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

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