The Concise Guide to PHARMACOLOGY 2023/24: Transporters.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16182. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Inhibition of GCN2 Reveals Synergy with Cell-Cycle Regulation and Proteostasis.

The integrated stress response is a signaling network comprising four branches, each sensing different cellular stressors, converging on the phosphorylation of eIF2α to downregulate global translation and initiate recovery. One of these branches includes GCN2, which senses cellular amino acid insufficiency and participates in maintaining amino acid homeostasis. Previous studies have shown that GCN2 is a viable cancer target when amino acid stress is induced by inhibiting an additional target. In this light, we screened numerous drugs for their potential to synergize with the GCN2 inhibitor TAP20. The drug sensitivity of six cancer cell lines to a panel of 25 compounds was assessed. Each compound was then combined with TAP20 at concentrations below their IC50, and the impact on cell growth was evaluated. The strongly synergistic combinations were further characterized using synergy analyses and matrix-dependent invasion assays. Inhibitors of proteostasis and the MEK-ERK pathway, as well as the pan-CDK inhibitors, flavopiridol, and seliciclib, were potently synergistic with TAP20 in two cell lines. Among their common CDK targets was CDK7, which was more selectively targeted by THZ-1 and synergized with TAP20. Moreover, these combinations were partially synergistic when assessed using matrix-dependent invasion assays. However, TAP20 alone was sufficient to restrict invasion at concentrations well below its growth-inhibitory IC50. We conclude that GCN2 inhibition can be further explored in vivo as a cancer target.

Intestinal Amino Acid Transport and Metabolic Health.

Amino acids derived from protein digestion are important nutrients for the growth and maintenance of organisms. Approximately half of the 20 proteinogenic amino acids can be synthesized by mammalian organisms, while the other half are essential and must be acquired from the nutrition. Absorption of amino acids is mediated by a set of amino acid transporters together with transport of di- and tripeptides. They provide amino acids for systemic needs and for enterocyte metabolism. Absorption is largely complete at the end of the small intestine. The large intestine mediates the uptake of amino acids derived from bacterial metabolism and endogenous sources. Lack of amino acid transporters and peptide transporter delays the absorption of amino acids and changes sensing and usage of amino acids by the intestine. This can affect metabolic health through amino acid restriction, sensing of amino acids, and production of antimicrobial peptides.

Do Amino Acid Antiporters Have Asymmetric Substrate Specificity?

Amino acid antiporters mediate the 1:1 exchange of groups of amino acids. Whether substrate specificity can be different for the inward and outward facing conformation has not been investigated systematically, although examples of asymmetric transport have been reported. Here we used LC-MS to detect the movement of 12C- and 13C-labelled amino acid mixtures across the plasma membrane of Xenopus laevis oocytes expressing a variety of amino acid antiporters. Differences of substrate specificity between transporter paralogs were readily observed using this method. Our results suggest that antiporters are largely symmetric, equalizing the pools of their substrate amino acids. Exceptions are the antiporters y+LAT1 and y+LAT2 where neutral amino acids are co-transported with Na+ ions, favouring their import. For the antiporters ASCT1 and ASCT2 glycine acted as a selective influx substrate, while proline was a selective influx substrate of ASCT1. These data show that antiporters can display non-canonical modes of transport.

Maternal heterozygosity of Slc6a19 causes metabolic perturbation and congenital NAD deficiency disorder in mice.

Nicotinamide adenine dinucleotide (NAD) is a key metabolite synthesised from vitamin B3 or tryptophan. Disruption of genes encoding NAD synthesis enzymes reduces NAD levels and causes congenital NAD deficiency disorder (CNDD), characterised by multiple congenital malformations. SLC6A19 (encoding B0AT1, a neutral amino acid transporter), represents the main transporter for free tryptophan in the intestine and kidney. Here, we tested whether Slc6a19 heterozygosity in mice limits the tryptophan available for NAD synthesis during pregnancy and causes adverse pregnancy outcomes. Pregnant Slc6a19+/- mice were fed diets depleted of vitamin B3, so that tryptophan was the source of NAD during gestation. This perturbed the NAD metabolome in pregnant Slc6a19+/- females, resulting in reduced NAD levels and increased rates of embryo loss. Surviving embryos were small and exhibited specific combinations of CNDD-associated malformations. Our results show that genes not directly involved in NAD synthesis can affect NAD metabolism and cause CNDD. They also suggest that human female carriers of a SLC6A19 loss-of-function allele might be susceptible to adverse pregnancy outcomes unless sufficient NAD precursor amounts are available during gestation. This article has an associated First Person interview with the first author of the paper.

Identification and characterization of a novel SNAT2 (SLC38A2) inhibitor reveals synergy with glucose transport inhibition in cancer cells.

SNAT2 (SLC38A2) is a sodium-dependent neutral amino acid transporter, which is important for the accumulation of amino acids as nutrients, the maintenance of cellular osmolarity, and the activation of mTORC1. It also provides net glutamine for glutaminolysis and consequently presents as a potential target to treat cancer. A high-throughput screening assay was developed to identify new inhibitors of SNAT2 making use of the inducible nature of SNAT2 and its electrogenic mechanism. Using an optimized FLIPR membrane potential (FMP) assay, a curated scaffold library of 33934 compounds was screened to identify 3-(N-methyl (4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide as a potent inhibitor of SNAT2. In two different assays an IC50 of 0.8-3 µM was determined. The compound discriminated against the close transporter homologue SNAT1. MDA-MB-231 breast cancer and HPAFII pancreatic cancer cell lines tolerated the SNAT2 inhibitor up to a concentration of 100 µM but in combination with tolerable doses of the glucose transport inhibitor Bay-876, proliferative growth of both cell lines was halted. This points to synergy between inhibition of glycolysis and glutaminolysis in cancer cells.

Impact of Inhibition of Glutamine and Alanine Transport on Cerebellar Glial and Neuronal Metabolism.

The cerebellum, or "little brain", is often overlooked in studies of brain metabolism in favour of the cortex. Despite this, anomalies in cerebellar amino acid homeostasis in a range of disorders have been reported. Amino acid homeostasis is central to metabolism, providing recycling of carbon backbones and ammonia between cell types. Here, we examined the role of cerebellar amino acid transporters in the cycling of glutamine and alanine in guinea pig cerebellar slices by inhibiting amino acid transporters and examining the resultant metabolism of [1-13C]d-glucose and [1,2-13C]acetate by NMR spectroscopy and LCMS. While the lack of specific inhibitors of each transporter makes interpretation difficult, by viewing results from experiments with multiple inhibitors we can draw inferences about the major cell types and transporters involved. In cerebellum, glutamine and alanine transfer is dominated by system A, blockade of which has maximum effect on metabolism, with contributions from System N. Inhibition of neural system A isoform SNAT1 by MeAIB resulted in greatly decreased metabolite pools and reduced net fluxes but showed little effect on fluxes from [1,2-13C]acetate unlike inhibition of SNAT3 and other glutamine transporters by histidine where net fluxes from [1,2-13C]acetate are reduced by ~50%. We interpret the data as further evidence of not one but several glutamate/glutamine exchange pools. The impact of amino acid transport inhibition demonstrates that the cerebellum has tightly coupled cells and that glutamate/glutamine, as well as alanine cycling, play a major role in that part of the brain.

Aristolochic acid-induced nephropathy is attenuated in mice lacking the neutral amino acid transporter B0AT1 (Slc6a19).

B0AT1 (Slc6a19) mediates absorption of neutral amino acids in the small intestine and in the kidneys, where it is primarily expressed in early proximal tubules (S1-S2). To determine the role of B0AT1 in nephropathy induced by aristolochic acid (AA), which targets the proximal tubule, littermate female B0AT1-deficient (Slc6a19-/-), heterozygous (Slc6a19+/-), and wild-type (WT) mice were administered AA (10 mg/kg ip) or vehicle every 3 days for 3 wk, and analyses were performed after the last injection or 3 wk later. Vehicle-treated mice lacking Slc6a19 showed normal body and kidney weight and plasma creatinine versus WT mice. The urinary glucose-to-creatinine ratio (UGCR) and urinary albumin-to-creatinine ratio (UACR) were two to four times higher in vehicle-treated Slc6a19-/- versus WT mice, associated with lesser expression of early proximal transporters Na+-glucose cotransporter 2 and megalin, respectively. AA caused tubular injury independently of B0AT1, including robust increases in cortical mRNA expression of p53, p21, and hepatitis A virus cellular receptor 1 (Havcr1), downregulation of related proximal tubule amino acid transporters B0AT2 (Slc6a15), B0AT3 (Slc6a18), and Slc7a9, and modest histological tubular damage and a rise in plasma creatinine. Absence of B0AT1, however, attenuated AA-induced cortical upregulation of mRNA markers of senescence (p16), inflammation [lipocalin 2 (Lcn2), C-C motif chemokine ligand 2 (Ccl2), and C-C motif chemokine receptor 2 (Ccr2)], and fibrosis [tissue inhibitor of metallopeptidase 1 (Timp1), transforming growth factor-ß1 (Tgfb1), and collagen type I-α1 (Col1a1)], associated with lesser fibrosis staining, lesser suppression of proximal tubular organic anion transporter 1, restoration of Na+-glucose cotransporter 2 expression, and prevention of the AA-induced fivefold increase in the urinary albumin-to-creatinine ratio observed in WT mice. The data suggest that proximal tubular B0AT1 is important for the physiology of renal glucose and albumin retention but potentially deleterious for the kidney response following AA-induced kidney injury.NEW & NOTEWORTHY Based on insights from studies manipulating glucose transport, the hypothesis has been proposed that inhibiting intestinal uptake or renal reabsorption of energy substrates has unique therapeutic potential to improve metabolic disease and kidney outcome in response to injury. The present study takes this idea to B0AT1, the major transporter for neutral amino acids in the intestine and kidney, and shows that its absence attenuates aristolochic acid-induced nephropathy.

Amino acid metabolism, transport and signalling in the liver revisited.

The liver controls the systemic exposure of amino acids entering via the gastro-intestinal tract. For most amino acids except branched chain amino acids, hepatic uptake is very efficient. This implies that the liver orchestrates amino acid metabolism and also controls systemic amino acid exposure. Although many amino acid transporters have been identified, cloned and investigated with respect to substrate specificity, transport mechanism, and zonal distribution, which of these players are involved in hepatocellular amino acid transport remains unclear. Here, we aim to provide a review of current insight into the molecular machinery of hepatic amino acid transport. Furthermore, we place this information in a comprehensive overview of amino acid transport, signalling and metabolism.

Amino Acid Homeostasis in Mammalian Cells with a Focus on Amino Acid Transport.

Amino acid homeostasis is maintained by import, export, oxidation, and synthesis of nonessential amino acids, and by the synthesis and breakdown of protein. These processes work in conjunction with regulatory elements that sense amino acids or their metabolites. During and after nutrient intake, amino acid homeostasis is dominated by autoregulatory processes such as transport and oxidation of excess amino acids. Amino acid deprivation triggers processes such as autophagy and the execution of broader transcriptional programs to maintain plasma amino acid concentrations. Amino acid transport plays a crucial role in the absorption of amino acids in the intestine, the distribution of amino acids across cells and organs, the recycling of amino acids in the kidney, and the recycling of amino acids after protein breakdown.

Amino acid transporters as modulators of glucose homeostasis.

Amino acids modulate glucose homeostasis. Cytosolic levels of amino acids are regulated by amino acid transporters, modulating insulin release, protein synthesis, cell proliferation, cell fate, and metabolism. In ß-cells, amino acid transporters modulate incretin-stimulated insulin release. In the liver, amino acid transporters provide glutamine and alanine for gluconeogenesis. Intestinal amino acid transporters facilitate the intake of amino acids causing protein restriction when inactive. Adipocyte development is regulated by amino acid transporters through activation of mechanistic target of rapamycin (mTORC1) and amino acid-related metabolites. The accumulation and metabolism of branched-chain amino acids (BCAAs) in muscle depends on transporters. The integration between amino acid metabolism and transport is critical for the maintenance and function of tissues and cells involved in glucose homeostasis.

Knockout of the Amino Acid Transporter SLC6A19 and Autoimmune Diabetes Incidence in Female Non-Obese Diabetic (NOD) Mice.

High protein feeding has been shown to accelerate the development of type 1 diabetes in female non-obese diabetic (NOD) mice. Here, we investigated whether reducing systemic amino acid availability via knockout of the Slc6a19 gene encoding the system B(0) neutral amino acid transporter AT1 would reduce the incidence or delay the onset of type 1 diabetes in female NOD mice. Slc6a19 gene deficient NOD mice were generated using the CRISPR-Cas9 system which resulted in marked aminoaciduria. The incidence of diabetes by week 30 was 59.5% (22/37) and 69.0% (20/29) in NOD.Slc6a19+/+ and NOD.Slc6a19-/- mice, respectively (hazard ratio 0.77, 95% confidence interval 0.41-1.42; Mantel-Cox log rank test: p = 0.37). The median survival time without diabetes was 28 and 25 weeks for NOD.Slc6a19+/+ and NOD.Slc6a19-/- mice, respectively (ratio 1.1, 95% confidence interval 0.6-2.0). Histological analysis did not show differences in islet number or the degree of insulitis between wild type and Slc6a19 deficient NOD mice. We conclude that Slc6a19 deficiency does not prevent or delay the development of type 1 diabetes in female NOD mice.

Quantitative modelling of amino acid transport and homeostasis in mammalian cells.

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell's endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Transporters.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15543. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Coordinated action of multiple transporters in the acquisition of essential cationic amino acids by the intracellular parasite Toxoplasma gondii.

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is 'trans-stimulated' by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites.

A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling.

Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of Xenopus laevis oocytes expressing various human amino acid transporters incubated in complex media to establish their comprehensive substrate profiles. For most transporters, amino acid selectivity matched reported substrate profiles. However, we could not detect substantial accumulation of cationic amino acids by SNAT4 and ATB0,+ in contrast to previous reports. In addition, comparative substrate profiles of two related sodium neutral amino acid transporters known as SNAT1 and SNAT2, revealed the latter as a significant leucine accumulator. As a consequence, SNAT2, but not SNAT1, was shown to be an effective activator of the eukaryotic cellular growth regulator mTORC1. We propose, that metabolomic profiling of membrane transporters in Xe nopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways.

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells.

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

Amino Acid Transporters as Targets for Cancer Therapy: Why, Where, When, and How.

Amino acids are indispensable for the growth of cancer cells. This includes essential amino acids, the carbon skeleton of which cannot be synthesized, and conditionally essential amino acids, for which the metabolic demands exceed the capacity to synthesize them. Moreover, amino acids are important signaling molecules regulating metabolic pathways, protein translation, autophagy, defense against reactive oxygen species, and many other functions. Blocking uptake of amino acids into cancer cells is therefore a viable strategy to reduce growth. A number of studies have used genome-wide silencing or knock-out approaches, which cover all known amino acid transporters in a large variety of cancer cell lines. In this review, these studies are interrogated together with other databases to identify vulnerabilities with regard to amino acid transport. Several themes emerge, such as synthetic lethality, reduced redundancy, and selective vulnerability, which can be exploited to stop cancer cell growth.

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution.

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.

Novel Chemical Scaffolds to Inhibit the Neutral Amino Acid Transporter B0AT1 (SLC6A19), a Potential Target to Treat Metabolic Diseases.

Lack of B0AT1 (SLC6A19) partially protects mice against the onset of non-alcoholic steatohepatitis (NASH). To achieve a similar outcome through pharmacological treatment, we improved previously identified inhibitors of B0AT1 by medicinal chemistry and identified second generation inhibitors by high through-put screening. Modified diarylmethine compounds inhibited B0AT1 with IC50 values ranging from 8-90 µM. A second generation of inhibitors was derived from high-throughput screening and showed higher affinity (IC50 of 1-15 µM) and strong selectivity against amino acid transporters with similar substrate specificity, such as ASCT2 (SLC1A5) and LAT1 (SLC7A5). All compounds were unrelated to B0AT1 substrates, but were likely to bind in the vicinity of the substrate binding site.