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We are currently witnessing a decline in the development of efficient new anticancer drugs, despite the salient efforts made on all fronts of cancer drug discovery. This trend presumably relates to the substantial heterogeneity and the inherent biological complexity of cancer, which hinder drug development success. Protein-protein interactions (PPIs) are key players in numerous cellular processes and aberrant interruption of this complex network provides a basis for various disease states, including cancer. Thus, it is now believed that cancer drug discovery, in addition to the design of single-targeted bioactive compounds, should also incorporate diversity-oriented synthesis (DOS) and other combinatorial strategies in order to exploit the ability of multi-functional scaffolds to modulate multiple protein-protein interactions (biological hubs). Throughout the review, we highlight the chemistry driven approaches to access diversity space for the discovery of small molecules that disrupt oncogenic PPIs, namely the p53-Mdm2, Bcl-2/Bcl-xL-BH3, Myc-Max, and p53-Mdmx/Mdm2 interactions. Molecules

Inhibition of platelet aggregation is indispensable for the treatment of acute arterial thrombotic episodes. We have previously reported the synthesis of a highly constrained cyclic peptide, that incorporates the -CDC- sequence, (S,S) PSRCDCR-NH(2), which potently inhibits aggregation and fibrinogen binding to human platelets in vitro. We have tested the safety and efficacy of the peptide on the electrically induced carotid artery thrombosis experimental rabbit model. The peptide's effects on carotid blood flow, thrombus weight, in vitro and ex vivo platelet aggregation, and bleeding and hemostatic parameters were evaluated. The peptide was administered via the femoral vein. Carotid blood flow was continuously monitored for 90 min after electrical thrombus formation. The peptide, at 12 mg/kg, prevented total artery occlusion and significantly preserved carotid artery's patency compared with placebo and eptifibatide. Furthermore, (S,S) PSRCDCR-NH(2) administration at 12 mg/kg reduced thrombus weight, whereas it inhibited ex vivo ADP, arachidonic acid (AA) and collagen-induced platelet aggregation. Moreover (S,S) PSRCDCR-NH(2) at 12 mg/kg presented significantly higher inhibitory effects on AA and collagen-induced ex vivo platelet aggregation compared to eptifibatide. The peptide at any dose did not affect the coagulation cascade, the bleeding times or the hemostatic response of the animals. Thus highly constrained cyclic peptides like (S,S) PSRCDCR-NH(2) that incorporate the -CDC- motif and fulfil certain conformational criteria represent novel compounds that potently inhibit thrombus formation, ex vivo platelet aggregation and carotid artery occlusion superiorly to other non-RGD peptides, such as YMESRADR, without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

The platelet integrin receptor alpha(IIb)beta(3) plays a critical role in thrombosis. We have shown previously that the octapeptide YMESRADR, corresponding to sequences 313 to 320 of the human alpha(IIb) subunit, inhibits human platelet activation and fibrinogen binding to alpha(IIb)beta(3), possibly interacting with the ligand. We investigated the effect of YMESRADR on electrically induced carotid artery thrombosis in New Zealand white rabbits. Peptide was administered via the femoral vein, starting 60 min before and continuing for 90 min after the electrical stimulation. Carotid blood flow was monitored for 90 min after the electrical stimulation. The peptide effects on platelet aggregation, in vitro and ex vivo, and on various coagulation, bleeding, and hemostatic parameters were evaluated. YMESRADR significantly inhibited rabbit platelet aggregation in vitro in a dose-dependent manner. It is important that peptide administration in vivo, at doses ranging from 3 to 15 mg/kg, prolonged the duration of the patency of the carotid artery, and no artery occlusion was observed until the end of the study (90 min after electrical stimulation). Furthermore, YMESRADR administration reduced platelet aggregation ex vivo and thrombus weight; however, these reductions reached statistical significance, compared with the control group, at the peptide doses of 12 and 15 mg/kg. YMESRADR did not affect any coagulation parameter studied and the hemostatic response observed in control animals. Thus, YMESRADR represents a novel antiplatelet agent that can inhibit thrombus formation effectively and carotid artery occlusion without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

Platelet-activating factor (PAF), the potent phospholipid mediator of inflammation, is involved in atherosclerosis. Platelet-activating factor-acetylhydrolase (PAF-AH), the enzyme that inactivates PAF bioactivity, possesses both acetylhydrolase and transacetylase activities. In the present study, we measured acetylhydrolase and transacetylase activities in human atherogenic aorta and nonatherogenic mammary arteries. Immunohistochemistry analysis showed PAF-AH expression in the intima and the media of the aorta and in the media of mammary arteries. Acetylhydrolase and transacetylase activities were (mean +/- SE, n = 38): acetylhydrolase of aorta, 2.8 +/- 0.5 pmol/min/mg of tissue; transacetylase of aorta, 3.3 +/- 0.7 pmol/min/mg of tissue; acetylhydrolase of mammary artery, 1.4 +/- 0.3 pmol/min/mg of tissue (P < 0.004 as compared with acetylhydrolase of aorta); transacetylase of mammary artery, 0.8 +/- 0.2 pmol/min/mg of tissue (P < 0.03 as compared with acetylhydrolase of mammary artery). Lyso-PAF accumulation and an increase in PAF bioactivity were observed in the aorta of some patients. Reverse-phase HPLC and electrospray ionization mass spectrometry analysis revealed that 1-O-hexadecyl-2 acetyl-sn glycero-3-phosphocholine accounted for 60% of the PAF bioactivity and 1-O-hexadecyl-2-butanoyl-sn-glycerol-3-phosphocholine for 40% of the PAF bioactivity. The nonatherogenic properties of mammary arteries may in part be due to low PAF formation regulated by PAF-AH activity. In atherogenic aortas, an imbalance between PAF-AH and transacetylase activity, as well as lyso-PAF accumulation, may lead to unregulated PAF formation and to progression of atherosclerosis. Journal of Lipid Research

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

The αIIb cytoplasmic domain of platelet integrin αIIbβ3 contains an unorganized acidic membrane-distal (1000)LEEDDEEGE(1008) region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000-1008) inhibits platelet aggregation induced by thrombin or by pal-K-989-995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain (989)KVGFFKR(995). We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-(1000)LEEDDE(1005) (pal-K-1000-1005) and palmitoyl-K-(1005)EEGE(1008) (pal-K-1005-1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000-1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000-1008, inhibited the association of talin with αIIbβ3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000-1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbβ3.