Dataset Information

Disulfide trapping to localize small-molecule agonists and antagonists for a G protein-coupled receptor.

ABSTRACT: A key question regarding the signaling mechanism for G protein-coupled receptors (GPCRs) is what triggers agonism versus antagonism. Peptide analogs derived from the chemokine, complement fragment 5 anaphylatoxin (C5a), can act as agonists or antagonists to the C5a receptor, a member of the GPCR family [Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J. & Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-4000]. Recently, we showed that two Cys residues engineered near a proposed binding site in the C5a receptor on transmembrane helices III and VI can selectively and reversibly trap short Cys-containing 3-mer peptides derived from C5a by disulfide bond formation [Buck, E. A., Bourne, H. & Wells, J. A. (November 18, 2004) J. Biol. Chem., 10.1074/jbc.C400500200]. Here, a library of 10,000 compounds, each containing an exchangeable thiol, was screened to identify specific small-molecule mimics that block binding of C5a. Some of the selected compounds acted as agonists and were as potent as the natural C5a ligand, and some acted as antagonists. A residue near these compounds, Ile-116 in helix III, functions as a "gatekeeper" to modulate these effects. A small substitution, Ile-116-Ala, enhanced affinity for some compounds and allowed antagonists to function as agonists; a larger substitution, Ile-116-Trp, decreased affinity and agonism. Thus, subtle changes in either the structure of the ligand or the receptor at the site between helix III, VI, and VII can switch the receptor on or off. This ligand binding and activation site may be similarly positioned in other members of the chemokine receptor family. Selective ligand trapping by reversible disulfide formation may serve to nucleate the development of small-molecule mimics.

SUBMITTER: Buck E

PROVIDER: S-EPMC549436 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Disulfide trapping to localize small-molecule agonists and antagonists for a G protein-coupled receptor.

Buck Elizabeth E Wells James A JA

Proceedings of the National Academy of Sciences of the United States of America 20050214 8

A key question regarding the signaling mechanism for G protein-coupled receptors (GPCRs) is what triggers agonism versus antagonism. Peptide analogs derived from the chemokine, complement fragment 5 anaphylatoxin (C5a), can act as agonists or antagonists to the C5a receptor, a member of the GPCR family [Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J. & Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-4000]. Recently, we showed that two Cys residues engineered near a proposed binding site in ...[more]

PMID: 15710877

Similar Datasets

Project description:BackgroundNovel small molecular ligands (SMLs) to the thyrotropin receptor (TSHR) have potential as improved molecular probes and as therapeutic agents for the treatment of thyroid dysfunction and thyroid cancer.MethodsTo identify novel SMLs to the TSHR, we developed a transcription-based luciferase-cAMP high-throughput screening system and we screened 48,224 compounds from a 100K library in duplicate.ResultsWe obtained 62 hits using the cut-off criteria of the mean±three standard deviations above the baseline. Twenty molecules with the greatest activity were rescreened against the parent CHO-luciferase cell for nonspecific activation, and we selected two molecules (MS437 and MS438) with the highest potency for further study. These lead molecules demonstrated no detectible cross-reactivity with homologous receptors when tested against luteinizing hormone (LH)/human chorionic gonadotropin receptor and follicle stimulating hormone receptor-expressing cells. Molecule MS437 had a TSHR-stimulating potency with an EC50 of 13×10(-8) M, and molecule MS438 had an EC50 of 5.3×10(-8) M. The ability of these small molecule agonists to bind to the transmembrane domain of the receptor and initiate signal transduction was suggested by their activation of a chimeric receptor consisting of an LHR ectodomain and a TSHR transmembrane. Molecular modeling demonstrated that these molecules bound to residues S505 and E506 for MS438 and T501 for MS437 in the intrahelical region of transmembrane helix 3. We also examined the G protein activating ability of these molecules using CHO cells co-expressing TSHRs transfected with luciferase reporter vectors in order to measure Gsα, Gβγ, Gαq, and Gα12 activation quantitatively. The MS437 and MS438 molecules showed potent activation of Gsα, Gαq, and Gα12 similar to TSH, but neither the small molecule agonists nor TSH showed activation of the Gβγ pathway. The small molecules MS437 and MS438 also showed upregulation of thyroglobulin (Tg), sodium iodine symporter (NIS), and TSHR gene expression.ConclusionsPharmacokinetic analysis of MS437 and MS438 indicated their pharmacotherapeutic potential, and their intraperitoneal administration to normal female mice resulted in significantly increased serum thyroxine levels, which could be maintained by repeated treatments. These molecules can therefore serve as lead molecules for further development of powerful TSH agonists.

Project description:Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active G?13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or ?2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic.

Dataset Information

Disulfide trapping to localize small-molecule agonists and antagonists for a G protein-coupled receptor.

Publications

Disulfide trapping to localize small-molecule agonists and antagonists for a G protein-coupled receptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets