypeptides such as polypeptides such as polylysin and protamine sulphate inhibit PDGF binding to its receptor, suggesting a role for ligand positive charge in the binding interaction. A receptor binding domain has been assigned to a region at the C-terminal end which is rich with basic amino acid, residues Lys-80—Cys-97 [39]. This region contains the sequence Val-78—Arg-79—Lys-80—Lys-81—Pro-82, (SEQ ID NO:14) which is conserved in both the A and B chains, and therefore may be involved in the binding of both chains to PDGF &agr;-receptor. A mutant PDGF-A chain in which the cationic sequence Arg-Lys-Lys has been replaced by the sequence Glu-Glu-Glu displays a marked reduction in both binding affinity for PDGF &agr;-receptor and mitogenic activity in fibroblast cells [7]. Initial studies with neutralizing monoclonal antibodies raised to PDGF-BB indicates that the segment between Thr-20 and Cys-43 represents a surface domain of PDGF-BB and contains amino acid residues involved in receptor binding [22].
Recently, the crystal structure of the homodimeric BB isoform of human recombinant PDGF has been determined [26]. The protein polypeptide chain is folded into two highly twisted anti-parallel pairs of &bgr;-strands and contains an unusual knotted arrangement of three intramolecular disulphide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites. The three loops are: loop I: Ile-25—Leu -38, loop II: Cys-53—Val-58 and loop III: Val-78—Lys-81.
Antibodies to PDGF would be extremely useful in the study of PDGF processing and biosynthesis. It has been difficult to make high avidity antibodies against PDGF, maybe because the molecule is conserved between species and only recently have monoclonal antibodies against PDGF, become available [22;34;12;38]. Rabbit and goat antisera to PDGF have been made to the two chains using protein purified from human platelets or recombinant protein or synthetic peptides, some showing chain specificity and neutralizing activity [28;17;13;37;30]. None of the antibodies raised to peptides however have been capable of recognising the native molecule or able to neutralize its biological activities.
PDGF has been implicated in many biological systems. Originally, the close similarity between PDGF and the transforming factor involved in SSV transformation led to the concept that over-production of the factor was involved in the development of human malignancies [14]. Examination of many tumour cell lines shows that the A and B chains are commonly expressed in such cell lines [15;24]. In general, aberrant expression of PDGF or of PDGF receptors is likely to be involved in the stimulation of the growth of certain tumours. In addition, over-activity of PDGF could also be part of the development of certain non-malignant disorders involving an excess of cell proliferation. Examples include atherosclerosis, where PDGF-induced stimulation of smooth muscle cell proliferation could contribute to the thickening of the intima of affected vessels [32], as well as chronic fibrotic processes, where PDGF could be involved in the stimulation of connective tissue cell proliferation. Ferns et al [8] showed that in a rat experimental model of angioplasty, polyclonal antibodies to PDGF administered intravenously inhibited smooth muscle cell accumulation in the intima of injured arteries, while administration of PDGF induced SMC proliferation in the media by 2-3 fold and, more significantly, increased SMC migration from the media to the intima by 20-fold [19].
However, PDGF does have a normal function. PDGF and PDGF receptors are expressed in embryonic tissues and in the placenta [23;18] which suggests a function for PDGF during development. A role for PDGF in neuronal development has also been proven [25] and PDGF and its receptors are present in the peripheral and central nervous systems [40;36]. PDGF is known to stimulate growth as well as chemotaxis of connective tissue cells and also chemotaxis of inflammatory cells, which suggests a role in wound healing [4;35]. Recently,

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