Silva, D. B. | Search bioRxiv

bioRxiv2023

The full spectrum of OCT1 (SLC22A1) mutations bridges transporter biophysics to drug pharmacogenomics

Membrane transporters play a fundamental role in the tissue distribution of endogenous compounds and xenobiotics and are major determinants of efficacy and side effects profiles. Polymorphisms within these drug transporters result in inter-individual variation in drug response, with some patients not responding to the recommended dosage of drug whereas others experience catastrophic side effects. For example, variants within the major hepatic Human organic cation transporter OCT1 (SLC22A1) can change endogenous organic cations and many prescription drug levels. To understand how variants mechanistically impact drug uptake, we systematically study how all known and possible single missense and single amino acid deletion variants impact expression and substrate uptake of OCT1. We find that human variants primarily disrupt function via folding rather than substrate uptake. Our study revealed that the major determinants of folding reside in the first 300 amino acids, including the first 6 transmembrane domains and the extracellular domain (ECD) with a stabilizing and highly conserved stabilizing helical motif making key interactions between the ECD and transmembrane domains. Using the functional data combined with computational approaches, we determine and validate a structure-function model of OCT1s conformational ensemble without experimental structures. Using this model and molecular dynamic simulations of key mutants, we determine biophysical mechanisms for how specific human variants alter transport phenotypes. We identify differences in frequencies of reduced function alleles across populations with East Asians vs European populations having the lowest and highest frequency of reduced function variants, respectively. Mining human population databases reveals that reduced function alleles of OCT1 identified in this study associate significantly with high LDL cholesterol levels. Our general approach broadly applied could transform the landscape of precision medicine by producing a mechanistic basis for understanding the effects of human mutations on disease and drug response.

biophysics↗

bioRxiv2022

Describing Inhibitor Specificity for the Amino Acid Transporter LAT1 from Metainference Simulations

The human L-type amino acid transporter 1 (LAT1; SLC7A5) is a membrane transporter of amino acids, thyroid hormones, and drugs such as the Parkinsons disease drug L-Dopa. LAT1 is found in the blood-brain-barrier (BBB), testis, bone marrow, and placenta, and its dysregulation has been associated with various neurological diseases such as autism and epilepsy as well as cancer. In this study, we combine metainference molecular dynamics (MD) simulations, molecular docking, and experimental testing, to characterize LAT1-inhibitor interactions. We first conducted a series of molecular docking experiments to identify the most relevant interactions between LAT1s substrate binding site and ligan ds, including both inhibitors and substrates. We then performed metainference MD simulations using cryo-EM structures in different conformations of LAT1 with the electron density map as a spatial restraint, to explore the inherent heterogeneity in the structures. We analyzed the LAT1 substrate binding site to map important LAT1-ligand interactions as well as newly described druggable pockets. Finally, this analysis guided the discovery of previously unknown LAT1 ligands using virtual screening and cellular uptake experiments. Our results improve our understanding of LAT1-inhibitor recognition, providing a framework for rational design of future lead compounds targeting this key drug target. Statement of SignificanceLAT1 is a membrane transporter of amino acids, thyroid hormones, and therapeutic drugs, that is primarily found in the BBB and placenta, as well as in tumor cells of several cancer types. We combine metainference MD simulations, molecular docking, and experimental testing, to characterize LAT1-inhibitor interactions. Our computational analysis predicts S66, G67, F252, G255, Y259, W405 are critical residues for inhibitor binding and druggable sub-pockets in the outward-occluded conformation that are ideal for LAT1 inhibitor discovery. Using virtual screening and functional testing, we discovered multiple LAT1 inhibitors with diverse scaffolds and binding modes. Our results improve our understanding of LAT1s structure and function, providing a framework for development of future therapeutics targeting LAT1 and other SLC transporters.

biophysics↗