Demonstration of the Innate Electrophilicity of 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor

Eng, Heather; Sharma, Raman; McDonald, Thomas S.; Edmonds, David J.; Fortin, Jean-Phillippe; Li, Xianping; Stevens, Benjamin D.; Griffith, David A.; Limberakis, Chris; Nolte, Whitney M.; Price, David A.; Jackson, Margaret; Kalgutkar, Amit S.

doi:10.1124/dmd.113.052183

Cited by 22 publications

(22 citation statements)

References 47 publications

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“…Ligands such as BETP and compound 2 are highly electrophilic and can form adducts with free cysteine residues ( Eng et al, 2013 ). This is a feature of the action of these molecules to form, in particular, the covalent interaction with C347 6.36b in ICL3/TM6 that is critical for both the intrinsic efficacy and the cooperative allosteric effect ( Nolte et al, 2014 ).…”

Section: Glucagon-like Peptide 1 Receptormentioning

confidence: 99%

Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes

Graaf

Donnelly

Wootten³

et al. 2016

Pharmacol Rev

290

295

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The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein–coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain–binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders.

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Section: Glucagon-like Peptide 1 Receptormentioning

confidence: 99%

Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes

Graaf

Donnelly

Wootten³

et al. 2016

Pharmacol Rev

290

295

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“…Small Molecule GLP-1R Modulators-Boc5, S4P, WB4 -24 and pyrimidine BETP were synthesized in-house with purities Ͼ95% using published procedures (37,38). These compounds were dissolved in 100% DMSO and diluted in buffer (final DMSO concentration was 1%) and added at 10 M final com- pound concentration.…”

Section: Methodsmentioning

confidence: 99%

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors

Zhao

Yin

Yang

et al. 2016

Journal of Biological Chemistry

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G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF 1 R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation.The class B or secretin family of GPCRs consists of 15 receptors for peptide hormones that include glucagon, glucagon-like peptides, parathyroid hormone, and calcitonin (Fig. 1A). These receptors are important drug targets for many human diseases including diabetes, neurodegeneration, cardiovascular diseases, and psychiatric disorders. The full-length receptors consist of two modular domains: a globular extracellular domain (ECD) 3 defined by three conserved disulfide bonds and a TMD that contains seven transmembrane helices (1-4). The ECD is responsible for the high affinity and specificity of hormone binding, and the TMD is required for receptor activation and signal coupling to downstream G proteins and other signaling effectors (4). Peptide hormone binding is thought to proceed through fast binding of its C terminus to the ECD followed by a slower association of the peptide N terminus with the receptor TMD (5), which leads to conformational changes in the receptor TMD and the receptor activation. The activated receptors are coupled primarily to stimulatory G proteins, resulting in elevation of the intracellular cAMP level.Structures of the ECDs of class B GPCRs in complex with their peptide ligands have been determined by x-ray crystallography (1, 6 -9) and NMR (10) and have provided useful information about structural mechanisms of ligand recognition and selectivity (2, 11). Only recently, crystal structures of t...

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“…Although structurally distinct, unfortunately both compounds are unstable and display poor pharmacokinetic properties that preclude potential clinical development ( 16 ). Not surprisingly, because of its electrophilic nature, BETP has been reported to conjugate with GSH in vitro ( 25 ).…”

Section: Introductionmentioning

confidence: 99%

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

Bueno

Showalter²,

Wainscott

et al. 2016

Journal of Biological Chemistry

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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 [3H]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.

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Demonstration of the Innate Electrophilicity of 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor

Cited by 22 publications

References 47 publications

Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes

Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors

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

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