<p> The Glucagon-like peptide 1 receptor (GLP-1R) belongs to the pharmaceutically important Class B family of G-protein coupled receptors (GPCRs) and its incretin peptide ligand GLP-1 analogs are adopted drugs for the treatment of type 2 diabetes (T2D). Despite remarkable anti-diabetic effects, Glucagon Like Peptide-1 (GLP-1) peptide-based drugs are limited by the need of injection or high cost oral formulation. On the other hand, developing non-peptide small molecule drugs targeting GLP-1R remains elusive likely due to the large nature of the orthosteric binding site on GLP-1R. A promising approach is to develop small molecule agonistic positive allosteric modulators (ago-PAMs) or positive allosteric modulators (PAMs) of GLP-1R by targeting the potential allosteric sites in the transmembrane (TM) domain of human GLP-1R. </p><p> As the first step of taking this approach, we constructed a three-dimensional structure model of the TM domain of human GLP-1R using homology modeling and conformational sampling techniques. Next, a potential allosteric binding site on the TM domain was predicted computationally. <i>In silico</i> screening of drug-like compounds against this predicted allosteric site has identified nine compounds as potential GLP-1R agonists. The independent agonistic activity of two compounds was subsequently confirmed using cyclic adenosine monophosphate (cAMP) response element (CRE)-based luciferase reporting system. One compound was also shown to stimulate insulin secretion through <i> in vitro</i> assay. In addition, this compound synergized with GLP-1 to activate human GLP-1R. </p><p> In 2017, the crystal structures of GLP-1R in its active state (PDB ID: 5VAI) became available. Hence, we have performed another round of <i> in silico</i> screening employing this structure. First, the potential ligand binding sites in 5VAI were identified using computational tools and <i> in silico</i> screening procedure as described above was carried out again. A new small 8 molecule with low molecular weight and logP was identified. <i> In vitro</i> studies of this compound confirmed that it acts as the ago-Positive Allosteric Modulator (PAM) of GLP-1R that improves GLP-1's affinity and efficacy towards GLP-1R. When used in combination with GLP-1, this compound improves insulin secretion than using GLP-1 alone. Site specific mutagenesis studies confirmed its binding site as predicted in the TM domain of GLP-1R. </p><p> Finally, this ago-PAM molecule was further optimized to improve its potency and specificity towards GLP-1R using structure-based optimization strategy and medicinal synthesis. The newly designed compound, whose molecular weight was less than the parental compound, was found to act as the PAM of GLP-1R and showed improvement in the specificity than the parental compound. Thus, this new compound could be further exploited in the drug development for T2D treatment. </p><p> These results demonstrated that allosteric regulation exists in GLP-1R and can be exploited for developing small molecule agonists. The success of this work will help pave the way for small molecule drug discovery targeting other Class B GPCRs through allosteric regulations.</p><p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:13861629 |
| Date | 25 April 2019 |
| Creators | Redij, Tejashree |
| Publisher | University of the Sciences in Philadelphia |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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