Estrogenic activity of E2-conjugated GLP-1 is mediated by intracellular endolysosomal acidification and estrone metabolism

Estrogenic activity of E2-conjugated GLP-1 is mediated by intracellular endolysosomal acidification and estrone metabolism

Objective: Recent modifications to glucagon-like peptide 1 (GLP-1), known for its insulinotropic and satiety-inducing effects, have focused on conjugating small molecules to enable selective delivery into GLP-1R+ tissues to achieve targeted synergy and improved metabolic outcomes. Despite continued...

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Main Authors: Callum Coupland, Na Sun, Ahmed Khalil, Özüm Ezgi Karaoglu, Arkadiusz Liskiewicz, Daniela Liskiewicz, Gerald Grandl, Seun Akindehin, Gandhari Maity, Bin Yang, Brian Finan, Patrick Knerr, Jonathan D. Douros, Axel Walch, Richard DiMarchi, Matthias H. Tschöp, Timo D. Müller, Aaron Novikoff
Format: Article
Language:English
Published: Elsevier 2025-06-01
Series:Molecular Metabolism
Subjects:
Glucagon-like peptide 1
Estradiol
Peptide conjugation
Pharmacology
Metabolomics
Bioluminescence resonance energy transfer (BRET)
Online Access:http://www.sciencedirect.com/science/article/pii/S2212877825000432
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Summary:Objective: Recent modifications to glucagon-like peptide 1 (GLP-1), known for its insulinotropic and satiety-inducing effects, have focused on conjugating small molecules to enable selective delivery into GLP-1R+ tissues to achieve targeted synergy and improved metabolic outcomes. Despite continued advancements in GLP-1/small molecule conjugate strategies, the intracellular mechanisms facilitating concurrent GLP-1R signaling and small molecule cargo release remain poorly understood. Methods: We evaluate an estradiol (E2)-conjugated GLP-1 (GLP-1-CEX/E2) for relative differences in GLP-1R signaling and trafficking, and elucidate endolysosomal dynamics that lead to estrogenic activity using various live-cell, reporter, imaging, and mass-spectrometry techniques. Results: We find GLP-1-CEX/E2 does not differentially activate or traffic the GLP-1R relative to its unconjugated GLP-1 backbone (GLP-1-CEX), but uniquely internalizes the E2 moiety and stimulates estrogenic signaling. Endolysosomal pH-dependent proteolytic activity likely mediates E2 moiety liberation, as evidenced by clear amplification in estrogenic activity following co-administration with lysosomal VATPase activator EN6. The hypothesized liberated metabolite from GLP-1-CEX/E2, E2-3-ether, exhibits partial estrogenic efficacy through ERα, and is predisposed toward estrone-3-sulfate conversion. Finally, we identify relative increases in intracellular E2, estrone, and estrone-3-sulfate following GLP-1-CEX/E2 incubation in GLP-1R+ cells, demonstrating proof-of-principle for desired cargo release. Conclusion: Together, our data suggest that GLP-1-CEX/E2 depends on GLP-1R trafficking and lysosome acidification for estrogenic efficacy, with a likely conversion of the liberated E2-3-ether metabolite into estrone-3-sulfate, resulting in a residual downstream flux into active estradiol. Our current findings aim to improve the understanding of small molecule targeting and the efficacy behind GLP-1/small molecule conjugates.
ISSN:2212-8778

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