Conserved allosteric inhibition mechanism in SLC1 transporters

Abstract

Excitatory Amino Acid Transporter 1 (EAAT1) is a plasma-membrane glutamate transporter belonging to the SLC1 family of solute carriers . It plays a key role in neurotransmitter transport and contributes to the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, a highly potent and non-competitive inhibitor of EAAT1. Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine, serine and alanine between intracellular and extracellular compartments in a Na+ dependent manner. ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2 with relatively low affinity. Guided by computational modeling using ligand docking and molecular dynamics (MD) simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double mutant transporter, 100% of the inhibitory effect of UCPH-101 on anion current could be restored, and the apparent affinity was increased (Ki = 9.3 mM), much closer to the EAAT1 value of 0.6 mM. Finally, we identify a novel non-competitive ASCT2 inhibitor, identified through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective inhibitors can be designed that target the ASCT2 allosteric binding site.

Comprehensive Characterization of LAT1 Cholesterol-Binding Sites

“…Additionally, designing small molecules that balance hydrophobicity for membrane permeability with adequate solubility and affinity is highly complex. Nevertheless, allosteric modulators have successfully targeted other transporters, including inhibitors for EAATs 64 and ASCT2 65 providing a proof-of-concept for this approach.…”

Computational Chemistry in Structure-Based Solute Carrier Transporter Drug Design: Recent Advances and Future Perspectives

“…In recent years, the strategy of combining structural biology techniques (i.e., X-ray and cryo-EM) with computational methods (i.e., computational modeling and high-throughput virtual screening) has been used to determine the structures of the target proteins and to identify potential allosteric sites. , The allosteric sites have been used to facilitate the identification of novel inhibitors from large compound libraries. However, allosteric sites always vary in size, shape, and location in targeting proteins.…”

Exploring Amino Acid Transporters as Therapeutic Targets for Cancer: An Examination of Inhibitor Structures, Selectivity Issues, and Discovery Approaches

“…* Indicates inhibitors with disputed inhibitory potency. For more information, see the main text [ 72 , 75 , 77 , 78 , 80 , 81 , 84 , 86 , 87 , 88 , 89 , 90 ].…”

“…Allosteric sites can be elusive to target, given that most knowledge is based on endogenous substrates that bind to the orthosteric binding site. However, as previously mentioned, recent studies have helped elucidate how ASCT2 might be inhibited allosterically [ 90 ]. Covalent inhibitors overcome the competitiveness problem by leading to a prolonged inhibition of the amino acid transporter because of covalently binding to the protein.…”