Structural and functional analysis of OATP and ABCB1-mediated transport of pentamidine analogs: An integrated in vitro and in silico approach.

Pentamidine is an FDA-approved antiparasitic drug used to treat African trypanosomiasis caused by Trypanosoma brucei, as well as Pneumocystis and Leishmania infections, however, its clinical utility is limited by poor blood-brain barrier penetration and severe side effects. In human cells, pentamidine uptake is primarily mediated by the organic cation transporter OCT1 and the organic anion-transporting polypeptide OATP1A2, while efflux occurs via the multidrug transporter P-glycoprotein (ABCB1). Here, we present a comprehensive experimental and computational characterization of six pentamidine analogs and their interactions with OATP1A2, OATP2B1, and ABCB1. Fluorescence-based uptake assays revealed that all analogs potently inhibit OATP1A2, with three compounds (4 - 6) exceeding the inhibitory potency of pentamidine. In contrast, inhibition of OATP2B1 was weaker and compound-specific, demonstrating clear transporter selectivity. Cell viability assays identified all analogs as OATP1A2 substrates, whereas in accordance with the uptake assay results, viability of OATP2B1-expressing cells was not altered by any of the tested compounds. Mechanistic studies confirmed apoptosis as the primary mode of cell death. ABCB1 overexpression substantially reduced cytotoxicity, highlighting a critical interplay between uptake and efflux transporters, and also indicating that function of ABCB1 may limit bioavailability of the compounds. Molecular docking and molecular dynamics simulations revealed that steric bulk and conformational rigidity of amidino substituents - most prominently in the N,N'-cyclohexylamidino derivative - underpin high-affinity OATP1A2 binding, while all compounds were accommodated within the ABCB1 substrate-binding cavity, consistent with efflux activity. Collectively, this study identifies OATP1A2 as a key determinant of pentamidine analog uptake and highlights the importance of transporter interplay in shaping intracellular drug exposure and pharmacological response.