LAUSR

Abstract Disclosure: A. Ulloa-Aguirre: None. E. Jardón-Valadez: None. T. Zariñán: None. R. Gutiérrez-Sagal: None. S. Nataraja: TocopheRx. H.N. Yu: CanWell Pharma Inc.. The FSHR plays an essential role in reproduction. Variations in the structure of this GPCR may result in impaired plasma membrane (PM) expression and function due to misfolding and intracellular retention of the defective receptor leading to reproductive failure. The present study took advantage of the recent cryo-electron microscopy (Cryo-EM) structure of the FSHR, further refined applying molecular dynamics simulations, to characterize the structural dynamics and functional features of a novel benzoindazolmethanone, small molecule allosteric agonist (CAN1405; CanWell Pharma Inc., Woburn, MA, USA) bound to the wild-type FSHR and its variants D408Y and I423T; we also analyzed its properties as a pharmacoperone to rescue PM expression of 13 trafficking-defective FSHR variants. Our refined MD simulations revealed key conformational changes and interactions within the transmembrane (TM) helices of FSHR, highlighting potential allosteric binding sites critical for receptor activation. CAN1405 showed promising binding affinity and efficacy to activate the FSHR but behaved as a biased agonist, selectively stimulating cAMP production but not beta arrestin- and PKA-dependent ERK1/2 phosphorylation in HEK293 FSHR+ cells. Three trafficking defective variants in the ectodomain of the FSHR (A189V; N191I; and D224V) and two variants located in the TM2 and 3 (A462P and P504S) failed to traffic to the PM in response to CAN1405. In contrast, in eight misfolded variants located in the TM2 (D408Y, A419T, and I423T), extracellular loop 2 (P519T), and TM6 (A575V, P587H, F591S, and L597I) CAN1405 rescued PM expression. Functional studies showed that after CAN1405 removal, all rescued FSHRs responded to the orthosteric agonist in terms of cAMP production and ERK1/2 phosphorylation. The conformational changes provoked by the D408Y and I423T substitutions on the FSHR, did not lead to functional selectivity in response to FSH when tested in HEK293 cells expressing the mutant FSHRs and with CRISPR-Cas9 silenced beta-arrestins. These findings provide a comprehensive structural and dynamic framework for understanding FSHR signal transduction and offer a promising therapeutic strategy for treating mutation-induced receptor dysfunction. In addition, this study underscores the synergistic potential of Cryo-EM and computational biophysics in drug discovery and precision medicine. [Study supported by grant IN208323 from PAPIIT-UNAM, Mexico (to A.U.-A.)]. Presentation: Sunday, July 13, 2025