Determination of Affinity and Residence Time of Potent Drug-Target Complexes by Label-free Biosensing.

Prolonged drug-target occupancy has become increasingly important in lead optimization, and biophysical assays that measure residence time are in high demand. Here we report a practical label-free assay methodology that provides kinetic and affinity measurements suitable for most target classes without long preincubations and over comparatively short sample contact times. The method, referred to as a "chaser" assay, has been applied to three sets of unrelated kinase/inhibitor panels in order to measure the residence times, where correlation with observed efficacy was suspected. A lower throughput chaser assay measured a residence time of 3.6 days ±3.4% (95% CI) and provided single digit pM sensitivity. A higher throughput chaser methodology enabled a maximum capacity of 108 compounds in duplicate/day with an upper residence time limit of 9 h given an assay dissociation time of 34 min.

Selective inhibitor of platelet-activating factor acetylhydrolases 1b2 and 1b3 that impairs cancer cell survival.

Platelet-activating factor acetylhydrolases (PAFAHs) 1b2 and 1b3 are poorly characterized serine hydrolases that form a complex with a noncatalytic protein (1b1) to regulate brain development, spermatogenesis, and cancer pathogenesis. Determining physiological substrates and biochemical functions for the PAFAH1b complex would benefit from selective chemical probes that can perturb its activity in living systems. Here, we report a class of tetrahydropyridine reversible inhibitors of PAFAH1b2/3 discovered using a fluorescence polarization-activity-based protein profiling (fluopol-ABPP) screen of the NIH 300,000+ compound library. The most potent of these agents, P11, exhibited IC50 values of ∼40 and 900 nM for PAFAH1b2 and 1b3, respectively. We confirm selective inhibition of PAFAH1b2/3 in cancer cells by P11 using an ABPP protocol adapted for in situ analysis of reversible inhibitors and show that this compound impairs tumor cell survival, supporting a role for PAFAH1b2/3 in cancer.

Therapeutic potential of monoacylglycerol lipase inhibitors.

Marijuana and aspirin have been used for millennia to treat a wide range of maladies including pain and inflammation. Both cannabinoids, like marijuana, that exert anti-inflammatory action through stimulating cannabinoid receptors, and cyclooxygenase (COX) inhibitors, like aspirin, that suppress pro-inflammatory eicosanoid production have shown beneficial outcomes in mouse models of neurodegenerative diseases and cancer. Both cannabinoids and COX inhibitors, however, have untoward effects that discourage their chronic usage, including cognitive deficits and gastrointestinal toxicity, respectively. Recent studies have uncovered that the serine hydrolase monoacylglycerol lipase (MAGL) links the endocannabinoid and eicosanoid systems together through hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) to provide the major arachidonic acid (AA) precursor pools for pro-inflammatory eicosanoid synthesis in specific tissues. Studies in recent years have shown that MAGL inhibitors elicit anti-nociceptive, anxiolytic, and anti-emetic responses and attenuate precipitated withdrawal symptoms in addiction paradigms through enhancing endocannabinoid signaling. MAGL inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. In cancer, MAGL inhibitors have been shown to have anti-cancer properties not only through modulating the endocannabinoid-eicosanoid network, but also by controlling fatty acid release for the synthesis of protumorigenic signaling lipids. Thus, MAGL serves as a critical node in simultaneously coordinating multiple lipid signaling pathways in both physiological and disease contexts. This review will discuss the diverse (patho)physiological roles of MAGL and the therapeutic potential of MAGL inhibitors in treating a vast array of complex human diseases.