Insights into the Transport Cycle of LAT1 and Interaction with the Inhibitor JPH203

Abstract

The large Amino Acid Transporter 1 (LAT1) is an interesting target in drug discovery since this transporter is overexpressed in several human cancers. Furthermore, due to its location in the blood-brain barrier (BBB), LAT1 is interesting for delivering pro-drugs to the brain. In this work, we focused on defining the transport cycle of LAT1 using an in silico approach. So far, studies of the interaction of LAT1 with substrates and inhibitors have not considered that the transporter must undergo at least four different conformations to complete the transport cycle. We built outward-open and inward-occluded conformations of LAT1 using an optimized homology modelling procedure. We used these 3D models and the cryo-EM structures in outward-occluded and inward-open conformations to define the substrate/protein interaction during the transport cycle. We found that the binding scores for the substrate depend on the conformation, with the occluded states as the crucial steps affecting the substrate affinity. Finally, we analyzed the interaction of JPH203, a high-affinity inhibitor of LAT1. The results indicate that conformational states must be considered for in silico analyses and early-stage drug discovery. The two built models, together with the available cryo-EM 3D structures, provide important information on the LAT1 transport cycle, which could be used to speed up the identification of potential inhibitors through in silico screening.

LAT1 (SLC7A5) catalyzes copper(histidinate) transport switching from antiport to uniport mechanism

“…Moreover, the Cu(His) 2 complex identifies a binding site close to the crucial residue for substrate gating, i.e., the F252 ( Figure 6 B), 2 the shortest contact being 2.87 Å, and matches the site of histidine binding previously described. 23 , 44 The Cu(His) 2 binding site is also not too far from residue K204, involved in the interaction with ATP 23 ( Figure 6 B). To deepen this aspect, the mutants F252A and K204Q were tested for their ability to mediate the transport of the Cu(His) 2 complex.…”

Structure-Based Design of Xanthine-Imidazopyridines and -Imidazothiazoles as Highly Potent and In Vivo Efficacious Tryptophan Hydroxylase Inhibitors

“…Transport assays were carried out in HBSS 24 h after transfection. After a triple wash with HBSS and the preincubation for 30 min with shaking, the test compound and JPH203 41 were dosed with 10 μM [3H]-leucine (probe substrate) at 10 μM to the corresponding apical compartments. The assay was performed in triplicate.…”

“…LCMS (method 3): R t = 0.997 min; MS (ESIpos) m/z = 442.2 [M + H] + . methyl) -8-((2methylimidazo[2,1-b]thiazol-6-yl)methyl)-1H-purine-2,6(3H,7H)dione (41). 39 (50 mg, 0.093 mmol) was dissolved in 4 mL MeOH and 2 mL acetone was added, followed by 23.4 mg (0.37 mmol) of NaCNBH 3 .…”

The Development of LAT1 Efflux Agonists as Mechanistic Probes of Cellular Amino Acid Stress

“…Indeed, a recent report by Bothwell et al suggested that LAT1 is a ‘harmonizer’ and not a driver of amino acid accumulation [ 11 ]. Other investigators have demonstrated the role of LAT1 in regulating mTOR [ 12 ] and how LAT1 uses different conformations to complete its transport cycle [ 13 ]. Due to its reversible modes, the uptake and export of radiolabeled leucine are accepted assays used to assess LAT1 function [ 14 ].…”