Conserved allosteric inhibition mechanism in SLC1 transporters
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eLife assessment
The goal of this study is to identify allosteric modulators of a SLC-1 amino acid transporter, ASCT2, which has been implicated in cancer progression. By combining computational and docking methods with functional measurements, this study provides solid evidence for specific aspects of allosteric SLC-1 inhibition mechanisms. The findings are important to transporter mechanism and pharmacology.
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Abstract
Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, apotent, 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 between intracellular and extracellular compartments . 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. Guided by computational modeling using ligand docking and molecular dynamics 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 could be restored, and the apparent affinity was increased ( K i = 4.3 μM), much closer to the EAAT1 value of 0.6 μM. Finally, we identify a novel non-competitive ASCT2 inhibitor, 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 ASCT2 allosteric inhibitors can be designed .
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Author Response
Reviewer #1 (Public Review):
The current study melds computational and docking methods with functional measurements in a systematic approach: first, they analyze the mechanism of inhibitor binding to EAAT2; second, they mutate ASCT to resemble EAAT and show that the general binding pocket and inhibition mechanism are conserved; third, they perform an in silico screen to identify compounds that bind to the WT ASCT binding pocket; fourth, they perform electrophysiological assays showing that this novel compound allosterically modulates ASCT function. This is a complete and comprehensive study with extensive experimental support for the major conclusions. The authors identify an allosteric ASCT inhibitor, and although only partial inhibition is achieved, this study serves as proof-of-concept that this site can be …
Author Response
Reviewer #1 (Public Review):
The current study melds computational and docking methods with functional measurements in a systematic approach: first, they analyze the mechanism of inhibitor binding to EAAT2; second, they mutate ASCT to resemble EAAT and show that the general binding pocket and inhibition mechanism are conserved; third, they perform an in silico screen to identify compounds that bind to the WT ASCT binding pocket; fourth, they perform electrophysiological assays showing that this novel compound allosterically modulates ASCT function. This is a complete and comprehensive study with extensive experimental support for the major conclusions. The authors identify an allosteric ASCT inhibitor, and although only partial inhibition is achieved, this study serves as proof-of-concept that this site can be targeted in diverse SLC-1 transporters as an allosteric inhibitory site.
We would like to thank Reviewer #1 for the encouraging comments.
Reviewer #2 (Public Review):
This study set out to explore the nature of a previously described non-competitive and selective inhibitor of the human glutamate transporter, EAAT1 and to explore if this mechanism was conserved across the glutamate transporter family. The non-competitive nature of UCHPH-101 inhibition of EAAT1 has previously been demonstrated with both functional analysis and structures of EAAT1. Here, the authors use detailed electrophysiology analysis to confirm this mechanism of inhibition and to demonstrate that the inhibitor slows the steps of the transport cycle associated with substrate translocation, rather than substrate or sodium ion binding. These findings agree with previous studies that have shown that the compound binds at the interface of the transport and scaffold domains in EAAT1, two domains that are required to move relative to each other for the transport process to occur. UCPH-101 also prevents the transporter from entering an anion-conducting state, which agrees with a recent structure and MD simulations of EAAT1 that demonstrate movements of the transport domain relative to the scaffold domain are required for the EAAT1 to move into the anion-conducting state and support the mechanism of UCPH-101 inhibition confirmed in this study (PMID: 35192345; PMID: 33597752).
While UCPH-101 has been shown to be selective for EAAT1 over other human glutamate transporter subtypes (notably EAAT2 and EAAT3), Dong et al., show that this inhibitor can also reduce transport by another member of the SLC1A family, a neutral amino acid exchanger, ASCT2. Using MD simulations and functional analysis, they show that UCPH-101 acts as a partial, low-affinity inhibitor of ASCT2 and identify two amino acid residues in the binding site that appear to be responsible for the different affinities for EAAT1 and ASCT2. Indeed, when these two residues are changed to the corresponding residues in EAAT1, UCPH-101 becomes a full inhibitor of ASCT2 with an increased affinity.
ASCT2 is a neutral amino acid transporter that can transport glutamine and it is known to be upregulated in several cancers. Thus, finding new compounds and novel ways to inhibit ASCT2 is worthy of investigation. In the last section of this study, the authors conduct a virtual screen of 3.8 million compounds to identify other compounds that could bind to this allosteric site in ASCT2. One compound was identified, and while it had relative low affinity it provides the basis for further exploration of this site.
We would like to thank Reviewer #2 for the thoughtful comments.
Reviewer #3 (Public Review):
Using whole-cell patch-clamp measurements, the authors nicely elaborate the competitive inhibition mechanism of UCPH-101 on EAAT1, concluding that it blocks conformational changes during transmembrane translocation, without inhibiting Na+/glutamate binding. The authors demonstrate that UCPH-101 binds to ASCT2 with strongly reduced affinity. Informed by sequence comparison between EAAT1 and ASCT2, the authors identify a pair of mutations, which makes the putative allosteric-binding pocket (which has been identified by crystallography earlier) in ASCT2 more similar to EAAT1 and restores the inhibitory effect of UCPH-101 in ASCT2. Overall, the electrophysiological experiments appear sound and convincing.
We appreciate the kind words.
Furthermore, using virtual screening against the UCPH-101 binding pocket in ASCT2, the authors identified a novel (non-UCPH-101-like) compound #302 that they experimentally demonstrate to also inhibit ASCT-2. However, the study lacks a detailed investigation of the inhibition mechanism of this compound and it remains unclear if #302 also mediates allosteric inhibition as the authors propose. Furthermore, the study lacks any experimental verification of the assumed binding site of #302.
We agree. Therefore, we have now added more detailed experiments on compound #302 inhibition mechanism, confirming allosteric inhibition (new Fig. G and I).
In addition, the study includes molecular-dynamics (MD) simulations on interactions of UCPH101 with EAAT1 and ASCT2. These simulations intend to support the interpretations of the electrophysiological experiments, i.e., relatively tight interactions of UCPH-101 with EAAT1 and weaker binding to ASCT2, which can be restored using two point-mutations in ASCT-2. Unfortunately, this is a relatively weak part of the study. Due to the lack of any convergence analysis, the statistical significance of the drawn conclusions remains unclear. Furthermore, since it is not reported how UCPH-101 has been parameterized, the chemical accuracy of these models is unclear.
We now add information on the UCPH-101 parametrization protocol, and we have extended the time of MD simulations. Also, we have created additional trajectories for the atom distances between amino acid substrate and ASCT2 side chain in the substrate binding site, providing another data point on convergence in the substrate binding site, which should be unaffected by UCPH-101 binding, according to the experimental data.
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eLife assessment
The goal of this study is to identify allosteric modulators of a SLC-1 amino acid transporter, ASCT2, which has been implicated in cancer progression. By combining computational and docking methods with functional measurements, this study provides solid evidence for specific aspects of allosteric SLC-1 inhibition mechanisms. The findings are important to transporter mechanism and pharmacology.
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Reviewer #1 (Public Review):
The current study melds computational and docking methods with functional measurements in a systematic approach: first, they analyze the mechanism of inhibitor binding to EAAT2; second, they mutate ASCT to resemble EAAT and show that the general binding pocket and inhibition mechanism are conserved; third, they perform an in silico screen to identify compounds that bind to the WT ASCT binding pocket; fourth, they perform electrophysiological assays showing that this novel compound allosterically modulates ASCT function. This is a complete and comprehensive study with extensive experimental support for the major conclusions. The authors identify an allosteric ASCT inhibitor, and although only partial inhibition is achieved, this study serves as proof-of-concept that this site can be targeted in diverse SLC-1 …
Reviewer #1 (Public Review):
The current study melds computational and docking methods with functional measurements in a systematic approach: first, they analyze the mechanism of inhibitor binding to EAAT2; second, they mutate ASCT to resemble EAAT and show that the general binding pocket and inhibition mechanism are conserved; third, they perform an in silico screen to identify compounds that bind to the WT ASCT binding pocket; fourth, they perform electrophysiological assays showing that this novel compound allosterically modulates ASCT function. This is a complete and comprehensive study with extensive experimental support for the major conclusions. The authors identify an allosteric ASCT inhibitor, and although only partial inhibition is achieved, this study serves as proof-of-concept that this site can be targeted in diverse SLC-1 transporters as an allosteric inhibitory site.
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Reviewer #2 (Public Review):
This study set out to explore the nature of a previously described non-competitive and selective inhibitor of the human glutamate transporter, EAAT1 and to explore if this mechanism was conserved across the glutamate transporter family. The non-competitive nature of UCHPH-101 inhibition of EAAT1 has previously been demonstrated with both functional analysis and structures of EAAT1. Here, the authors use detailed electrophysiology analysis to confirm this mechanism of inhibition and to demonstrate that the inhibitor slows the steps of the transport cycle associated with substrate translocation, rather than substrate or sodium ion binding. These findings agree with previous studies that have shown that the compound binds at the interface of the transport and scaffold domains in EAAT1, two domains that are …
Reviewer #2 (Public Review):
This study set out to explore the nature of a previously described non-competitive and selective inhibitor of the human glutamate transporter, EAAT1 and to explore if this mechanism was conserved across the glutamate transporter family. The non-competitive nature of UCHPH-101 inhibition of EAAT1 has previously been demonstrated with both functional analysis and structures of EAAT1. Here, the authors use detailed electrophysiology analysis to confirm this mechanism of inhibition and to demonstrate that the inhibitor slows the steps of the transport cycle associated with substrate translocation, rather than substrate or sodium ion binding. These findings agree with previous studies that have shown that the compound binds at the interface of the transport and scaffold domains in EAAT1, two domains that are required to move relative to each other for the transport process to occur. UCPH-101 also prevents the transporter from entering an anion-conducting state, which agrees with a recent structure and MD simulations of EAAT1 that demonstrate movements of the transport domain relative to the scaffold domain are required for the EAAT1 to move into the anion-conducting state and support the mechanism of UCPH-101 inhibition confirmed in this study (PMID: 35192345; PMID: 33597752).
While UCPH-101 has been shown to be selective for EAAT1 over other human glutamate transporter subtypes (notably EAAT2 and EAAT3), Dong et al., show that this inhibitor can also reduce transport by another member of the SLC1A family, a neutral amino acid exchanger, ASCT2. Using MD simulations and functional analysis, they show that UCPH-101 acts as a partial, low-affinity inhibitor of ASCT2 and identify two amino acid residues in the binding site that appear to be responsible for the different affinities for EAAT1 and ASCT2. Indeed, when these two residues are changed to the corresponding residues in EAAT1, UCPH-101 becomes a full inhibitor of ASCT2 with an increased affinity.
ASCT2 is a neutral amino acid transporter that can transport glutamine and it is known to be upregulated in several cancers. Thus, finding new compounds and novel ways to inhibit ASCT2 is worthy of investigation. In the last section of this study, the authors conduct a virtual screen of 3.8 million compounds to identify other compounds that could bind to this allosteric site in ASCT2. One compound was identified, and while it had relative low affinity it provides the basis for further exploration of this site.
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Reviewer #3 (Public Review):
Using whole-cell patch-clamp measurements, the authors nicely elaborate the competitive inhibition mechanism of UCPH-101 on EAAT1, concluding that it blocks conformational changes during transmembrane translocation, without inhibiting Na+/glutamate binding. The authors demonstrate that UCPH-101 binds to ASCT2 with strongly reduced affinity. Informed by sequence comparison between EAAT1 and ASCT2, the authors identify a pair of mutations, which makes the putative allosteric-binding pocket (which has been identified by crystallography earlier) in ASCT2 more similar to EAAT1 and restores the inhibitory effect of UCPH-101 in ASCT2. Overall, the electrophysiological experiments appear sound and convincing.
Furthermore, using virtual screening against the UCPH-101 binding pocket in ASCT2, the authors identified a …
Reviewer #3 (Public Review):
Using whole-cell patch-clamp measurements, the authors nicely elaborate the competitive inhibition mechanism of UCPH-101 on EAAT1, concluding that it blocks conformational changes during transmembrane translocation, without inhibiting Na+/glutamate binding. The authors demonstrate that UCPH-101 binds to ASCT2 with strongly reduced affinity. Informed by sequence comparison between EAAT1 and ASCT2, the authors identify a pair of mutations, which makes the putative allosteric-binding pocket (which has been identified by crystallography earlier) in ASCT2 more similar to EAAT1 and restores the inhibitory effect of UCPH-101 in ASCT2. Overall, the electrophysiological experiments appear sound and convincing.
Furthermore, using virtual screening against the UCPH-101 binding pocket in ASCT2, the authors identified a novel (non-UCPH-101-like) compound #302 that they experimentally demonstrate to also inhibit ASCT-2. However, the study lacks a detailed investigation of the inhibition mechanism of this compound and it remains unclear if #302 also mediates allosteric inhibition as the authors propose. Furthermore, the study lacks any experimental verification of the assumed binding site of #302.
In addition, the study includes molecular-dynamics (MD) simulations on interactions of UCPH-101 with EAAT1 and ASCT2. These simulations intend to support the interpretations of the electrophysiological experiments, i.e., relatively tight interactions of UCPH-101 with EAAT1 and weaker binding to ASCT2, which can be restored using two point-mutations in ASCT-2. Unfortunately, this is a relatively weak part of the study. Due to the lack of any convergence analysis, the statistical significance of the drawn conclusions remains unclear. Furthermore, since it is not reported how UCPH-101 has been parameterized, the chemical accuracy of these models is unclear.
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