Abstract
Inhibition of inflammasome and pyroptotic pathways are promising strategies for clinical treatment of autoimmune and inflammatory disorders. MCC950, a potent inhibitor of the NLR-family inflammasome pyrin domain-containing 3 (NLRP3) protein, has shown encouraging results in animal models for a range of conditions; however, until now, no off-targets have been identified. Herein, we report the design, synthesis, and application of a novel photoaffinity alkyne-tagged probe for MCC950 (IMP2070) which shows direct engagement with NLRP3 and inhibition of inflammasome activation in macrophages. Affinity-based chemical proteomics in live macrophages identified several potential off-targets, including carbonic anhydrase 2 (CA2) as a specific target of IMP2070, and independent cellular thermal proteomic profiling revealed stabilization of CA2 by MCC950. MCC950 displayed noncompetitive inhibition of CA2 activity, confirming carbonic anhydrase as an off-target class for this compound. These data highlight potential biological mechanisms through which MCC950 and derivatives may exhibit off-target effects in preclinical or clinical studies.
Highlights
Inhibition of inflammasome and pyroptotic pathways are promising strategies for clinical treatment of autoimmune and inflammatory disorders
Activating mutations in NLR-family inflammasome pyrin domain-containing 3 (NLRP3) are directly linked to autoinflammatory fever syndromes, and deregulated NLRP3/ caspase-1 signaling is linked to inflammation
While MCC950 as a clinical candidate was reportedly halted in Phase II clinical trials, possibly due to off-target effects at high doses, derivatives of MCC950 remain in clinical trials, and it is critical to understand the off-target mechanisms of this class of molecules
Summary
Shenoy − Medical Research Council Centre for Molecular Bacteriology and Infection, Department of Infectious Disease, Imperial College London, London SW7 2AZ, United Kingdom; The Francis Crick Institute, London NW1 1AT, United Kingdom; Email: a.shenoy@ imperial.ac.uk. Kennedy − Department of Chemistry, Molecular Sciences Research Hub and Institute of Chemical Biology Centre for Doctoral Training, Imperial College London, London W12 0BZ, United Kingdom; orcid.org/ 0000-0003-1638-1701. Tristan Kovacǐ č − Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, London W12 0BZ, United Kingdom. Ravi Singh − Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, London W12 0BZ, United Kingdom; orcid.org/0000-0003-4344-4085. Ward − Department of Chemistry, Molecular Sciences Research Hub and Institute of Chemical Biology Centre for Doctoral Training, Imperial College London, London W12 0BZ, United Kingdom.
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