Identification of free fatty acid receptor 2 agonists using virtual screening.

Structure- and ligand-based virtual-screening methods (docking, 2D- and 3D-similarity searching) were analyzed for their effectiveness in virtual screening against FFAR2. To evaluate the performance of these methods, retrospective virtual screening was performed. Statistical quality of the methods was evaluated by BEDROC and RIE. The results revealed that electrostatic similarity search protocol using EON (ET combo) outperformed all other protocols with outstanding enrichment of >95% in top 1% and 2% of the dataset with an AUC of 0.958. Interestingly, the hit lists that are obtained from different virtual-screening methods are generally highly complementary to hits found from electrostatic similarity searching. These results suggest that considering electrostatic similarity searching first increases the chance of identifying more (and more diverse) active compounds from a virtual-screening campaign. Accordingly, prospective virtual screening using electrostatic similarity searching was used to identify novel FFAR2 ligands. The discovered compounds provide new chemical matter starting points for the initiation of a medicinal chemistry campaign.

Design, synthesis and biological evaluation of indane derived GPR40 agoPAMs.

GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet ß-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet ß-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM). Efforts were made to identify a suitable GPR40 AgoPAM tool molecule to investigate mechanism of action and de-risk liver toxicity of GPR40 AgoPAMs due to reactive acyl-glucuronide (AG) metabolites.

GPR120 suppresses adipose tissue lipolysis and synergizes with GPR40 in antidiabetic efficacy.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

GPR40 reduces food intake and body weight through GLP-1.

G protein-coupled receptor 40 (GPR40) partial agonists lower glucose through the potentiation of glucose-stimulated insulin secretion, which is believed to provide significant glucose lowering without the weight gain or hypoglycemic risk associated with exogenous insulin or glucose-independent insulin secretagogues. The class of small-molecule GPR40 modulators, known as AgoPAMs (agonist also capable of acting as positive allosteric modulators), differentiate from partial agonists, binding to a distinct site and functioning as full agonists to stimulate the secretion of both insulin and glucagon-like peptide-1 (GLP-1). Here we show that GPR40 AgoPAMs significantly increase active GLP-1 levels and reduce acute and chronic food intake and body weight in diet-induced obese (DIO) mice. These effects of AgoPAM treatment on food intake are novel and required both GPR40 and GLP-1 receptor signaling pathways, as demonstrated in GPR40 and GLP-1 receptor-null mice. Furthermore, weight loss associated with GPR40 AgoPAMs was accompanied by a significant reduction in gastric motility in these DIO mice. Chronic treatment with a GPR40 AgoPAM, in combination with a dipeptidyl peptidase IV inhibitor, synergistically decreased food intake and body weight in the mouse. The effect of GPR40 AgoPAMs on GLP-1 secretion was recapitulated in lean, healthy rhesus macaque demonstrating that the putative mechanism mediating weight loss translates to higher species. Together, our data indicate effects of AgoPAMs that go beyond glucose lowering previously observed with GPR40 partial agonist treatment with additional potential for weight loss.

Improvement of hERG-ROMK index of spirocyclic ROMK inhibitors through scaffold optimization and incorporation of novel pharmacophores.

SAR in the previously described spirocyclic ROMK inhibitor series was further evolved from lead 4 by modification of the spirocyclic core and identification of novel right-side pharmacophores. In this process, it was discovered that the spiropyrrolidinone core with the carbonyl group α to the spirocenter was preferred for potent ROMK activity. Efforts aimed at decreasing hERG affinity within the series led to the discovery of multiple novel right-hand pharmacophores including 3-methoxythiadiazole, 2-methoxypyrimidine, and pyridazinone. The most promising candidate is pyridazinone analog 32 that showed an improved functional hERG/ROMK potency ratio and preclinical PK profile. In vivo evaluation of 32 demonstrated blood pressure lowering effects in the spontaneously hypertensive rat model.

A non-canonical mechanism of GPCR activation.

The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists-ligands that trigger GPCR activation-function by causing rearrangement of the GPCR's transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations which disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets.

Label-free LC-MS based assay to characterize small molecule compound binding to cells.

Study of small molecule binding to live cells provides important information on the characterization of ligands pharmacologically. Here we developed and validated a label-free, liquid chromatography-mass spectrometry (LC-MS) based cell binding assay, using centrifugation to separate binders from non-binders. This assay was applied to various target classes, with particular emphasis on those for which protein-based binding assay can be difficult to achieve. In one example, to study a G protein coupled receptor (GPCR), we used one antagonist as probe and multiple other antagonists as competitor ligands. Binding of the probe was confirmed to be specific and saturable, reaching a fast equilibrium. Competition binding analysis by titration of five known ligands suggested a good correlation with their inhibition potency. In another example, this assay was applied to an ion channel target with its agonists, of which the determined binding affinity was consistent with functional assays. This versatile method allows quantitative characterization of ligand binding to cell surface expressed targets in a physiologically relevant environment.

Extracellular loop C of NPC1L1 is important for binding to ezetimibe.

Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [(3)H]AS. Chimeric and mutational studies indicate that high-affinity binding of [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes.

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

Cell-Based In Vitro Assay Automation: Balancing Technology and Data Reproducibility/Predictability.

G-protein-coupled receptors (GPCRs) are modulated by many marketed drugs, and as such, they continue to be key targets for drug discovery and development. Many GPCR targets at Merck Research Laboratories (MRL) are profiled using homogenous time-resolved fluorescence (HTRF) inositol monophosphate (IP-1) cell-based functional assays using adherent cells in 384-well microplates. Due to discrepancies observed across several in vitro assays supporting lead optimization structure-activity relationship (SAR) efforts, different assay paradigms were evaluated for removing growth medium from the assay plates prior to compound addition and determination of IP-1 accumulation. Remarkably, employing the noncontact centrifugation BlueWasher method leads to left-shifted potencies across multiple structural classes and rescues "false negatives" relative to the traditional manual evacuation method. Further, assay performance is improved, with the minimum significant ratio of challenging chemotypes dropping from ~5-6 to <3. While the impact of BlueWasher on a broad range of our GPCR targets remains to be determined, for highly protein-bound small molecules, it provides a path toward improving assay reproducibility across scientists and sites as well as reducing replicates in SAR assay support.

ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors.

Voltage-gated sodium (Na(V)1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na(V)1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na(V)1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na(V)1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na(V)1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na(V)1.7 channels, inhibiting Na(V)1.7 with an IC(50) value of 0.3 nM, compared with IC(50) values of 30 to 150 nM for other heterologously expressed Na(V)1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Abeta-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ((125)I-ProTx-II) binds with high affinity (K(d) = 0.3 nM) to recombinant hNa(V)1.7 channels. Binding of (125)I-ProTx-II is insensitive to the presence of other well characterized Na(V)1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the (125)I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na(V)1.7-selective blockers.

Madin-Darby canine kidney II cells: a pharmacologically validated system for NPC1L1-mediated cholesterol uptake.

Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [(3)H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 x 10(5) [(3)H]AS sites/cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC(50) blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.

A high-capacity membrane potential FRET-based assay for the sodium-coupled glucose co-transporter SGLT1.

Secondary active glucose transport is mediated by at least four members of the solute-linked carrier 5 gene family (sodium/glucose transporter [SGLT] 1-4). Human genetic disorders of SGLTs including glucose-galactose malabsorption and familial renal glucosuria have increased attention on members of this family of transporters as putative drug targets. Using human SGLT1 (hSGLT1) as a paradigm, we developed a functional assay that should be adaptable to ultra-high-throughput operation and to other SGLTs. Human embryonic kidney (HEK) 293 cells stably expressing hSGLT1 (hSGLT1/HEK293 cells) display a Na(+)-dependent, phlorizin-sensitive alpha-methyl-D-[(14)C]glucopyranoside flux with expected kinetic parameters. In electrophysiological studies with hSGLT1/HEK293 cells, substrate-dependent changes in membrane potential were observed, consistent with the electrogenic operation of hSGLT1. With the use of voltage-sensitive dyes, a membrane potential, fluorescence resonance energy transfer-based functional assay on a voltage/ion probe reader platform has been established for SGLT1. This high-capacity functional assay displays similar characteristics in terms of substrate specificity and phlorizin sensitivity to those determined by more traditional approaches and should provide a means to identify novel and selective SGLT inhibitors.

Structure-Activity Relationship of Novel and Selective Biaryl-Chroman GPR40 AgoPAMs.

A series of biaryl chromans exhibiting potent and selective agonism for the GPR40 receptor with positive allosteric modulation of endogenous ligands (AgoPAM) were discovered as potential therapeutics for the treatment of type II diabetes. Optimization of physicochemical properties through modification of the pendant aryl rings resulted in the identification of compound AP5, which possesses an improved metabolic profile while demonstrating sustained glucose lowering.

GPR40 partial agonist MK-2305 lower fasting glucose in the Goto Kakizaki rat via suppression of endogenous glucose production.

GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects in vivo. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 ex vivo and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia.

Design and Synthesis of Novel, Selective GPR40 AgoPAMs.

GPR40 is a G-protein-coupled receptor expressed primarily in pancreatic islets and intestinal L-cells that has been a target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion only in the presence of elevated blood glucose levels, minimizing the risk of hypoglycemia. GPR40 agoPAMs have shown superior efficacy to partial agonists as assessed in a glucose tolerability test (GTT). Herein, we report the discovery and optimization of a series of potent, selective GPR40 agoPAMs. Compound 24 demonstrated sustained glucose lowering in a chronic study of Goto Kakizaki rats, showing no signs of tachyphylaxis for this mechanism.

GPR40 partial agonists and AgoPAMs: Differentiating effects on glucose and hormonal secretions in the rodent.

GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.

Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40.

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Design, Synthesis, and Evaluation of Novel and Selective G-protein Coupled Receptor 120 (GPR120) Spirocyclic Agonists.

Type 2 diabetes mellitus (T2DM) is an ever increasing worldwide epidemic, and the identification of safe and effective insulin sensitizers, absent of weight gain, has been a long-standing goal of diabetes research. G-protein coupled receptor 120 (GPR120) has recently emerged as a potential therapeutic target for treating T2DM. Natural occurring, and more recently, synthetic agonists have been associated with insulin sensitizing, anti-inflammatory, and fat metabolism effects. Herein we describe the design, synthesis, and evaluation of a novel spirocyclic GPR120 agonist series, which culminated in the discovery of potent and selective agonist 14. Furthermore, compound 14 was evaluated in vivo and demonstrated acute glucose lowering in an oral glucose tolerance test (oGTT), as well as improvements in homeostatic measurement assessment of insulin resistance (HOMA-IR; a surrogate marker for insulin sensitization) and an increase in glucose infusion rate (GIR) during a hyperinsulinemic euglycemic clamp in diet-induced obese (DIO) mice.