Abstract
Vascular damage due to trauma or disease exposes circulating platelets to collagen in the subendothelial matrix. This is a critical event in the formation of a hemostatic plug or an occluding thrombus because collagen is not only a substrate for platelet adhesion but is also a strong platelet agonist. Platelets possess two physiologic collagen receptors: glycoprotein VI, a member of the immunoglobin superfamily, and the integrin α2β1. To design small molecule inhibitors of the interaction of platelets with collagen, we focused on α2β1 as a target because murine models of α2β1 deficiency display normal bleeding times and only a slight decrease in platelet activation by collagen and because the small number of reported patients with congenital α2β1 deficiency demonstrated only a mild bleeding diathesis. Thus, α2β1 antagonists could be effective anti-thrombotic agents with minimal toxicity, especially when combined with other anti-platelet drugs. We have developed a class of compounds that target the I-like domain of the β1 subunit, an allosteric site that regulates collagen binding to α2β1 by preventing the conversion of α2β1 from an inactive (low affinity) to an active (high affinity) conformation. This class of compounds is based on a proline-substituted 2,3-diaminopropionic acid scaffold. Structure-activity relationship studies of the scaffold have focused on optimization of the proline moiety, the urea functionality, and the sulfonyl group and have resulted in the development of potent inhibitors of α2β1-mediated platelet adhesion to collagen with IC50’s in the high picomolar to low nanomolar range. In particular, optimization of the proline moiety lead to compounds with high potency: transitioning from proline (DB496, IC50 of 29–62 nM) to a thiazolidine (SB68A) improved the IC50 to 2–8 nM; adding a methyl group at the 2 position of the thiazolidine (SB68B) slightly improved the IC50 to 1–12 nM; adding two methyl groups at the 5 position of the thiazolidine (SW4-161) resulted in a lead compound with an IC50 of 0.33–8 nM. As expected, the compounds had no effect on the binding of isolated α2 I-domains to collagen, consistent with their I-like domain mode of activity. Further, they were specific for α2β1-mediated platelet adhesion to collagen because they had no impact on ADP-stimulated platelet aggregation when added at 2 μM, a concentration more than 100-fold greater than the IC50 for inhibition of platelet adhesion to collagen. The compounds were also strong inhibitors of murine platelet adhesion to collagen and when tested in the ferric chloride-initiated murine carotid artery injury model, displayed activity similar to aspirin. Thus, 71% of untreated mice in this thrombosis model developed occlusive thrombi that remained stable for the 30 min duration of the assay, whereas stable thrombi developed in only 32% of mice treated with 1g/kg aspirin orally and in 41% of mice receiving 60 mg/kg CSW4-161intravenously. In summary, we have developed a class of potent inhibitors of the integrin α2β1 that demonstrate both in vitro and in vivo anti-platelet activity. Further development of this class of compounds may result in novel and relatively non-toxic anti-thrombotic agents.
Identification and Mechanistic Studies of a Novel Ubiquitin E1 Inhibitor
