Allosteric Modulation of Endogenous Metabolites as an Avenue for Drug Discovery

Wootten, Denise; Savage, Emilia Elizabeth; Valant, Céline; May, Lauren T.; Sloop, Kyle W.; Ficorilli, James; Showalter, Aaron D.; Willard, Francis S.; Christopoulos, Arthur; Sexton, Patrick M.

doi:10.1124/mol.112.079319

Cited by 72 publications

(99 citation statements)

References 31 publications

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“…To confirm that this cooperativity was not probe dependent, we also examined the allosteric modulation of the GLP-1 metabolite, GLP-1(9-36)NH 2 ( Fig. S11) (36). These data demonstrate that the allosteric modulation of the GLP-1R occurs within a single monomeric unit and does not require dimerization of the receptor.…”

Section: Allosteric Modulation Of the Glp-1r Occurs Within A Single Rmentioning

confidence: 84%

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

Harikumar

Wootten

Pinon

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The glucagon-like peptide-1 receptor (GLP-1R) is a family B G proteincoupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent highaffinity binding to GLP-1(7-36)NH 2 but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.allosteric modulation | biased signaling | G protein-coupled receptors | family B GPCRs G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface proteins and play crucial roles in virtually every physiological process. Their widespread abundance, yet selective distribution, and ability to couple to a variety of signaling and effector systems make them extremely attractive targets for drug development (1). Recently, there has been increasing interest in the stoichiometry of receptors involved in GPCR signaling complexes and how this may impact on receptor function and drug discovery (2-4).With the exception of family C GPCRs, where obligate dimerization can occur (4), the role of oligomerization in GPCR function has remained controversial (2-8), and this has been the subject of a number of recent reviews (9-11). Although there is an increasing body of evidence supporting dimerization of GPCRs as a widespread feature of GPCR biology, including numerous studies on family A GPCRs, whether these are stable, transient, constitutive, or ligand dependent, and how they impact on receptor function and drug discovery are less clear, and general rules for oligomeric behavior are not evident (9-16). Even where effects on signaling are studied, these are generally linked to a single pathway and the role of dimerization in the control of receptor engagement and preference for distinct intracellular signaling intermediates (i.e., signal bias) is virtually unstudied.For family B GPCRs, which encompass many ther...

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Section: Allosteric Modulation Of the Glp-1r Occurs Within A Single Rmentioning

confidence: 84%

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

Harikumar

Wootten

Pinon

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…In contrast, BETP, a small molecule GLP-1R modulator that does not bind the ECD, signals through a distinct mechanism that appears to function in parallel to GLP-1 (Refs. 32, 34, and 35) and that induces a distinct ("biased") downstream signaling response (34). Surprisingly, the ability of BETP but not GLP-1 to stimulate GLP-1R signaling depended on Cys-347 in ICL3 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Boc5 and S4P largely, but not completely, displaced labeled GLP-1 from GLP-1R and may, therefore, also interact with the TMD (27). In contrast, the pyrimidine BETP (31) was not competed by exendin(9 -39) or GLP-1 and is, therefore, thought to allosterically activate GLP-1R (31)(32)(33)(34). Recent work has demonstrated that BETP functions by forming covalent adducts with Cys-347 in the intracellular loop 3 (ICL3) of GLP-1R, which may mimic a physiological covalent modification (35).…”

Section: Small Molecule Glp-1r Modulators Mimic Endogenous Peptide Homentioning

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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“…Recent work demonstrated that coincubation with BETP markedly enhances the activity of truncated metabolite of GLP-1(7-36)NH 2 , i.e., GLP-1(9-36)NH 2 , at the GLP-1R (Wootten et al, 2012). Here, we took advantage of the latter procedure to test whether the GSH conjugate of BETP (i.e., M1) retains positive allosteric modulator properties of BETP.…”

Section: Resultsmentioning

confidence: 99%

“…4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (compound B or BETP) (Fig. 1) is one such analog that has been identified as a positive allosteric modulator of the naturally occurring inactive GLP-1 metabolite, GLP-1(9-36)NH 2 , while showing little modulation of the active, circulating form, i.e., GLP-1(7-36)NH 2 (Sloop et al, 2010;Wootten et al, 2012). Likewise, the insulinotropic effect of oxyntomodulin [glucagon(1-37)], a low-affinity full agonist of the GLP-1R, was also markedly enhanced in the rat i.v.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small-molecule activator of the glucagonlike peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following i.v. (but not oral) administration. To explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after i.v. and oral dosing. Failure to detect BETP in circulation after oral administration of a 10-mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon i.v. administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma, and liver microsomes from rodents and humans indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of NADP was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH-and GSH-supplemented liver microsomes and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R.

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Allosteric Modulation of Endogenous Metabolites as an Avenue for Drug Discovery

Cited by 72 publications

References 31 publications

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

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

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

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