A Small Molecule Ligand of the Glucagon-like Peptide 1 Receptor Targets Its Amino-terminal Hormone Binding Domain

Abstract

The glucagon-like peptide 1 receptor (GLP-1R) belongs to a distinct subgroup of G protein-coupled peptide hormone receptors (class B) that has been difficult to target by small molecule drugs. Here, we report that a non-peptide compound, T-0632, binds with micromolar affinity to the human GLP-1R and blocks GLP-1-induced cAMP production. Furthermore, the observation that T-0632 has almost 100-fold selectivity for the human versus the highly homologous rat GLP-1R provided an opportunity to map determinants of non-peptide binding. Radioligand competition experiments utilizing a series of chimeric human/rat GLP-1R constructs revealed that partial substitution of the amino terminus of the rat GLP-1R with the corresponding sequence from the human homolog was sufficient to confer high T-0632 affinity. Follow-up analysis of receptors where individual candidate amino acids had been exchanged between the human and rat GLP-1Rs identified a single residue that explained species selectivity of non-peptide binding. Replacement of tryptophan 33 in the human GLP-1R by serine (the homologous amino acid in the rat GLP-1R) resulted in a 100-fold loss of T-0632 affinity, whereas the converse mutation in the rat GLP-1R led to a reciprocal gain-of-function phenotype. These observations suggest that in a class B receptor, important determinants of non-peptide affinity reside within the extracellular amino-terminal domain. Compound T-0632 may mimic, and thereby interfere with, the putative "pseudo-tethering" mechanism by which the amino terminus of class B receptors initiates the binding of cognate hormones.

A Small-Molecule Oral Agonist of the Human Glucagon-like Peptide-1 Receptor

“… 22 , 43 Overall, our findings were reminiscent of a previous report describing the 100-fold reduction in the binding affinity of the small-molecule GLP-1R antagonist T-0632 in the W33S mutant of the human GLP-1R. 45 It was postulated that the binding of T-0632 stabilizes a closed, inactive conformation of the GLP-1R, which involves W33. 46 …”

Structural insights into the activation of GLP-1R by a small molecule agonist

“…1e), which was also observed in binding to non-peptide antagonist T-0632, and agonist peptide 5. 5,10 The difference of W33 in humans and cognate residue S33 in rat GLP-1R determines the species specificity of small molecule ligands as RGT1383 only activates the human GLP-1R whereas GLP-1 can activate GLP-1R from both human and rat (Fig. 1e).…”

G-Protein coupled receptors: structure and function in drug discovery

“…6). 63 Some known allosteric modulators of family B GPCRs include NovoNordisk compounds 1-6: 63 T-0632, which blocks the GLP-1 induced cAMP production 63,64 (GLP 1 receptor); DMP696, which blocks the CRF-stimulated adenylyl cyclase activity in cell line expressing CRF 1 receptor; 63,65 NBI 27914, which blocks the CRF 1 receptor; 63,66 NBI 35965; 63 antarlamin 63 (CRF 1 receptor). 63 Cooperativity is a thermodynamic term which has varying meanings in different biochemical contexts.…”