# Functional rescue and AI analysis of a human inactivating GPCR mutation using a small molecule

## TL;DR

A small molecule repurposed as a pharmacological chaperone rescues a human GPCR mutation in mice, with AI used to analyze treatment effects.

## Contribution

A repurposed GPCR ligand rescues a human LHR mutation in mice, combined with AI to classify treatment outcomes.

## Key findings

- Female mutant mice showed irregular estrous cycles and infertility due to LHR mutation.
- Pharmacological chaperone treatment restored LH signaling and estrous cyclicity in mutant females.
- AI accurately classified control, mutant, and rescued cells based on calcium signaling patterns.

## Abstract

G protein-coupled receptors (GPCRs) carry out the majority of cellular transmembrane signaling. Many pathologies have underlying GPCR mutations, most of which cause misfolding and GPCR cell surface trafficking failure. Large libraries of existing small molecule GPCR ligands could be repurposed as pharmacological chaperones (PCs) which restore mutant GPCR folding and function, presenting an exciting alternative to complex gene repair, yet such in vivo studies are limited. Therefore, as proof-of-concept, we use one such known ligand/PC, Org42599/Org43553, to show functional rescue in mice bearing an inactivating human luteinizing hormone receptor (LHR) mutation. Mutant males had delayed puberty and Leydig cell LHR signaling impairment, however, fertility was unaffected. Mutant females had irregular estrous cycles, anovulation, abrogated ovarian LHR signaling, and complete infertility. PC treatment of mutant females restored LH signaling and estrous cyclicity. To characterize treatment efficacy, we developed an AI algorithm that reliably identified inherent differences among experimental groups, enabling functional analysis of the treatment effect in vivo. Our data set the stage to integrate AI analysis with GPCR-targeting PC molecules to treat diverse GPCR-based diseases.

The repurposed small molecule ligand of a GPCR functionally rescues a human mutation in a novel preclinical mouse model. Artificial intelligence-based machine learning was employed to develop a preclinical classifier.

Female mice with a humanized mutation in the luteinizing hormone receptor exhibit irregular estrous cycles, absence of ovulation, and abrogated calcium signaling.A known ligand of the luteinizing hormone receptor, repurposed as a pharmacological chaperone, functionally rescued the mutant receptor in vivo.Artificial intelligence accurately classified the control, mutant, and rescued cells, detecting differences even in the pattern of spontaneous calcium signals.The mathematical model revealed unprecedented heterogeneity in the response of target cells.

Female mice with a humanized mutation in the luteinizing hormone receptor exhibit irregular estrous cycles, absence of ovulation, and abrogated calcium signaling.

A known ligand of the luteinizing hormone receptor, repurposed as a pharmacological chaperone, functionally rescued the mutant receptor in vivo.

Artificial intelligence accurately classified the control, mutant, and rescued cells, detecting differences even in the pattern of spontaneous calcium signals.

The mathematical model revealed unprecedented heterogeneity in the response of target cells.

The repurposed small molecule ligand of a GPCR functionally rescues a human mutation in a novel preclinical mouse model. Artificial intelligence-based machine learning was employed to develop a preclinical classifier.