Targeting SLC transporters: small molecules as modulators and therapeutic opportunities.

Solute carrier (SLCs) transporters mediate the transport of a broad range of solutes across biological membranes. Dysregulation of SLCs has been associated with various pathologies, including metabolic and neurological disorders, as well as cancer and rare diseases. SLCs are therefore emerging as key targets for therapeutic intervention with several recently approved drugs targeting these proteins. Unlocking this large and complex group of proteins is essential to identifying unknown SLC targets and developing next-generation SLC therapeutics. Recent progress in experimental and computational techniques has significantly advanced SLC research, including drug discovery. Here, we review emerging topics in therapeutic discovery of SLCs, focusing on state-of-the-art approaches in structural, chemical, and computational biology, and discuss current challenges in transporter drug discovery.

l-Type amino acid transporter 1 activity of 1,2,3-triazolyl analogs of l-histidine and l-tryptophan.

A series of 1,2,3-triazole analogs of the amino acids l-histidine and l-tryptophan were modeled, synthesized and tested for l-type amino acid transporter 1 (LAT1; SLC7A5) activity to guide the design of amino acid-drug conjugates (prodrugs). These triazoles were conveniently prepared by the highly convergent Huisgen 1,3-dipolar cycloaddition (Click Chemistry). Despite comparable predicted binding modes, triazoles generally demonstrated reduced cell uptake and LAT1 binding potency relative to their natural amino acid counterparts. The structure-activity relationship (SAR) data for these triazoles has important ramifications for treating cancer and brain disorders using amino acid prodrugs or LAT1 inhibitors.

Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters.

The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (Tms) of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg), agmatine, l-arginine methyl ester, and l-arginine amide. The resulting Tms indicated stabilization of AdiC variants upon ligand binding, in which Tms and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

Mapping Functionally Important Residues in the Na+/Dicarboxylate Cotransporter, NaDC1.

Transporters from the SLC13 family couple the transport of two to four Na+ ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na+/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue VcINDY in an inward-facing conformation with one sodium ion bound at the Na1 site. In the study presented here, we modeled the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1, respectively) using an outward-facing model of VcINDY as a template and identified residues in or near the putative Na2 and Na3 cation binding sites. Guided by the structural models in both conformations, we performed site-directed mutagenesis in rbNaDC1 for residues proposed to be in the Na+ or substrate binding sites. Cysteine substitution of T474 in the predicted Na2 binding site results in an inactive protein. The M539C mutant has a low apparent affinity for both sodium and lithium cations, suggesting that M539 may form part of the putative Na3 binding site. The Y432C and T86C mutants have increased Km values for succinate, supporting their proposed location in the outward-facing substrate binding site. In addition, cysteine labeling by MTSEA-biotin shows that Y432C is accessible from the outside of the cell, and the accessibility changes in the presence or absence of Na+. The results of this study improve our understanding of substrate and ion recognition in the mammalian members of the SLC13 family and provide a framework for developing conformationally specific inhibitors against these transporters.

Chemical Modulation of the Human Oligopeptide Transporter 1, hPepT1.

In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is located across the apical membranes of the small intestine and kidney, where it serves as a high-capacity low-affinity transporter of a broad range of di- and tripeptides. hPepT1 is also overexpressed in the colon of inflammatory bowel disease (IBD) patients, where it mediates the transport of harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for prodrug substrates interacting with intracellular proteins or inhibitors blocking the transport of toxic bacterial products. In this study, we construct multiple structural models of hPepT1 representing different conformational states that occur during transport and inhibition. We then identify and characterize five ligands of hPepT1 using computational methods, such as virtual screening and QM-polarized ligand docking (QPLD), and experimental testing with uptake kinetic measurements and electrophysiological assays. Our results improve our understanding of the substrate and inhibitor specificity of hPepT1. Furthermore, the newly discovered ligands exhibit unique chemotypes, providing a framework for developing tool compounds with optimal intestinal absorption as well as future IBD therapeutics against this emerging drug target.

Structure activity relationships of benzylproline-derived inhibitors of the glutamine transporter ASCT2.

The glutamine transporter ASCT2 has been identified as a promising target to inhibit rapid growth of cancer cells. However, ASCT2 pharmacology is not well established. In this report, we performed a systematic structure activity analysis of a series of substituted benzylproline derivatives. Substitutions on the phenyl ring resulted in compounds with characteristics of ASCT2 inhibitors. Apparent binding affinity increased with increasing hydrophobicity of the side chain. In contrast, interaction of the ASCT2 binding site with specific positions on the phenyl ring was not observed. The most potent compound inhibits the ASCT2 anion conductance with a Ki of 3µM, which is in the same range as that of more bulky and higher molecular weight inhibitors recently reported by others. The experimental results are consistent with computational analysis based on docking of the inhibitors against an ASCT2 homology model. The benzylproline scaffold provides a valuable tool for further improving binding potency of future ASCT2 inhibitors.

Mutations in the Na(+)/citrate cotransporter NaCT (SLC13A5) in pediatric patients with epilepsy and developmental delay.

Mutations in the SLC13A5 gene that codes for the Na(+)/citrate cotransporter, NaCT, are associated with early onset epilepsy, developmental delay and tooth dysplasia in children. In the present study we identify additional SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and treatment strategies. There are currently no effective treatments, but some anti-epileptic drugs targeting the GABA system reduce seizure frequency. Acetazolamide, a carbonic anhydrase inhibitor and atypical anti-seizure medication decreases seizures in 4 patients. In contrast to previous reports, the ketogenic diet and fasting produce worsening of symptoms. The effects of the mutations on NaCT transport function and protein expression were examined by transient transfections of COS-7 cells. There was no transport activity from any of the mutant transporters, although some of the mutant transporter proteins were present on the plasma membrane. The structural model of NaCT suggests that these mutations can affect helix packing or substrate binding. We tested various treatments, including chemical chaperones and low temperatures, but none improve transport function in the NaCT mutants. Interestingly, coexpression of NaCT and the mutants results in decreased protein expression and activity of the wild-type transporter, indicating functional interaction. In conclusion, our study has identified additional SLC13A5 mutations in patients with chronic epilepsy starting in the neonatal period, with the mutations producing inactive Na(+)/citrate transporters.

Ligand Discovery for the Alanine-Serine-Cysteine Transporter (ASCT2, SLC1A5) from Homology Modeling and Virtual Screening.

The Alanine-Serine-Cysteine transporter ASCT2 (SLC1A5) is a membrane protein that transports neutral amino acids into cells in exchange for outward movement of intracellular amino acids. ASCT2 is highly expressed in peripheral tissues such as the lung and intestines where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. ASCT2 also plays an important role in the development of a variety of cancers such as melanoma by transporting amino acid nutrients such as glutamine into the proliferating tumors. Therefore, ASCT2 is a key drug target with potentially great pharmacological importance. Here, we identify seven ASCT2 ligands by computational modeling and experimental testing. In particular, we construct homology models based on crystallographic structures of the aspartate transporter GltPh in two different conformations. Optimization of the models' binding sites for protein-ligand complementarity reveals new putative pockets that can be targeted via structure-based drug design. Virtual screening of drugs, metabolites, fragments-like, and lead-like molecules from the ZINC database, followed by experimental testing of 14 top hits with functional measurements using electrophysiological methods reveals seven ligands, including five activators and two inhibitors. For example, aminooxetane-3-carboxylate is a more efficient activator than any other known ASCT2 natural or unnatural substrate. Furthermore, two of the hits inhibited ASCT2 mediated glutamine uptake and proliferation of a melanoma cancer cell line. Our results improve our understanding of how substrate specificity is determined in amino acid transporters, as well as provide novel scaffolds for developing chemical tools targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders.

LAT-1 activity of meta-substituted phenylalanine and tyrosine analogs.

The transporter protein Large-neutral Amino Acid Transporter 1 (LAT-1, SLC7A5) is responsible for transporting amino acids such as tyrosine and phenylalanine as well as thyroid hormones, and it has been exploited as a drug delivery mechanism. Recently its role in cancer has become increasingly appreciated, as it has been found to be up-regulated in many different tumor types, and its expression levels have been correlated with prognosis. Substitution at the meta position of aromatic amino acids has been reported to increase affinity for LAT-1; however, the SAR for this position has not previously been explored. Guided by newly refined computational models of the binding site, we hypothesized that groups capable of filling a hydrophobic pocket would increase binding to LAT-1, resulting in improved substrates relative to parent amino acid. Tyrosine and phenylalanine analogs substituted at the meta position with halogens, alkyl and aryl groups were synthesized and tested in cis-inhibition and trans-stimulation cell assays to determine activity. Contrary to our initial hypothesis we found that lipophilicity was correlated with diminished substrate activity and increased inhibition of the transporter. The synthesis and SAR of meta-substituted phenylalanine and tyrosine analogs is described.

LAT1 activity of carboxylic acid bioisosteres: Evaluation of hydroxamic acids as substrates.

Large neutral amino acid transporter 1 (LAT1) is a solute carrier protein located primarily in the blood-brain barrier (BBB) that offers the potential to deliver drugs to the brain. It is also up-regulated in cancer cells, as part of a tumor's increased metabolic demands. Previously, amino acid prodrugs have been shown to be transported by LAT1. Carboxylic acid bioisosteres may afford prodrugs with an altered physicochemical and pharmacokinetic profile than those derived from natural amino acids, allowing for higher brain or tumor levels of drug and/or lower toxicity. The effect of replacing phenylalanine's carboxylic acid with a tetrazole, acylsulfonamide and hydroxamic acid (HA) bioisostere was examined. Compounds were tested for their ability to be LAT1 substrates using both cis-inhibition and trans-stimulation cell assays. As HA-Phe demonstrated weak substrate activity, its structure-activity relationship (SAR) was further explored by synthesis and testing of HA derivatives of other LAT1 amino acid substrates (i.e., Tyr, Leu, Ile, and Met). The potential for a false positive in the trans-stimulation assay caused by parent amino acid was evaluated by conducting compound stability experiments for both HA-Leu and the corresponding methyl ester derivative. We concluded that HA's are transported by LAT1. In addition, our results lend support to a recent account that amino acid esters are LAT1 substrates, and that hydrogen bonding may be as important as charge for interaction with the transporter binding site.

Structure-Based Identification of Inhibitors for the SLC13 Family of Na(+)/Dicarboxylate Cotransporters.

In mammals, citric acid cycle intermediates play a key role in regulating various metabolic processes, such as fatty acid synthesis and glycolysis. Members of the sodium-dependent SLC13 transporter family mediate the transport of di- and tricarboxylates into cells. SLC13 family members have been implicated in lifespan extension and resistance to high-fat diets; thus, they are emerging drug targets for aging and metabolic disorders. We previously characterized key structural determinants of substrate and cation binding for the human NaDC3/SLC13A3 transporter using a homology model. Here, we combine computational modeling and virtual screening with functional and biochemical testing, to identify nine previously unknown inhibitors for multiple members of the SLC13 family from human and mouse. Our results reveal previously unknown substrate selectivity determinants for the SLC13 family, including key residues that mediate ligand binding and transport, as well as promiscuous and specific SLC13 small molecule ligands. The newly discovered ligands can serve as chemical tools for further characterization of the SLC13 family or as lead molecules for the future development of potent inhibitors for the treatment of metabolic diseases and aging. Our results improve our understanding of the structural components that are important for substrate specificity in this physiologically important family as well as in other structurally related transport systems.

Determinants of substrate and cation transport in the human Na+/dicarboxylate cotransporter NaDC3.

Metabolic intermediates, such as succinate and citrate, regulate important processes ranging from energy metabolism to fatty acid synthesis. Cytosolic concentrations of these metabolites are controlled, in part, by members of the SLC13 gene family. The molecular mechanism underlying Na(+)-coupled di- and tricarboxylate transport by this family is understood poorly. The human Na(+)/dicarboxylate cotransporter NaDC3 (SLC13A3) is found in various tissues, including the kidney, liver, and brain. In addition to citric acid cycle intermediates such as α-ketoglutarate and succinate, NaDC3 transports other compounds into cells, including N-acetyl aspartate, mercaptosuccinate, and glutathione, in keeping with its dual roles in cell nutrition and detoxification. In this study, we construct a homology structural model of NaDC3 on the basis of the structure of the Vibrio cholerae homolog vcINDY. Our computations are followed by experimental testing of the predicted NaDC3 structure and mode of interaction with various substrates. The results of this study show that the substrate and cation binding domains of NaDC3 are composed of residues in the opposing hairpin loops and unwound portions of adjacent helices. Furthermore, these results provide a possible explanation for the differential substrate specificity among dicarboxylate transporters that underpin their diverse biological roles in metabolism and detoxification. The structural model of NaDC3 provides a framework for understanding substrate selectivity and the Na(+)-coupled anion transport mechanism by the human SLC13 family and other key solute carrier transporters.

Pro-apoptotic meiogynin A derivatives that target Bcl-xL and Mcl-1.

The biological evaluation of a natural sesquiterpene dimer meiogynin A 1, is described as well as that of five non-natural analogues. Although active on a micromolar range on the inhibition of Bcl-xL/Bak and Mcl-1/Bid interaction, meiogynin A 1 is not cytotoxic on three cell lines that overexpress Bcl-xL and Mcl-1. Contrarily, one of its analogues 6 with an inverted configuration on the side chain and an aromatic moiety replacing the cyclohexane ring was active on both target proteins, cytotoxic on a micromolar range and was found to induce apoptosis through a classical pathway.

Virtual screening of the SAMPL4 blinded HIV integrase inhibitors dataset.

Several combinations of docking software and scoring functions were evaluated for their ability to predict the binding of a dataset of potential HIV integrase inhibitors. We found that different docking software were appropriate for each one of the three binding sites considered (LEDGF, Y3 and fragment sites), and the most suitable two docking protocols, involving Glide SP and Gold ChemScore, were selected using a training set of compounds identified from the structural data available. These protocols could successfully predict respectively 20.0 and 23.6 % of the HIV integrase binders, all of them being present in the LEDGF site. When a different analysis of the results was carried out by removing all alternate isomers of binders from the set, our predictions were dramatically improved, with an overall ROC AUC of 0.73 and enrichment factor at 10 % of 2.89 for the prediction obtained using Gold ChemScore. This study highlighted the ability of the selected docking protocols to correctly position in most cases the ortho-alkoxy-carboxylate core functional group of the ligands in the corresponding binding site, but also their difficulties to correctly rank the docking poses.

Impact of spa therapy on physical activity, sleep and heart rate variability among individuals with fibromyalgia: Results of an ancillary study.

Spa therapy is recommended to manage symptoms of fibromyalgia, but the physiological mechanisms underlying this improvement have been poorly studied. In an original study, we explored the effect of a 3-week rheumatology spa treatment for fibromyalgia patients on quality of life and with a symptom severity questionnaire. We present here the results of an ancillary study which explored three secondary criteria using objective measurement methods: diurnal actimetry for physical activity analysis, nocturnal actimetry for sleep analysis and heart rate variability. Eighty-three fibromyalgia patients were randomized to participate in an immediate 3-week rheumatological spa therapy, either a start within 6 weeks after inclusion (interventional group, n = 39) or a delayed, start 6 months after inclusion (control group, n = 44). Patients were asked to wear an actimeter (n = 56) to assess diurnal physical activity and sleep quality and a 24-h Holter ECG (n = 60) to assess nocturnal heart rate variability at baseline, 3 months and 6 months after inclusion. Time spent in sedentary and light physical activity was reduced to ∼30 min at 6 months in the interventional group (P = 0.027). Sleep quality and heart rate variability were not improved. Spa therapy made it possible to reduce sedentary activities in patients' daily life for up to 6 months afterwards, concomitant with the improvement in quality of life, pain and fatigue as highlighted in the original Thermalgi study.

Physical Activity in Long COVID: A Comparative Study of Exercise Rehabilitation Benefits in Patients with Long COVID, Coronary Artery Disease and Fibromyalgia.

Exercise in long COVID is poorly studied. Nevertheless, exerciserehabilitation could improve cardiorespiratory, muscular and autonomic functions. We aimed to investigate improvement in physical and autonomic performances of long COVID patients (n = 38) after a 4-week exercise rehabilitation program (3 sessions/week) compared to two control groups composed of coronary artery disease (n = 38) and fibromyalgia patients (n = 38), two populations for whom exercise benefits are well known. Efficacy of exercise training was assessed by a cardiopulmonary exercise test, a handgrip force test, and a supine heart rate variability recording at rest before and after the rehabilitation program. Cardiorespiratory and muscular parameters were enhanced after exercise rehabilitation in the three groups (p < 0.001). No significant difference was observed for the autonomic variables. Through this comparative study with control groups, we confirm and reinforce the interest of caring for long COVID patients without post-exertional symptom exacerbation by exercise rehabilitation of both strength and endurance training, by personalizing the program to the patient and symptoms.

The Acti-Pair program helps men with prostate cancer increase physical activity with peer support: a mixed method pilot study.

Background: Although the health benefits of physical activity (PA) are recognized, prostate cancer patients do not follow PA recommendations. Barriers to PA, whether physical, environmental or organizational, are known. Furthermore, even when these barriers are overcome, this achievement is not systematically accompanied by lifestyle change. Many strategies have shown to be effective in increasing patient adherence to PA. This study aims to assess the feasibility and the viability of the Acti-Pair program which combines three strategies: peer support, a personalized and realistic PA project, and support from health and adapted physical activity professionals in a local context. Methods and analysis: We conducted a pilot study utilizing a mixed qualitative and quantitative methodology, employing feasibility and viability assessments. Quantitative assessments included recruitment, retention adherence rates, process and potential effectiveness (PA and motivation) indicators; while qualitative methods were used to evaluate the program's practicality, suitability and usefulness. Indicators of potential effectiveness were assessed before and after the intervention using a Wilcoxon test for matched data. Qualitative data were collected through semistructured interviews conducted by two researchers with various program stakeholders. The study lasted for 3 years. Results: Twenty-four patients were recruited over a 25-month period. Forty-two percent of patients completed the program 3 months after the beginning. We recruited 14 peers and trained nine peers over a 10-month period. The program was coordinated extensively by adapted PA professionals, while health professionals were involved in recruiting patients and peers. Self-reporting of moderate to vigorous PA was increased after the Acti-Pair program initiation [42.86 (30.76) at baseline to 53.29 (50.73)]. Intrinsic motivation significantly increased after participation in the Acti-Pair program [1.76 (1.32) before the intervention vs. 2.91 (1.13) after the intervention]. The key player to support the Acti-Pair program in the field has been the PA support system. The main challenge has been the difficulty of health professionals in promoting PA. Discussion: This pilot study has shown that the Acti-Pair program is feasible and viable. It will allow us to extend the peer support intervention to other contexts and assess the effectiveness of this intervention and its generalization.

Paralog-dependent isogenic cell assay cascade generates highly selective SLC16A3 inhibitors.

Despite being considered druggable and attractive therapeutic targets, most of the solute carrier (SLC) membrane transporters remain pharmacologically underexploited. One of the reasons for this is a lack of reliable chemical screening assays, made difficult by functional redundancies among SLCs. In this study we leveraged synthetic lethality between the lactate transporters SLC16A1 and SLC16A3 in a screening strategy that we call paralog-dependent isogenic cell assay (PARADISO). The system involves five isogenic cell lines, each dependent on various paralog genes for survival/fitness, arranged in a screening cascade tuned for the identification of SLC16A3 inhibitors. We screened a diversity-oriented library of ∼90,000 compounds and further developed our hits into slCeMM1, a paralog-selective and potent SLC16A3 inhibitor. By implementing chemoproteomics, we showed that slCeMM1 is selective also at the proteome-wide level, thus fulfilling an important criterion for chemical probes. This study represents a framework for the development of specific cell-based drug discovery assays.

Management of Long COVID-The CoviMouv' Pilot Study: Importance of Adapted Physical Activity for Prolonged Symptoms Following SARS-CoV2 Infection.

Context: After a COVID-19 infection, some patients have persistent symptoms, the most common is fatigue. To prevent it from becoming chronic (post-COVID-19 syndrome), early management before 3 months could be useful. Exercise and education are recommended. Objective: To assess fatigue in patients with prolonged symptoms after COVID-19 infection and who received a mixed program of remote adapted physical activity and therapeutic education. The secondary objective was to evaluate the efficacy and safety of this training method thanks to aerobic and anaerobic parameters. Methods: "CoviMouv': From Coaching in Visual to Mouv in real" is a nonrandomized controlled pilot study. Patients in telerehabilitation followed 12 remote exercise sessions and 3 therapeutic education workshops. Patients on traditional rehabilitation followed their program with a community-based physiotherapist. Results: Fatigue was reduced after the one-month intervention in both groups (p = 0.010). The majority of aerobic parameters were significantly improved, e.g., maximal oxygen uptake (p = 0.005), walking distance (p = 0.019) or hyperventilation values (p = 0.035). The anaerobic parameter was not improved (p = 0.400). No adverse event was declared. Discussion: Telerehabilitation is a good alternative when a face-to-face program is not possible. This care at an early stage of the disease could help prevent the chronicity of post-COVID-19 symptoms and the installation of vicious circles of physical deconditioning. A larger study would be necessary.

Targeting Solute Carrier Transporters through Functional Mapping.

Solute carrier (SLC) transporters are emerging drug targets. Identifying the molecular determinants responsible for their specific and selective transport activities and describing key interactions with their ligands are crucial steps towards the design of potential new drugs. A general functional mapping across more than 400 human SLC transporters would pave the way to the rational and systematic design of molecules modulating cellular transport.