Development of small-molecule inhibitors of the initiating proteases, C1r and C1s, of the classical complement pathway

Abstract

Complement is a proteolytic cascade that upon activation plays a key effector role in the innate immune system and acts to prime the adaptive immune response. During normal homeostatic events, complement is tightly regulated for its roles in immune complex clearance, lysis of target cells, opsonization, and recruitment of leukocytes and monocytes to target areas. Several endogenous regulators are responsible for the control of complement activation, however when dysregulation occurs, aberrant complement activation has been linked to autoimmune, proinflammatory, and neurodegenerative diseases, including Alzheimer’s disease. Inhibition of the classical complement component C1 may ameliorate hallmarks of autoimmune and inflammatory disease. The serine proteases within the C1 complex, C1r and C1s, are promising therapeutic targets for structure-based small-molecule drug development. We investigated the activity of a series of small-molecule compounds identified in a large-scale fragment library screen and those from a cheminformatics computational docking screen in which hit compounds were predicted to bind the C1r or C1s proteases. Using surface plasmon resonance and ELISA-based assays for hit validation, we analyzed the binding affinities and the inhibitory IC50’s of several compounds predicted to bind and inhibit the activation of C1r or C1s in a dose-dependent manner. In this study, we have identified four lead compounds (cmp-1611, cmp-1663, cmp-1696, cmp-1827) and their 10 active structural analogues that target and inhibit C1r activation. Given their abilities to bind and inhibit C1r and favorable physicochemical properties, our lead compounds may provide a starting point for optimizing affinity and specificity necessary for developing novel routes of therapeutic upstream complement inhibition.