Small-molecule inhibitors of integrin α ₂ β ₁ that prevent pathological thrombus formation via an allosteric mechanism

Abstract

There is a grave need for safer antiplatelet therapeutics to prevent heart attack and stroke. Agents targeting the interaction of platelets with the diseased vessel wall could impact vascular disease with minimal effects on normal hemostasis. We targeted integrin ␣2␤1, a collagen receptor, because its overexpression is associated with pathological clot formation whereas its absence does not cause severe bleeding. Structure-activity studies led to highly potent and selective small-molecule inhibitors. Responses of integrin ␣2␤1 mutants to these compounds are consistent with a computational model of their mode of inhibition and shed light on the activation mechanism of I-domain-containing integrins. A potent compound was proven efficacious in an animal model of arterial thrombosis, which demonstrates in vivo efficacy for inhibition of this platelet receptor. These results suggest that targeting integrin ␣2␤1 could be a potentially safe, effective approach to long-term therapy for cardiovascular disease.I-domain ͉ platelet ͉ thrombosis ͉ integrin ͉ alpha(2) beta(1)

Inhibition of Integrin α2β1 Ameliorates Glomerular Injury

“…We used a high-affinity small-molecular weight inhibitor, namely the thiazolidine-modified compound 15, that was originally designed to inhibit integrin a2b1 on platelets by locking the integrin a2b1 in the inactive low-affinity conformation. 16 Compound 15 is a member of a family of nonpeptide inhibitors that can block platelet adhesion to collagen I under both static conditions and flow, inhibit thrombus formation in a mouse model of arterial damage, and block integrin a2b1-mediated angiogenesis in vivo in a zebrafish model. 16,18 Another integrin a2b1 inhibitor, namely the sulfonamide derivative BTT-3016, prevented platelets aggregation in an integrin a2b1-dependent manner and reduced thrombus formation after vascular injury.…”

“…16 Compound 15 is a member of a family of nonpeptide inhibitors that can block platelet adhesion to collagen I under both static conditions and flow, inhibit thrombus formation in a mouse model of arterial damage, and block integrin a2b1-mediated angiogenesis in vivo in a zebrafish model. 16,18 Another integrin a2b1 inhibitor, namely the sulfonamide derivative BTT-3016, prevented platelets aggregation in an integrin a2b1-dependent manner and reduced thrombus formation after vascular injury. 23 Targeting integrin a2b1 is considered safe, because although its overexpression is associated with pathologic clot formation, its deletions do not cause severe pathologic bleeding 24,25 and its inhibition prolongs bleeding time in a manner comparable with aspirin.…”

“…Selective high-affinity small-molecule inhibitors of integrin a2b1, such as the thiazolidine-modified compound 15, have been designed and shown to allosterically inhibit integrin activation and adhesion of human platelets to fibrillar collagen and block thrombus formation in a murine model of arterial damage. 16 Additionally, another thiazolidinemodified small-molecule inhibitor of integrin a2b1, SMI496, was shown to block collagen-induced angiogenesis in vitro and interfere with zebrafish embryo angiogenesis in vivo. 18 To assess whether inhibition of integrin a2b1 by compound 15 results in decreased collagen IV synthesis, WT and integrin a2KO mesangial cells were incubated with this compound, and the levels of collagen were analyzed after 24 hours.…”

“…For the experiments with the small-molecule integrin a2b1 inhibitor thiazolidine-modified compound 15 (synthesized as previously described 16 ), WT BALB/c mice received a single intravenous injection of adriamycin (10 mg/kg), and then, they were either treated with PBS or treated every second day with compound 15 (20 mg/kg intraperitoneal injection). Mice were sacrificed 2 weeks after adriamycin injection.…”

“…15 In both models, the integrin a2-null mice developed significantly less proteinuria and reduced glomerulosclerosis. To confirm the beneficial effects of inhibiting integrin a2b1-dependent signaling in the setting of glomerular injury and more importantly, to translate these findings to a more clinically relevant setting, we pharmacologically inhibited integrin a2b1 with a selective high-affinity, nonpeptide small-molecule inhibitor 16 immediately after injecting BALB/c mice with adriamycin. Treatment with this inhibitor significantly reduced proteinuria, glomerular injury, and collagen deposition.…”