Inhibition of ABC transporters is a promising approach to overcome multidrug resistance in cancer. Herein, we report the characterization of a potent ABCG2 inhibitor, namely, chromone 4a (C4a). Molecular docking and in vitro assays using ABCG2 and P-glycoprotein (P-gp) expressing membrane vesicles of insect cells revealed that C4a interacts with both transporters, while showing selectivity toward ABCG2 using cell-based transport assays. C4a inhibited the ABCG2-mediated efflux of different substrates and molecular dynamic simulations demonstrated that C4a binds in the Ko143-binding pocket. Liposomes and extracellular vesicles (EVs) of Giardia intestinalis and human blood were used to successfully bypass the poor water solubility and delivery of C4a as assessed by inhibition of the ABCG2 function. Human blood EVs also promoted delivery of the well-known P-gp inhibitor, elacridar. Here, for the first time, we demonstrated the potential use of plasma circulating EVs for drug delivery of hydrophobic drugs targeting membrane proteins.

Evidence across G protein-coupled receptor systems indicates that subtle modifications in three-dimensional ligand architecture can profoundly influence receptor signaling. However, the contribution of conformational constraints at the Trp4 position, of the urotensin II-related peptide (URP), to signal transduction and functional selectivity at the human urotensin II receptor (hUTR) remains poorly understood. Here, we revisited a series of previously described conformationally constrained URP analogs and comprehensively characterized their signaling profiles at the hUTR. Signaling outputs of [D-Tiq4]URP, [L-Tpi4]URP, [D-Aia4]URP, and [D-Phg4]URP were evaluated using bioluminescence resonance energy transfer (BRET)-based assays to monitor Gaq, Ga12, DAG production, PKN1 activation and β-arrestin1/2 recruitment. Ligand bias was quantified using the operational model of partial agonism Log(t/KA). Despite displaying nanomolar binding affinities (ranging from 9 to 60 nM) and reaching maximal contractile responses comparable or superior to the reference URP, the analogs exhibited markedly distinct signaling signatures. [L-Tpi4]URP showed partial agonism across all pathways except Gaq, despite binding affinity comparable to URP. [D-Aia4]URP produced a more pronounced reduction in signaling outputs compared to URP, with a pronounced reduction in potency for Gaq activation. In contrast, the smaller analogs [D-Phg4]URP and [D-Tiq4]URP evoked potent Gaq activation. Notably, [D-Tiq4]URP was the only analog that preserved β-arrestin recruitment at levels comparable to URP. Collectively, these findings demonstrate that conformational restriction at Trp4 can differentially modulate hUTR signaling while preserving receptor recognition, highlighting this position as a critical structural determinant of functional selectivity and ligand-directed signaling.

One of the major challenges of studying biomarkers in tumor samples is the low quantity and quality of isolated RNA, DNA, and proteins. Additionally, the extraction methods ideally should obtain macromolecules from the same tumor biopsy, allowing better-integrated data interpretation. In this work, an in-house, low-cost, mixed-type tissue crusher combining blade and beating principles was made and the simultaneous isolation of macromolecules from human cells and tissues was achieved using TRIzol. RT-qPCR, genotyping, SDS-PAGE, and Western blot analysis were used to validate the approach. For tissue samples, RNA, DNA, and proteins resulted in an average yield of 677 ng/mg, 225 ng/mg, and 1.4 µg/mg, respectively. The same approach was validated using cell lines. The isolated macromolecule validation included the detection of mRNA levels of ATP-binding cassette (ABC) transporters through RT-qPCR, genotyping of TNFR1 (rs767455), and protein visualization through SDS-PAGE following Coomassie blue staining and Western blot. This work contributed to filling a gap in knowledge about TRIzol efficiency for the simultaneous extraction of RNA, DNA, and proteins from a single human tissue sample. A low-cost, high yield, and quality method was validated using target biomarkers of multidrug resistance mechanisms. This approach might be advantageous for future biomarker studies using different tissue specimens.