Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here we describe a method to covalently label carrier proteins from PK and NRP synthases using the enzymatic transfer of a modified coenzyme A analog by a 4'-phosphopantetheinyltransferase. Using this method, carrier proteins can be loaded with single fluorescent or affinity reporters, providing novel entry for protein visualization, Western blot identification, and affinity purification. Application of these methods provides an ideal tool to track and quantify metabolically engineered pathways. Such techniques are valuable to measure protein expression, solubility, activity, and native posttranslational modification events in heterologous systems.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.

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