The incretin co-agonist tirzepatide requires GIPR for hormone secretion from human islets.

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) mediate insulin responses that are proportionate to nutrient intake to facilitate glucose tolerance1. The GLP-1 receptor (GLP-1R) is an established drug target for the treatment of diabetes and obesity2, whereas the therapeutic potential of the GIP receptor (GIPR) is a subject of debate. Tirzepatide is an agonist at both the GIPR and GLP-1R and is a highly effective treatment for type 2 diabetes and obesity3,4. However, although tirzepatide activates GIPR in cell lines and mouse models, it is not clear whether or how dual agonism contributes to its therapeutic benefit. Islet beta cells express both the GLP-1R and the GIPR, and insulin secretion is an established mechanism by which incretin agonists improve glycemic control5. Here, we show that in mouse islets, tirzepatide stimulates insulin secretion predominantly through the GLP-1R, owing to reduced potency at the mouse GIPR. However, in human islets, antagonizing GIPR activity consistently decreases the insulin response to tirzepatide. Moreover, tirzepatide enhances glucagon secretion and somatostatin secretion in human islets. These data demonstrate that tirzepatide stimulates islet hormone secretion from human islets through both incretin receptors.

Mutual Cooperativity of Three Allosteric Sites on the Dopamine D1 Receptor.

An amine-containing molecule called Compound A has been reported by a group from Bristol-Myers Squibb to act as a positive allosteric modulator (PAM) at the dopamine D1 receptor. We synthesized the more active enantiomer of Compound A (BMS-A1) and compared it with the D1 PAMs DETQ and MLS6585, which are known to bind to intracellular loop 2 and the extracellular portion of transmembrane helix 7, respectively. Results from D1/D5 chimeras indicated that PAM activity of BMS-A1 tracked with the presence of D1 sequence in the N-terminal/extracellular region of the D1 receptor, a unique location compared with either of the other PAMs. In pairwise combinations, BMS-A1 potentiated the small allo-agonist activity of each of the other PAMs, while the triple PAM combination (in the absence of dopamine) produced a cAMP response about 64% of the maximum produced by dopamine. Each of the pairwise PAM combinations produced a much larger leftward shift of the dopamine EC50 than either single PAM alone. All three PAMs in combination produced a 1000-fold leftward shift of the dopamine curve. These results demonstrate the presence of three non-overlapping allosteric sites that cooperatively stabilize the same activated state of the human D1 receptor. SIGNIFICANCE STATEMENT: Deficiencies in dopamine D1 receptor activation are seen in Parkinson disease and other neuropsychiatric disorders. In this study, three positive allosteric modulators of the dopamine D1 receptor were found to bind to distinct and separate sites, interacting synergistically with each other and dopamine, with the triple combination causing a 1000-fold leftward shift of the response to dopamine. These results showcase multiple opportunities to modulate D1 tone and highlight new pharmacological approaches for allosteric modulation of G-protein-coupled receptors.

LY3437943, a novel triple glucagon, GIP, and GLP-1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept.

With an increasing prevalence of obesity, there is a need for new therapies to improve body weight management and metabolic health. Multireceptor agonists in development may provide approaches to fulfill this unmet medical need. LY3437943 is a novel triple agonist peptide at the glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). In vitro, LY3437943 shows balanced GCGR and GLP-1R activity but more GIPR activity. In obese mice, administration of LY3437943 decreased body weight and improved glycemic control. Body weight loss was augmented by the addition of GCGR-mediated increases in energy expenditure to GIPR- and GLP-1R-driven calorie intake reduction. In a phase 1 single ascending dose study, LY3437943 showed a safety and tolerability profile similar to other incretins. Its pharmacokinetic profile supported once-weekly dosing, and a reduction in body weight persisted up to day 43 after a single dose. These findings warrant further clinical assessment of LY3437943.

Structural determinants of dual incretin receptor agonism by tirzepatide.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.

Discovery of an Orally Efficacious Positive Allosteric Modulator of the Glucagon-like Peptide-1 Receptor.

The identification of LSN3318839, a positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), is described. LSN3318839 increases the potency and efficacy of the weak metabolite GLP-1(9-36)NH2 to become a full agonist at the GLP-1R and modestly potentiates the activity of the highly potent full-length ligand, GLP-1(7-36)NH2. LSN3318839 preferentially enhances G protein-coupled signaling by the GLP-1R over ß-arrestin recruitment. Ex vivo experiments show that the combination of GLP-1(9-36)NH2 and LSN3318839 produces glucose-dependent insulin secretion similar to that of GLP-1(7-36)NH2. Under nutrient-stimulated conditions that release GLP-1, LSN3318839 demonstrates robust glucose lowering in animal models alone or in treatment combination with sitagliptin. From a therapeutic perspective, the biological properties of LSN3318839 support the concept that GLP-1R potentiation is sufficient for reducing hyperglycemia.

Nuisance compounds in cellular assays.

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity ("nuisance compounds") are routinely encountered in cellular assays, including phenotypic and high-content screening assays. Much is known regarding compound-dependent assay interferences in cell-free assays. However, despite the essential role of cellular assays in chemical biology and drug discovery, there is considerably less known about nuisance compounds in more complex cell-based assays. In our view, a major obstacle to realizing the full potential of chemical biology will not just be difficult-to-drug targets or even the sheer number of targets, but rather nuisance compounds, due to their ability to waste significant resources and erode scientific trust. In this review, we summarize our collective academic, government, and industry experiences regarding cellular nuisance compounds. We describe assay design strategies to mitigate the impact of nuisance compounds and suggest best practices to efficiently address these compounds in complex biological settings.

A synthetic method to assay adhesion-family G-protein coupled receptors. Determination of the G-protein coupling profile of ADGRG6(GPR126).

G-protein coupled receptors (GPCRs) are the largest family of membrane-spanning receptors in metazoans and mediate diverse biological processes such as chemotaxis, vision, and neurotransmission. Adhesion GPCRs represent an understudied class of GPCRs. Adhesion GPCRs (ADGRs) are activated by an intrinsic proteolytic mechanism executed by the G-protein autoproteolysis inducing domain that defines this class of GPCRs. It is hypothesized that agonist ligands modulate the proteolyzed receptor to regulate the activity of a tethered agonist peptide that is an intramolecular activator of ADGRs. The mechanism of activation of ADGRs in physiological settings is unclear and the toolbox for interrogating ADGR physiology in cellular models is limited. Therefore, we generated a novel enterokinase-activated tethered ligand system for ADGRG6(GPR126). Enterokinase addition to cells expressing a synthetic ADGRG6 protein induced potent and efficacious signal transduction through heterotrimeric G-protein coupled second messenger pathways including cyclic nucleotide production, intracellular calcium mobilization, and GPCR-pathway linked reporter gene induction. These studies support the hypothesis that ADGRG6(GPR126) is coupled to multiple heterotrimeric G-proteins: including Gαs, Gαq, and Gα12. This novel assay method is robust, specific, and compatible with numerous cell pharmacology approaches. We present a new tool for determination of the biological function of ADGRs and the identification of ligands that engage these receptors.

A High-Throughput Assay for the Pancreatic Islet Beta-Cell Potassium Channel: Use in the Pharmacological Characterization of Insulin Secretagogues Identified from Phenotypic Screening.

Phenotypic screening is a neoclassical approach for drug discovery. We conducted phenotypic screening for insulin secretion enhancing agents using INS-1E insulinoma cells as a model system for pancreatic beta-cells. A principal regulator of insulin secretion in beta-cells is the metabolically regulated potassium channel Kir6.2/SUR1 complex. To characterize hit compounds, we developed an assay to quantify endogenous potassium channel activity in INS-1E cells. We quantified ligand-regulated potassium channel activity in INS-1E cells using fluorescence imaging and thallium flux. Potassium channel activity was metabolically regulated and coupled to insulin secretion. The pharmacology of channel opening agents (diazoxide) and closing agents (sulfonylureas) was used to validate the applicability of the assay. A precise high-throughput assay was enabled, and phenotypic screening hits were triaged to enable a higher likelihood of discovering chemical matter with novel and useful mechanisms of action.

Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over ß-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein-coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.

Selective Phosphodiesterase 1 Inhibitor BTTQ Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats: Role of Peripheral Vasodilation.

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1-11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and, thus, peripheral resistance. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (BTTQ) on the cardiovascular system. Normal Sprague Dawley, spontaneously hypertensive (SHR), and Dahl salt-sensitive rats were used to test in vivo the efficacy of the compound. Phosphodiesterase radiometric enzyme assay revealed that BTTQ inhibited all three isoforms of PDE1 in nanomolar concentration, while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study conducted on mesenteric arteries revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriols reflected by the rise in the temperature. Furthermore, BTTQ proved a high efficacy in lowering the blood pressure about 9, 36, and 24 mmHg in normal Sprague Dawley, SHR and, Dahl salt-sensitive rats, respectively, compared to the vehicle-treated group. Moreover, additional blood pressure lowering of about 22 mmHg could be achieved when BTTQ was administered on top of ACE inhibitor lisinopril, a current standard of care in the treatment of hypertension. Therefore, PDE1 inhibition induced efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Administration of BTTQ was also associated with increased heart rate in both models of hypertension as well as in the normotensive rats. Thus, PDE1 appears to be an attractive therapeutic target for the treatment of resistant hypertension, while tachycardia needs to be addressed by further compound structural optimization.

Synthetic protease-activated class B GPCRs.

G-protein coupled receptors (GPCRs) are the ligand detection machinery of a majority of extracellular signaling systems in metazoans. Novel chemical and biological tools to probe the structure-function relationships of GPCRs have impacted both basic and applied GPCR research. To better understand the structure-function of class B GPCRs, we generated receptor-ligand fusion chimeric proteins that can be activated by exogenous enzyme application. As a prototype, fusion proteins of the glucagon-like peptide-1 receptor (GLP-1R) with GLP-1(7-36) and exendin-4(1-39) peptides incorporating enterokinase-cleavable N-termini were generated. These receptors are predicted to generate fusion protein neo-epitopes upon proteolysis with enterokinase that are identical to the N-termini of GLP-1 agonists. This system was validated by measuring enterokinase-dependent GLP-1R mediated cAMP accumulation, and a structure-activity relationship for both linker length and peptide sequence was observed. Moreover, our results show this approach can be used in physiologically relevant cell systems, as GLP-1R-ligand chimeras were shown to induce glucose-dependent insulin secretion in insulinoma cells upon exposure to enterokinase. This approach suggests new strategies for understanding the structure-function of peptide-binding GPCRs.

Tirzepatide is an imbalanced and biased dual GIP and GLP-1 receptor agonist.

Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and nonalcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor than the GLP-1 receptor, corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIP receptor but shows bias at the GLP-1 receptor to favor cAMP generation over ß-arrestin recruitment, coincident with a weaker ability to drive GLP-1 receptor internalization compared with GLP-1. Experiments in primary islets reveal ß-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting that the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIP receptor, combined with distinct signaling properties at the GLP-1 receptor, together may account for the promising efficacy of this investigational agent.

Structural insights into probe-dependent positive allosterism of the GLP-1 receptor.

Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a 'molecular glue'. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric Gs. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Our findings expand protein-protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.

Discovery and pharmacology of the covalent GLP-1 receptor (GLP-1R) allosteric modulator BETP: A novel tool to probe GLP-1R pharmacology.

The glucagon-like peptide-1 receptor (GLP-1R) is a significant therapeutic target for small molecule drug discovery given the therapeutic impact of peptide agonists in the diabetes sphere. We review the discovery and subsequent characterization of the small molecule GLP-1R allosteric modulator 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP). BETP is a covalent modulator of the GLP-1R, and we discuss the pharmacological implications and possible structural basis of this novel mode of action. We highlight the insights into class B G-protein coupled receptor pharmacology and biology provided by studies conducted with BETP. These include the descriptions of exquisite allosteric modulator probe dependence and biased signaling in vitro and in vivo. We conclude with an analysis of the utility of BETP as a chemical probe for the GLP-1R.

Activation of the GLP-1 receptor by a non-peptidic agonist.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.

The current state of GPCR-based drug discovery to treat metabolic disease.

One approach of modern drug discovery is to identify agents that enhance or diminish signal transduction cascades in various cell types and tissues by modulating the activity of GPCRs. This strategy has resulted in the development of new medicines to treat many conditions, including cardiovascular disease, psychiatric disorders, HIV/AIDS, certain forms of cancer and Type 2 diabetes mellitus (T2DM). These successes justify further pursuit of GPCRs as disease targets and provide key learning that should help guide identifying future therapeutic agents. This report reviews the current landscape of GPCR drug discovery with emphasis on efforts aimed at developing new molecules for treating T2DM and obesity. We analyse historical efforts to generate GPCR-based drugs to treat metabolic disease in terms of causal factors leading to success and failure in this endeavour. LINKED ARTICLES: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.

Beyond Glucagon-like Peptide-1: Is G-Protein Coupled Receptor Polypharmacology the Path Forward to Treating Metabolic Diseases?

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G-protein coupled receptor (GPCR) that has proven to be an effective target for developing medicines that treat type 2 diabetes mellitus (T2DM). GLP-1R agonists improve T2DM by enhancing glucose-stimulated insulin secretion, delaying gastric transit, decreasing glucagon levels, and reducing body weight due to anorexigenic actions. The therapeutic successes of these agents helped inspire the design of new multifunctional molecules that are GLP-1R agonists but also activate receptors linked to pathways that enhance insulin sensitization and/or energy expenditure. Herein, these agents are discussed in the context of polypharmacological approaches that may enable even further improvement in treatment outcomes. Moreover, we revisit classical polypharmaceutical GPCR approaches and how they may be utilized for treatment of T2DM. To determine optimal combination regimens, changes in drug discovery practices are likely needed because compensatory mechanisms appear to underlie progression of T2DM and limit the ability of current therapies to induce disease regression or remission.

Regulation of Endogenous (Male) Rodent GLP-1 Secretion and Human Islet Insulin Secretion by Antagonism of Somatostatin Receptor 5.

Incretin and insulin responses to nutrient loads are suppressed in persons with diabetes, resulting in decreased glycemic control. Agents including sulfonylureas and dipeptidyl peptidase-4 inhibitors (DPP4i) partially reverse these effects and provide therapeutic benefit; however, their modes of action limit efficacy. Because somatostatin (SST) has been shown to suppress insulin and glucagonlike peptide-1 (GLP-1) secretion through the Gi-coupled SST receptor 5 (SSTR5) isoform in vitro, antagonism of SSTR5 may improve glycemic control via intervention in both pathways. Here, we show that a potent and selective SSTR5 antagonist reverses the blunting effects of SST on insulin secretion from isolated human islets, and demonstrate that SSTR5 antagonism affords increased levels of systemic GLP-1 in vivo. Knocking out Sstr5 in mice provided a similar increase in systemic GLP-1 levels, which were not increased further by treatment with the antagonist. Treatment of mice with the SSTR5 antagonist in combination with a DPP4i resulted in increases in systemic GLP-1 levels that were more than additive and resulted in greater glycemic control compared with either agent alone. In isolated human islets, the SSTR5 antagonist completely reversed the inhibitory effect of exogenous SST-14 on insulin secretion. Taken together, these data suggest that SSTR5 antagonism should increase circulating GLP-1 levels and stimulate insulin secretion (directly and via GLP-1) in humans, improving glycemic control in patients with diabetes.

Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles.

Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5'-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9-36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [(3)H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and characterization of small molecule GLP-1R ligands as possible therapeutics.

Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays.

Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.