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Abstract
The initiating protease of the complement classical pathway, C1r, represents an upstream and pathway-specific intervention point for complement-related autoimmune and inflammatory diseases. Yet, C1r-targeted therapeutic development is currently underrepresented relative to other complement targets. In this study, we developed a fragment-based drug discovery approach using surface plasmon resonance (SPR) and molecular modeling to identify and characterize novel C1r-binding small-molecule fragments. SPR was used to screen a 2000-compound fragment library for binding to human C1r. This led to the identification of 24 compounds that bound C1r with equilibrium dissociation constants ranging between 160–1700 µM. Two fragments, termed CMP-1611 and CMP-1696, directly inhibited classical pathway-specific complement activation in a dose-dependent manner. CMP-1611 was selective for classical pathway inhibition, while CMP-1696 also blocked the lectin pathway but not the alternative pathway. Direct binding experiments mapped the CMP-1696 binding site to the serine protease domain of C1r and molecular docking and molecular dynamics studies, combined with C1r autoactivation assays, suggest that CMP-1696 binds within the C1r active site. The group of structurally distinct fragments identified here, along with the structure–activity relationship profiling of two lead fragments, form the basis for future development of novel high-affinity C1r-binding, classical pathway-specific, small-molecule complement inhibitors.
Highlights
The complement system is a primary arm of innate immunity involved in recognizing and eliminating infectious agents, marking and removing cellular debris, maintaining homeostasis, and triggering inflammation [1]
Whereas the alternative pathway is constitutively activated via a spontaneous hydrolytic process known as ‘tick-over’, the lectin pathway and classical pathway are defined by the relative activities of pathway-associated pattern recognition proteins
Because C1r is a serine protease, we acquired a small serine–protease inhibitor’ compounds (SPI) library that consisted of both small molecules and fragments (164–575 Da) with scaffold similarity to known protease inhibitors
Summary
The complement system is a primary arm of innate immunity involved in recognizing and eliminating infectious agents, marking and removing cellular debris, maintaining homeostasis, and triggering inflammation [1]. Complement activation occurs through one of three canonical pathways known as the classical pathway, lectin pathway, or alternative pathway (Figure 1A) [2,3,4]. Whereas the alternative pathway is constitutively activated via a spontaneous hydrolytic process known as ‘tick-over’, the lectin pathway and classical pathway are defined by the relative activities of pathway-associated pattern recognition proteins. Independent of the initiating event, all three pathways lead to the activation of the central molecule of the cascade, complement component C3. Cleavage of C3 into C3a and C3b by enzymatic complexes, known as C3 convertases C4b2b), results in complement amplification on, and opsonization of, target surfaces. C3 activation initiates the distal reactions of the complement cascade through cleavage of C5 into C5a and C5b
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