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 plasma platelet-activating factor acetylhydrolase (pPAFAH, a.k.a. PLA2G7) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains. However, there are no commercially available, selective, and in vivo-active inhibitors for investigation of pPAFAH biology. As such, we initiated a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high throughput screening (HTS) campaign to identify potential inhibitors of pPAFAH and three PAFAH family members: PAFAH2, PAFAHb2, and PAFAH1b3.
The pPAFAH HTS campaign revealed a large number (~5000) of potential lead compounds, but secondary gel-based screening of ~150 cherry picked top inhibitors quickly revealed the carbamate as a promising scaffold for inhibitor design. Given that carbamate inhibitors for serine hydrolase enzymes have previously been found to have tunable potency and selectivity and good activity in vivo, we endeavored to derive a pPAFAH-selective probe from the carbamate scaffold. In this effort, we were aided by the recent identification of a lead pPAFAH carbamate inhibitor from a small in-house library. By combining elements of both the HTS carbamate hits and our in-house lead, the medchem optimized probe (ML256, SID 125269079) is highly potent against its target enzyme (IC50 = 31 nM mouse isoform; 6 nM human isoform), and is active in situ and in vivo, showing excellent oral bioavailability and blood-brain barrier penetration. ML256 is at least 322-fold selective for all other brain serine hydrolases (~20) assessed by gel-based competitive activity-based protein profiling (ABPP), and is selective for other PAFAH enzymes as determined by both gel-based screening and multidimensional LC-MS/MS analysis (ABPP-MudPIT). ML256 inhibits pPAFAH by irreversible carbamoylation of the active site serine. The complete properties, characterization, and synthesis of ML256 are detailed in this report.