The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target for metabolic disorders, particularly type 2 diabetes and obesity. Although current treatments are effective, their unavoidable side effects continue to drive the search for novel therapeutic strategies. Ago-allosteric modulators (ago-PAMs), which act as agonists on their own while enhancing the affinity and efficacy of orthosteric agonists, represent a promising avenue to overcome limitations associated with traditional peptide-based therapies. However, the molecular mechanisms by which ago-PAMs modulate GLP-1R activation remain poorly understood. In this work, we selected compound 2, a validated ago-PAM of GLP-1R, as a probe to explore these mechanisms at the atomic level. Using molecular dynamics (MD) simulations, we elucidate how compound 2 stabilizes the active conformation of GLP-1R through allosteric binding and reveal distinct pathways by which it enhances the binding of both peptide and non-peptide orthosteric agonists. Enhanced sampling simulations further provided a comprehensive conformational landscape of GLP-1R activation, identifying two intermediate states that bridge inactive and active conformations. Compound 2 was found to bias the receptor toward active-like ensembles, consistent with its intrinsic agonist activity. Together, our findings provide mechanistic insights into ago-allosteric modulation of GLP-1R, offering useful information for the rational design of small-molecule modulators with improved therapeutic profiles.
