Utilizing ortholog and paralog differences to identify mechanisms of polyspecificity of the organic cation transporter OCT1

• Applicant: Dr. Marleen J. Meyer-Tönnies
• Subject Area: Pharmacology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 521444400

Organic cation transporter 1 (OCT1) is strongly expressed in the sinusoidal membrane of human hepatocytes. OCT1 is polyspecific. Its substrates are structurally highly variable drugs and endogenous substances. The underlying mechanisms that confer this polyspecificity are unknown. Revealing these mechanisms will help us predict new substrates and inhibitors, to better evaluate substrate-specific effects of common OCT1 genetic variants, and the physiological relevance of OCT1 in general. OCT1 orthologs show strong, substrate-dependent differences in their transport kinetics that can be used to analyze the mechanisms of OCT1 polyspecificity. In our previous work, we observed up to 10-fold differences in transport affinity between human and mouse OCT1. Using human-mouse chimeric proteins followed by site-directed mutagenesis, we were able to identify several amino acids responsible for these major extremes in transport kinetics. In this project, we will extend this strategy to further orthologs and to the paralogs of human OCT1. This will enable us to analyze 272 additional amino acid positions (49% of the whole protein) and should help us identify additional substrate-specific interactions with OCT1. In a hypothesis-free approach, we will screen for differences in transport kinetics of selected substrates between nine mammalian OCT1 orthologs and the three human OCT paralogs. Then, we will use chimeric proteins, followed by site-directed mutagenesis to pinpoint single amino acids that confer the observed differences. Finally, we will compare the transport of structurally highly similar compounds to identify moieties that are important for the substrate-specific interactions with OCT1. We will use 23 substrates belonging to the classes of β2-adrenergics, biguanides, tropane alkaloids, and n-tetraakylammonia compounds. In an independent hypothesis-driven approach, we will compare the effects of already known key amino acids between selected OCT1 orthologs and paralogs. Beyond better understanding the mechanisms of OCT1 polyspecificity, this project will also (i) enable upscaling of OCT1 data from preclinical animal models to humans, paving the way for including OCT1 in preclinical drug development. It will (ii) deliver data about the role of OCT1 and its paralogs in the pharmacokinetics of drugs used in veterinary medicine, (iii) enable a better translation of findings from the novel OCT3 cryo-EM structure to OCT1, and (iv) may help to extend the existing data from 3D structural models to other substrates and species.

• DFG Programme: Research Grants