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Targeting the Initiator Protease of the Classical Pathway of Complement Using Fragment-Based Drug Discovery.

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.

SUBMITTER: Rushing BR

PROVIDER: S-EPMC7504721 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Targeting the Initiator Protease of the Classical Pathway of Complement Using Fragment-Based Drug Discovery.

Rushing Blake R BR Rohlik Denise L DL Roy Sourav S Skaff D Andrew DA Garcia Brandon L BL

Molecules (Basel, Switzerland) 20200903 17

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 fragment ...[more]

PMID: 32899120

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Project description:The complement, as part of the innate immune system, represents the first line of defense against Gram-negative bacteria invading the bloodstream. The complement system is a zymogen cascade that ultimately assemble into the so-called membrane attack complex (MAC), which lyses Gram-negative bacteria upon insertion into the outer membrane. Traditionally, serum has been used as complement source, for example to study the bactericidal activity of monoclonal antibodies or antibodies raised upon vaccination. Due to the significant donor to donor variability, as well as susceptibility of complement factors to handling and storage conditions, assay reproducibility using human serum is low. Moreover, the presence of pre-existing antibodies and antimicrobial compounds are confounding factors. To remove antibodies from human serum, we applied ?/?-light chain specific affinity chromatography, however the method severely reduced the complement activity due to the depletion of complement components. Therefore, we attempted to reconstitute human complement-namely the alternative (rAP) and the classical (rCP) pathways-from purified complement factors. We found that adding C1-inhibitor to the mixture was essential to maintain a stable and functional C1 and thus to generate an active rCP. We further confirmed the functionality of the rCP by testing the complement-dependent bactericidal activity of a human monoclonal antibody, A1124 against an E. coli clinical isolate belonging to the ST131 clonal complex, and that of a polyclonal IVIg against a laboratory E. coli strain (MG1655) not expressing LPS O-antigen and capsule. Although the alternative pathway did not have any bactericidal activity by itself, it enhanced MAC deposition induced by rCP and increased the overall bactericidal activity against the ST131 E. coli strain. In conclusion, we report for the first time the successful in vitro reconstitution of the classical pathway of the human complement to establish a serum-free, complement dependent bactericidal assay. This system offers high level of standardization and could support the study of the complement in different research fields.

Dataset Information

Targeting the Initiator Protease of the Classical Pathway of Complement Using Fragment-Based Drug Discovery.

Publications

Targeting the Initiator Protease of the Classical Pathway of Complement Using Fragment-Based Drug Discovery.

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