Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo as determined by activity-based protein profiling and raises brain anandamide levels for up to 24 hrs, resulting in profound cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

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Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (kinact/Ki and IC50 values of 40300 M−1 s−1 and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.

Protein kinases mediate signal transduction through phosphorylation of their protein substrates. 1 Up to one-third of proteins in a cell are phosphorylated, 2 and a major goal of phosphoproteomics is to characterize phosphorylation mediated signaling cascades by identifying phosphorylated proteins. This feat is analytically challenging because most phosphoproteins are of low abundance and substoichiometrically phosphorylated. Further, once a phosphoprotein is identified, it is difficult to integrate the role of the phosphorylation event into signal transduction networks without knowledge of the upstream kinase. Affinity purification techniques, such as strong cation exchange (SCX), 3 immobilized metal ion affinity chromatography (IMAC), 4 chemical tagging of phosphorylated residues with biotin, 5 and phosphomotif specific antibodies 6 can enrich phosphopeptides or proteins, but the information regarding the kinase responsible for phosphate transfer is uncoupled from the phosphorylation event. We previously have described methodology that allows selective labeling of direct kinase substrates, using analogue specific (as) kinases and orthogonal unnatural nucleotides (A*TP and A*TPγS); 7 however, it remains challenging to biochemically isolate the labeled substrates, impeding their identification.Here, we report a technique that combines direct substrate labeling with immunoaffinity purification (Schemes 1 and 2). To label the substrates of a given kinase, an as allele is used to enzymatically label substrates with A*TPγS. The selectively introduced thiophosphate is then chemically derivatized to construct a bio-orthogonal affinity tag. This approach is similar to other bio-orthogonal tagging strategies using ketones 8 or azides, 9 except thiophosphate cannot be selectively tagged in a single chemical step. For example, an alkylating agent will label both thiophosphate and other cellular nucleophiles, but we envisioned that an antibody could discriminate the thiophosphate alkylation products from other undesired alkylation products. The alkylating agent p-nitrobenzylmesylate (PNBM) was selected to construct the epitope because we predicted antibodies could recognize the product of thiophosphate alkylation over other nitrobenzyl alkylated amino acid residues, based on unique size and charge. Also, several high affinity antibodies have been raised against haptens containing pnitrophenyl moieties, 10 increasing the chances of eliciting an antibody capable of immunoprecipitation. Antibodies raised against hapten 4 are likely to be sequence independent because the binding determinants are relatively distant from the peptide backbone.Utilizing hapten 4, polyclonal antibodies (IgY and IgG) were raised in chickens and rabbits, respectively. 11 To enrich for specific binders, immune antibodies were purified on an affinity column containing immobilized hapten 4. Chicken IgY antibodies (α-3-IgY) performed best shokat@cmp.ucsf The α-3-IgY antibodies only successfully recognized H1 that had been thiophosphorylated and PNBM alky...