Characterization of substrates and inhibitors of the human heterodimeric transporter 4F2hc-LAT1 using purified protein and the scintillation proximity radioligand binding assay

Abstract

Amino acids have diverse and essential roles in many cellular functions such as in protein synthesis, metabolism and as precursors of different hormones. Translocation of amino acids and derivatives thereof across biological membranes is mediated by amino acid transporters. 4F2hc-LAT1 is a heterodimeric amino acid transporter that is composed of two subunits belonging to the SLC3 (4F2hc) and SLC7 (LAT1) solute carrier families. The ancillary protein 4F2hc is responsible for the correct trafficking and regulation of the transporter LAT1. Preclinical studies have identified 4F2hc-LAT1 as a valid anticancer target due to its importance in tumor progression. The scintillation proximity assay (SPA) is a valuable radioligand binding assay that allows the identification and characterization of ligands of membrane proteins. Here, we present a SPA ligand binding study using purified recombinant human 4F2hc-LAT1 protein and the radioligand [³H]L-leucine as tracer. Binding affinities of different 4F2hc-LAT1 substrates and inhibitors determined by SPA are comparable with previously reported K _m and IC ₅₀ values from 4F2hc-LAT1 cell-based uptake assays. In summary, the SPA is a valuable method for the identification and characterization of ligands of membrane transporters including inhibitors. In contrast to cell-based assays, where the potential interference with other proteins such as endogenous transporters persists, the SPA uses purified protein making target engagement and characterization of ligands highly reliable.

Keywords: JPH203; LAT1; Pichia pastoris; SLC7; amino acid transporter; inhibitor; membrane protein; scintillation proximity assay.

FIGURE 1

Determination of the human 4F2hc-LAT1 binding specificity for selected amino acids by SPA. All twenty proteogenic L-amino acids and D-leucine were used as competitors. A competitor concentration of 250 µM was used, which corresponds to ten times the K_m of 4F2hc-LAT1 for L-leucine (Kantipudi et al., 2020). Residual binding of the radioligand [³H]L-leucine in the presence of competitors was normalized with respect to control samples without competitors (Ctrl). Means with SD from normalized data of three independent experiments (each at least in triplicate) are displayed. The numbers above the bars represent the mean values in %.

FIGURE 2

Determination of dissociation (K _D) and inhibition constants (K _i) of selected substrates and inhibitors for human 4F2hc-LAT1 by SPA. Competitive binding curves for determination of K _D [L-leucine (A), orange], and K _i values [L-histidine (B), light blue; BCH (C), violet; JPH203 (D), green; T3 (E), blue and T4 (F), dark pink]. Determined K _D and K _i values, and 95% confidence intervals (CIs) are indicated. Mean ± standard deviation of normalized data from three independent experiments, each at least in triplicate, are shown. If not visible, error bars are smaller than symbols.

FIGURE 3

SPA binding competition experiments for l-leucine and selected structural analogues, e.g., with varying aliphatic chain lengths and number of methyl groups. Purified 4F2hc-LAT1 and [³H]L-leucine were used for SPA as described in Materials and Methods. The competitor concentration was 250 µM. Residual binding in the presence of competitors was normalized with respect to control samples without competitors (Ctrl). Means with SD from normalized data of three independent experiments (each at least in triplicate) are shown. The numbers next to the bars represent the mean values in %.

FIGURE 4

SPA binding competition experiments for l-histidine and selected structural analogues, i.e., molecules with a methyl group at different positions in the molecule. Purified 4F2hc-LAT1 and [³H]L-leucine were used for SPA as described in Materials and Methods. The competitor concentration was 250 µM. Residual binding in the presence of competitors was normalized with respect to control samples without competitors (Ctrl). Means with SD from normalized data of three independent experiments (each at least in triplicate) are shown. The numbers next to the bars represent the mean values in %.