Discovery and preclinical characterization of SB-01, a novel allosteric inhibitor of human factor XIa

Abstract Human factor XI (hFXI) is a key protein in the intrinsic coagulation pathway that amplifies clot formation under pathological conditions while having minimal impact on hemostasis. This unique profile makes hFXI an attractive target for anticoagulation. This approach is supported by strong clinical and genetic evidence: individuals with congenital FXI deficiency exhibit reduced thrombosis risk without spontaneous bleeding, while elevated FXI levels are associated with a dose-dependent increase in thrombosis. Although recent drug development has focused on small-molecule orthosteric inhibitors of FXIa, such as asundexian and milvexian, an alternative strategy of allosterically disrupting the catalytic triad offers a potentially superior mechanism. Allosteric sites are often less conserved than orthosteric sites, which can lead to enhanced selectivity and the possibility of partial antagonism, an outcome not achievable with traditional orthosteric inhibitors. Here, we present a novel, synthetic small molecule, SB-01, which is structurally and functionally distinct from other hFXIa inhibitors currently in development. In vitro, SB-01 potently inhibited hFXIa with a 130 nM potency and ~90% efficacy. This inhibition was highly selective, as SB-01 demonstrated 30–800-fold greater potency against hFXIa compared to other related serine proteases (FIIa​, FXa​, FIXa​, FXIIa​, and P.Kal). We further confirmed the allosteric mechanism of SB-01. Kinetic analyses revealed a dose-dependent reduction in Vmax​ and a slight increase in KM​, suggesting a mixed-inhibition profile. To definitively ascertain allosterism, we first demonstrated that covalently blocking the active site only marginally increased affinity of SB-01 for hFXIa. Using spectrofluorimetry, we determined the affinity of SB-01 for hFXI to be ~80 nM. Furthermore, stoichiometric analysis of the hFXI::SB-01 co-complex revealed a 4:1 ratio, suggesting that allosteric binding occurs at both sites within the dimeric form of the target. Given these promising in vitro results, we evaluated SB-01 in preclinical models of arterial and venous thrombosis. Intravenous administration of SB-01 (6 mg/kg) significantly reduced thrombus formation in a FeCl3​-induced arterial thrombosis model. Similarly, in a rat thromboplastin-induced vena cava thrombosis model, SB-01 significantly reduced thrombus formation at doses of 3 and 6 mg/kg. Importantly, we also evaluated SB-01 in a mouse lung cancer model, a patient population at high risk for venous thromboembolism (VTE). In this model, pre-treatment with SB-01 significantly reduced both thrombus weight and the incidence of thrombosis. Crucially, SB-01 showed a clean safety profile with no significant effects on body weight, bleeding time, human ether-à-go-go-related gene (hERG) activity, or CYP450 inhibition. In conclusion, this study introduces SB-01 as a highly promising, structurally unique, and potent allosteric inhibitor of hFXIa. Its high selectivity and proven efficacy in multiple preclinical thrombosis models, coupled with an excellent safety profile, position it as a strong candidate for pre-IND studies and future human clinical trials.