Sarah Römer

• Organic cation transporter 1 (OCT1), located in the sinusoidal membrane of hepatocytes, mediates hepatic uptake and elimination of weak bases and cationic compounds like metformin, morphine or fenoterol. With over 200 known substrates, OCT1 exhibits broad polyspecificity. Despite its discovery over 30 years ago, the molecular mechanisms enabling this polyspecific transport are not completely understood. Recent cryo-electron microscopy (cryo-EM) structures provide snapshots of OCT1 in multiple conformational states. However, these static structures involve only a few ligands, and functional studies have focused on just two substrates, limiting insight into the precise mechanisms underlying general transport mechanisms and OCT1 polyspecificity. This work aimed to deepen the understanding of the structural determinants underlying general transport mechanisms and polyspecificity of OCT1. To this end, first, alanine scanning mutagenesis was performed on residues highlighted by cryo-EM structures as potentially involved in substrate transport, followed by functional analysis in HEK293 cells. By applying extended mutagenesis, ligand structure walking, and molecular dynamics (MD) simulations, the specific contribution of each residue—whether to general transport mechanisms or direct substrate interaction—was evaluated. Second, hypothesis-free approaches such as species comparisons and the analysis of substrate-dependent differences in inhibition potency were conducted to identify mechanisms underlying polyspecificity that are not apparent from cryo-EM structural analysis alone. Uptake of a broad substrate spectrum in alanine mutants revealed highly diverse contribution of residues flanking the substrate binding pocket to transport. While mutations at E386, R439, and W354 completely abolished transport, I446 showed no specific involvement. Importantly substrate-specific effects were observed. They ranged from retained uptake of single substrates in the Y361A mutant to distinct, substrate-dependent involvement in transport for W217A and F244A. Combining functional validation with MD simulations, the YER motif (Y361, E386, R439) was identified to contribute to the closure of the outer gate of the binding pocket, whereas W354 enables inner gate opening and stabilizes the outward-open state. In contrast, W217 and F244 shape the binding pocket and stabilize substrate binding through π–π interactions, with their contribution in transport decreasing as substrate size and lipophilicity increase. Generally, small substrates like metformin were strongly affected by each mutant, suggesting that interactions across multiple residues are required. These interactions are significantly affected by the altered size of the binding pocket upon mutation, which probably prevents sufficient triggering of the transport mechanism. Supporting that uptake of metformin is highly sensitive to alterations within the binding pocket, a comparative analysis of human and mouse OCT1 revealed species-specific differences in metformin uptake. These differences were linked to transmembrane helices (TMH) 2 and TMH3—helices indirectly influencing metformin binding sites or transport mechanics. Consequently, these minor structural differences resulted in an estimated 11-fold higher metformin concentration in mice than in human liver. This highlights the importance of accounting for such interspecies differences during preclinical drug development to ensure accurate translation. Using a cocktail of eight substrates (victim drugs), differences in the substrate-dependent inhibition of clinically relevant inhibitors (perpetrators) were identified. These differences led to varying predictions of drug-drug interaction (DDI) risk depending on the victim drug used—a mechanism to be considered in future preclinical DDI studies involving OCTs. Moreover, substrate-dependent differences in inhibition led to the identification of two distinct inhibitor groups that affected substrate transport in opposing ways, providing an additional tool to analyze polyspecificity. In summary, understanding OCT1 polyspecificity requires consideration of four key factors: First, the mechanisms underlying conformational changes that must be triggered by the substrate. Second, direct substrate–residue interactions that stabilize the substrate within the binding pocket. Third, indirect contributions from residues that shape the size and geometry of the pocket, creating steric constraints that determine substrate selectivity. And fourth, dynamic effects from TMHs not directly involved in binding, which modulate conformational flexibility and thus facilitate or restrict the conformational transitions necessary for transport. Overall, this underscores the value of understanding OCT1’s molecular transport mechanisms to improve preclinical development by enabling better prediction of new molecular entities (NMEs) and their potential to act as substrates or inhibitors of OCT1—thereby improving DDI predictions and consequently enhancing drug safety.