Estropipate

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.

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.

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.

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.

OATP2B1, encoded by SLCO2B1, is a drug transporter expressed widely throughout the body in tissues such as the intestine and liver. Genetic variation of this transporter may lead to altered disposition of OATP2B1 substrate drugs, but especially the effects of rare variants are poorly understood. The aim of this study was to characterize the effects of naturally occurring missense single nucleotide variants of SLCO2B1 (c.601G>A, c.935G>A, c.953C>T, c.1175C>, c.1457C>T, c.1559G>C, c.1596C>A, and the c.601G>A + c.935G>A haplotype) on the in vitro functionality of OATP2B1. To characterize transport activity, cellular uptake of dibromofluorescein, 5-carboxyfluorescein, estrone sulfate, and rosuvastatin was compared in OATP2B1 reference- and variant-expressing HEK293 cells. The abundance of OATP2B1 variants in HEK293 crude membrane preparations was quantified with LC-MS/MS-based quantitative targeted absolute proteomics analysis. Variant c.1559G>C impaired OATP2B1-mediated uptake of all tested substrates almost completely, but protein abundance was not reduced to the same extent. Other studied variants had comparable or only modestly reduced protein abundance and transport function compared to reference OATP2B1. These results can be utilized to understand findings from clinical pharmacogenetic studies. More importantly, the results can aid in predicting the consequences of rare variants, such as the loss-of-function variant c.1559G>C, which can be difficult to detect in clinical studies.