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Optimization and characterization of a triazole urea inhibitor for platelet-activating factor acetylhydrolase type 2 (PAFAH2).


Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.
2011 Mar 31 [updated 2013 Mar 07].


Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders. Phospholipids in particular are susceptible to oxidative damage, and it is thought that the cytosolic enzyme type II platelet-activating factor acetylhydrolase (PAFAH2) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains. In support of this theory, over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death [1]. However, no selective inhibitors of PAFAH2 are known for investigation of PAFAH2 biology. We initiated a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) HTS campaign to identify potential inhibitors of PAFAH2 (AIDs 492956 and 493030). The assay also served as a counterscreen for inhibitor discovery for the related enzyme, plasma PAFAH (pPAFAH; AIDs 463082, 463230). Interestingly, the triazole urea SID 7974398—a top lead in the lysophospholipase (LYPLA1) inhibitor screen from which we derived a dual inhibitor of LYPLA1/LYPLA2 (ML211) and inhibitor of ABHD11 (ML226)—was also a top hit in the PAFAH2 HTS assay. Given that triazole ureas were previously found to have tunable potency and selectivity, low cytotoxicity, and good activity in situ, we endeavored to derive a PAFAH2-selective probe from the triazole urea scaffold. The medchem optimized probe (ML225, SID 103913572) is highly potent against its target enzyme (IC50 = 3 nM), and is active in situ at sub-nanomolar concentrations. ML225 is at least 333-fold selective for all other serine hydrolases (~20) assessed by gel-based competitive activity-based protein profiling, and is selective for other PAFAH enzymes. ML225 inhibits PAFAH2 by carbamoylating the active site serine. The complete properties, characterization, and synthesis of ML225 are detailed in this Probe Report.

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