The Glucagon-like peptide 1 receptor (GLP-1R) is a class B1 G-protein-coupled receptor (GPCR) that is widely expressed and helps mediate insulin secretion, gastric emptying, and satiety. Currently, GLP-1R peptide agonists are used as standard of care for the treatment of T2DM and more recently for obesity. These GLP-1R peptide agonists require frequent subcutaneous injections or strict dosing guidelines thus increasing the need to develop an oral small-molecule GLP-1R agonist. Here we describe the identification of a novel small molecule GLP-1R agonist (GS-4571) which stimulated a potent cAMP response in pancreatic β-cells (EndoC-BH1) and selective agonism against human and monkey GLP-1R versus other class B GPCRs. In an intraperitoneal glucose tolerance test (IPGTT) it demonstrated improved glucose tolerance in humanized GLP-1R mice. Further studies, in obese cynomolgus monkeys, GS-4571 demonstrated similar improvements in glucose tolerance after a single dose treatment. Once-daily oral administration of GS-4571 in obese diabetic cynomolgus monkeys not only showed improved glycemic control but also showed a reduction in energy intake which led to an increase in weight loss over 36 days. Together these data support the continued development of GS-4571 into the clinic as a novel orally bioavailable GLP-1R small molecule agonist. Disclosure J. Vogel: None. M. Seung: None. B. Marchand: Employee; Gilead Sciences, Inc. Stock/Shareholder; Gilead Sciences, Inc., Pfizer Inc., Arrowhead Pharmaceuticals, Inc., Kronos Bio, Inc., Allogene Therapeutics, Inc., AbCellera Biologics Inc., Arbutus Biopharma Corporation. N. Bhangre: Employee; Gilead Sciences, Inc. Research Support; Gilead Sciences, Inc. Stock/Shareholder; Gilead Sciences, Inc. J. Mukherjee: None. S.E. Ammann: Employee; Gilead Sciences, Inc. M. Armstrong: Employee; Gilead Sciences, Inc. G. Brizgys: Employee; Gilead Sciences, Inc. E. Chin: Employee; Gilead Sciences, Inc. C. Chou: None. J. Cottell: Employee; Gilead Sciences, Inc. S.D. Schroeder: Employee; Gilead Sciences, Inc. R. Thomas-Tran: Employee; Gilead Sciences, Inc. Z. Yang: None. M. Peters: Employee; Gilead Sciences, Inc. G. Budas: Employee; Gilead Sciences, Inc. M.L. Mitchell: Employee; Gilead Sciences, Inc. M. Chojnacka: None. S. Jermain: None. J. Hung: Employee; Gilead Sciences, Inc. S. Kulkarni: None. Q. Yue: None. D.W. Lin: Employee; Gilead Sciences, Inc. D.A. Miranda: None.

The ability of HIV to integrate into the host genome and establish latent reservoirs is the main hurdle preventing an HIV cure. LEDGINs are small-molecule integrase inhibitors that target the binding pocket of LEDGF/p75, a cellular cofactor that substantially contributes to HIV integration site selection.

GS-9695 and GS-9822 are next-generation noncatalytic site integrase inhibitors (NCINIs) with significantly improved potency against human immunodeficiency virus compared with previous drugs such as BI-224436. Development stopped due to vacuolation of the bladder urothelium seen in cynomolgus monkey but not in rat; this lesion was absent in equivalent preclinical studies with BI-224436 (tested in dog and rat). Lesions were unlikely to be attributable to target because NCINIs specifically target viral integrase protein and no mammalian homologue is known. Secondary pharmacology studies, mitochondrial toxicity studies, immunophenotyping, and analysis of proteins implicated in cell-cell interactions and/or bladder integrity (E-cadherin, pan-cytokeratin, uroplakins) failed to offer any plausible explanation for the species specificity of the lesion. Because it was characterized by inflammation and disruption of urothelial morphology, we investigated physicochemical changes in the bladder of cynomolgus monkey (urinary pH 5.5-7.4) that might not occur in the bladder of rats (urinary pH 7.3-8.5). In measurements of surface activity, GS-9822 showed an unusual transition from a monolayer to a bilayer at the air/water interface with decreasing pH, attributed to the strong association between drug molecules in adjacent bilayer leaflets and expected to be highly disruptive to the urothelium. Structural analysis of GS-9822 and GS-9695 showed zwitterionic characteristics over the range of pH expected in cynomolgus monkey but not rat urine. This exotic surface behavior is unlikely with BI-224436 since it would transition from neutral to cationic (never zwitterionic) with decreasing pH. These data provide useful insights to guide discovery and development of NCINIs, related compounds, and zwitterions.

Novel reaction and work-up conditions were developed for the unprecedented Henry reaction using nitrocyclopropane in the key step towards the synthesis of HIV-1 protease inhibitors. This procedure may find application in the preparation of diverse compounds of potential biological interest. (C) 2017 Elsevier Ltd. All rights reserved.

A new class of highly potent NS5A inhibitors with an unsymmetric benzimidazole-difluorofluorene-imidazole core and distal [2.2.1]azabicyclic ring system was discovered. Optimization of antiviral potency and pharmacokinetics led to the identification of 39 (ledipasvir, GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has an extended plasma half-life of 37-45 h in healthy volunteers and produces a rapid >3 log viral load reduction in monotherapy at oral doses of 3 mg or greater with once-daily dosing in genotype 1a HCV-infected patients. 39 has been shown to be safe and efficacious, with SVR12 rates up to 100% when used in combination with direct-acting antivirals having complementary mechanisms.

tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.

We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers that express two different epitope tags that are recognized by their respective antibodies, coupled to distinct fluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitro enzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN is dependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols, had no effect on IN dimer formation. By studying mutants of IN, we determined that cysteines in IN appear to be dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD) of lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor that interacts with the IN dimer interface, were tested in this IN dimerization assay. These peptides, which compete with LEDGF for binding to IN, displayed an intriguing equilibrium binding dose-response curve characterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modeling of this binding system revealed that these LEDGF-derived peptides promote IN dimerization and block subunit exchange between IN dimers. This dose-response behavior was also observed with a small molecule that interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel IN dimerization assay revealed that peptide and small molecule inhibitors of the IN-LEDGF interaction also stabilize IN dimers and promote their formation.

Introduction of a unique phosphonate moiety at the PI position of the TMC-126 (3) scaffold provided a series of novel HIV-1 protease inhibitors (Pis) with an improved resistance profile against highly resistant variants. Optimization of the linker and phosphonate moieties lead to the identification of GS-8374 (1). Compound I is a potent and orally bioavailable HIV-1 PI with a superior resistance profile. Synthesis and characterization of 1 are reported.

A novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core were optimized for pharmacokinetics (PK) in rats. Several associations between physicochemical properties and PK were identified and exploited to guide the design of compounds. In addition, a simple new metric that may aid in the prediction of bioavailability for compounds with higher polar surface area is described (3*HBD-cLogP).