Lead identification and pre-clinical studies on allosteric inhibitors of coagulation factor XIa

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Anticoagulants are the mainstay in the treatment of thrombotic diseases, such as heart attack and stroke, and may also be used in other diseases such as cancer and COVID-19. Although several anticoagulants including heparins, warfarin, and direct oral anticoagulants (DOACs) are used in the clinic, each agent suffers from major and minor bleeding adverse effects. As of now, a safe anticoagulant that inhibits coagulation without bleeding risk has not been developed as yet. Under the NHLBI's R01 mechanism, we earlier discovered that the heparin-binding exosite 2 of human factor XIa (hFXIa) is remarkably different from other homologous coagulation proteases (e.g., hFIIa, hFXa and others). We developed a novel design strategy that relies on anionic sulfates and aromatic rings to effect highly selective recognition of hFXIa's exosite 2 resulting in inhibition of enzyme activity. We developed a highly promising allosteric inhibitor, named sulfated D-chiro-inositol (SCI), which was synthesized in four steps, displayed >100-fold selectivity for hFXIa; bound plasma FXIa in exosite 2 with an affinity of 20–60 nM even when the enzyme's active site was covalently blocked; and inhibited in vivo arterial and venous thrombosis in the rat at 250 µg per animal (~1 mg/kg) without enhancing tail bleeding. SCI was tolerated at doses as high as 25 mg/rat suggesting a therapeutic window of ~100. SCI is a highly promising anticoagulant; yet its pharmacokinetics is not the best. Using cues from heparin-based drugs, we hypothesize that optimizing the number and position of sulfate groups on the D-chiro inositol scaffold, while also screening its various stereoisomers would improve hFXIa affinity, inhibition potency, PK, pharmacodynamics (PD), and chemistry, manufacturing and controls (CMC) properties before embarking on IND-enabling studies. The current proposal focuses on studying a library of 20 rationally designed SCI analogs with the goal of identifying at least one LEAD AGENT for advanced stage pre-clinical development. The specific aims are 1) synthesis, anticoagulation efficacy, and bleeding of 20 putative factor XIa inhibitors based on the SCI structure; 2) in vivo antithrombotic efficacy, DMPK, in vitro and in vivo toxicity of inhibitors; and 3) scaled-up non-GMP synthesis, CMC, dose escalation efficacy, and PD studies to identify one or two lead molecules. SCI has been claimed in a US patent (#9,758,459 B2 titled `Allosteric modulators of factor XIa as anticoagulant agents') with the PI as one of the inventors. Quantitative milestones with regard to synthesis, in vitro & in vivo efficacy, in vitro and in vivo toxicity will be used to guide the transition from the R61 to R33 Phase. Alternatively, the best 1 (or 2) analogs of SCI would be identified as the most promising agent for further IND-enabling studies starting from the 20 designed SCI analogs.