Investigation of Hits against the GLP-1 Receptor Identified by Affinity Selection Mass Spectrometry: Hit Expansion, Structure-Activity Relationships, and Selectivity Profiling

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Abstract:

A prior high-throughput affinity selection mass spectrometry (ASMS) screen of 50,000 compounds against the GLP-1 receptor expressed in copolymer-based nanodiscs using the NativeMP^TM platform by Cube Biotech identified 34 confirmed hit compounds. Here we report the hit expansion and characterization of three distinct chemical series emerging from that screen. Structurally similar analogs were acquired from commercial sources. Three chemical scaffolds were characterized: a pyrazolinone/indazolone lactam series (20 analogs), an aminopyrimidine series (16 analogs), and a benzimidazole-amide series (11 analogs). Key structure-activity findings included: a 5–100Å~ potency advantage for direct C4-aryl over aryl-ketone substituents in the lactam series; a 26-fold regiochemistry penalty for meta- vs. para-substitution on the pendant phenyl; dominant halogenation effects in the aminopyrimidine series (Br ≈ CF₃ > Cl >> H); and a 3Å~ activity gain from a constrained piperidine linker in the benzimidazole-amide series. Selective GLP1R binders were subsequently profiled against two related off-targets GIPR and GCGR all in nanodisc format prepared by Cube Biotech. GCGR was the dominant cross-reactivity challenge (39% of compounds preferred GCGR). Halogenated compounds showed 3Å~ better selectivity on average, and competition assays confirmed orthosteric binding within each series. This work illustrates the utility of nanodisc-based ASMS for rapid, label-free hit expansion and selectivity differentiation of membrane protein binders.

Conclusion:

Small molecule engagement of the GLP-1 receptor using affinity selection mass spectrometry (ASMS) proved to be a productive starting point for drug discovery, with a 50,000 compound diversity screen identifying three structurally distinct hit series that could be expanded and characterized in depth. Hit expansion across 52 analogs established clear structure-activity relationships within 3 scaffolds. Selectivity profiling against the closely related GIPR and GCGR confirmed that GLP1R-selective binding is achievable (22 of 44 compounds showed net GLP1R preference) but that GCGR cross-reactivity, driven by ~48% transmembrane sequence identity, represents the primary differentiability challenge. Taken together, this work demonstrates that nanodisc-based ASMS is a powerful and efficient platform for the iterative characterization of membrane protein binders, from primary hit identification through analog profiling and selectivity differentiation, without the need for labeled compounds, surface immobilization, or extensive assay development, and that it is directly applicable to other GPCRs and membrane-embedded targets where selectivity among structurally homologous family members is a key challenge.

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