Soloxolone N-3-(Dimethylamino)propylamide Restores Drug Sensitivity of Tumor Cells with Multidrug-Resistant Phenotype via Inhibition of P-Glycoprotein Efflux Function

Multidrug resistance (MDR) remains a significant challenge in cancer therapy, primarily due to the overexpression of transmembrane drug transporters, with P-glycoprotein (P-gp) being a central focus. Consequently, the development of P-gp inhibitors has emerged as a promising strategy to combat MDR. Given the P-gp targeting potential of soloxolone amides previously predicted by us by an absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, the aim of the current study was to experimentally verify their P-gp inhibitory and MDR reversing activities in vitro. Screening of soloxolone amides as modulators of P-gp using molecular docking and cellular P-gp substrate efflux assays revealed the ability of compound <b>4</b> bearing a <i>N</i>-3-(dimethylamino)propylamide group to interact with the active site of P-gp and inhibit its transport function. Blind and site-specific molecular docking accompanied by a kinetic assay showed that <b>4</b> directly binds to the P-gp transmembrane domain with a binding energy similar to that of zosuquidar, a third-generation P-gp inhibitor (ΔG = -10.3 kcal/mol). In vitro assays confirmed that compound <b>4</b> enhanced the uptake of Rhodamine 123 (Rho123) and doxorubicin (DOX) by the P-gp-overexpressing human cervical carcinoma KB-8-5 (by 10.2- and 1.5-fold, respectively (<i>p</i> < 0.05, unpaired <i>t</i>-test)) and murine lymphosarcoma RLS40 (by 15.6- and 1.75-fold, respectively (<i>p</i> < 0.05, unpaired <i>t</i>-test)) cells at non-toxic concentrations. In these cell models, <b>4</b> showed comparable or slightly higher activity than the reference inhibitor verapamil (VPM), with the most pronounced effect of the hit compound in Rho123-loaded RLS40 cells, where <b>4</b> was 2-fold more effective than VPM. Moreover, <b>4</b> synergistically restored the sensitivity of KB-8-5 cells to the cytotoxic effect of DOX, demonstrating MDR reversal activity. Based on the data obtained, <b>4</b> can be considered as a drug candidate to combat the P-gp-mediated MDR of tumor cells and semisynthetic triterpenoids, with amide moieties in general representing a promising scaffold for the development of novel therapeutics for tumors with low susceptibility to antineoplastic agents.