The Plasmodium Lactate/H+ Transporter PfFNT Is Essential and Druggable In Vivo.

Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l-lactate/H+ from the cell. This transporter is a member of the strictly microbial formate-nitrite transporter (FNT) family and a novel putative drug target. Small, drug-like FNT inhibitors potently block lactate transport and kill Plasmodium falciparum parasites in culture. The protein structure of Plasmodium falciparum FNT (PfFNT) in complex with the inhibitor has been resolved and confirms its previously predicted binding site and its mode of action as a substrate analog. Here, we investigated the mutational plasticity and essentiality of the PfFNT target on a genetic level, and established its in vivo druggability using mouse malaria models. We found that, besides a previously identified PfFNT G107S resistance mutation, selection of parasites at 3 × IC50 (50% inhibitory concentration) gave rise to two new point mutations affecting inhibitor binding: G21E and V196L. Conditional knockout and mutation of the PfFNT gene showed essentiality in the blood stage, whereas no phenotypic defects in sexual development were observed. PfFNT inhibitors mainly targeted the trophozoite stage and exhibited high potency in P. berghei- and P. falciparum-infected mice. Their in vivo activity profiles were comparable to that of artesunate, demonstrating strong potential for the further development of PfFNT inhibitors as novel antimalarials.

Structural aspects of the glucose and monocarboxylate transporters involved in the Warburg effect.

Cancer cells shift their glucose catabolism from aerobic respiration to lactic fermentation even in the presence of oxygen, and this is known as the "Warburg effect". To accommodate the high glucose demands and to avoid lactate accumulation, the expression levels of human glucose transporters (GLUTs) and human monocarboxylate transporters (MCTs) are elevated to maintain metabolic homeostasis. Therefore, inhibition of GLUTs and/or MCTs provides potential therapeutic strategies for cancer treatment. Here, we summarize recent advances in the structural characterization of GLUTs and MCTs, providing a comprehensive understanding of their transport and inhibition mechanisms to facilitate further development of anticancer therapies.

Structure and mechanism of the SGLT family of glucose transporters.

Glucose is a primary energy source in living cells. The discovery in 1960s that a sodium gradient powers the active uptake of glucose in the intestine1 heralded the concept of a secondary active transporter that can catalyse the movement of a substrate against an electrochemical gradient by harnessing energy from another coupled substrate. Subsequently, coupled Na+/glucose transport was found to be mediated by sodium-glucose cotransporters2,3 (SGLTs). SGLTs are responsible for active glucose and galactose absorption in the intestine and for glucose reabsorption in the kidney4, and are targeted by multiple drugs to treat diabetes5. Several members within the SGLT family transport key metabolites other than glucose2. Here we report cryo-electron microscopy structures of the prototypic human SGLT1 and a related monocarboxylate transporter SMCT1 from the same family. The structures, together with molecular dynamics simulations and functional studies, define the architecture of SGLTs, uncover the mechanism of substrate binding and selectivity, and shed light on water permeability of SGLT1. These results provide insights into the multifaceted functions of SGLTs.

Structural characterization of the Plasmodium falciparum lactate transporter PfFNT alone and in complex with antimalarial compound MMV007839 reveals its inhibition mechanism.

Plasmodium falciparum, the deadliest causal agent of malaria, caused more than half of the 229 million malaria cases worldwide in 2019. The emergence and spreading of frontline drug-resistant Plasmodium strains are challenging to overcome in the battle against malaria and raise urgent demands for novel antimalarial agents. The P. falciparum formate-nitrite transporter (PfFNT) is a potential drug target due to its housekeeping role in lactate efflux during the intraerythrocytic stage. Targeting PfFNT, MMV007839 was identified as a lead compound that kills parasites at submicromolar concentrations. Here, we present 2 cryogenic-electron microscopy (cryo-EM) structures of PfFNT, one with the protein in its apo form and one with it in complex with MMV007839, both at 2.3 Å resolution. Benefiting from the high-resolution structures, our study provides the molecular basis for both the lactate transport of PfFNT and the inhibition mechanism of MMV007839, which facilitates further antimalarial drug design.

A Holistic Evolutionary and 3D Pharmacophore Modelling Study Provides Insights into the Metabolism, Function, and Substrate Selectivity of the Human Monocarboxylate Transporter 4 (hMCT4).

Monocarboxylate transporters (MCTs) are of great research interest for their role in cancer cell metabolism and their potential ability to transport pharmacologically relevant compounds across the membrane. Each member of the MCT family could potentially provide novel therapeutic approaches to various diseases. The major differences among MCTs are related to each of their specific metabolic roles, their relative substrate and inhibitor affinities, the regulation of their expression, their intracellular localization, and their tissue distribution. MCT4 is the main mediator for the efflux of L-lactate produced in the cell. Thus, MCT4 maintains the glycolytic phenotype of the cancer cell by supplying the molecular resources for tumor cell proliferation and promotes the acidification of the extracellular microenvironment from the co-transport of protons. A promising therapeutic strategy in anti-cancer drug design is the selective inhibition of MCT4 for the glycolytic suppression of solid tumors. A small number of studies indicate molecules for dual inhibition of MCT1 and MCT4; however, no selective inhibitor with high-affinity for MCT4 has been identified. In this study, we attempt to approach the structural characteristics of MCT4 through an in silico pipeline for molecular modelling and pharmacophore elucidation towards the identification of specific inhibitors as a novel anti-cancer strategy.

SLC16 Family: From Atomic Structure to Human Disease.

The solute carrier 16 (SLC16) family represents a diverse group of membrane proteins mediating the transport of monocarboxylates across biological membranes. Family members show a variety of functional roles ranging from nutrient transport and intracellular pH regulation to thyroid hormone homeostasis. Changes in the expression levels and transport function of certain SLC16 transporters are manifested in severe health disorders including cancer, diabetes, and neurological disorders. L-Lactate-transporting SLC16 family members play essential roles in the metabolism of certain tumors and became validated drug targets. This review illuminates the SLC16 family under a new light using structural information obtained from a SLC16 homolog. Furthermore, the role of these transporters in cancer metabolism and how their inhibition can contribute to anticancer therapy are discussed.

Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates.

Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0-3.3 Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.

Identification of the essential extracellular aspartic acids conserved in human monocarboxylate transporters 1, 2, and 4.

Human monocarboxylate transporters (hMCTs) 1-4 transport monocarboxylates, such as l-lactate and pyruvate, as well as H+ across the plasma membrane. hMCT1, 2, and 4 play important roles in energy balance, pH homeostasis. However, the molecular mechanism of these transporters, especially their pH dependency, remains unknown. The aim of this study was to identify the residues involved in the pH dependence of hMCT1, 2, and 4. Firstly, we focused on the effects of extracellular acids of hMCT1. l-Lactate uptake assay and site-directed mutagenesis revealed that the aspartic acid of hMCT1 (hMCT1 D414) was an important residue conserved in MCT1, 2, and 4 (hMCT2 D398 and hMCT4 D379). Because the functional characteristic of hMCT2-mediated l-lactate transport has not been reported, we built a hMCT2-expressing system using Xenopus laevis oocytes. The transport activity of hMCT2 was enhanced by co-expression with embigin, an ancillary protein, and kinetic analysis of hMCT2-mediated l-lactate uptake revealed that the apparent Km value (0.32 ± 0.02 mM) was lower than that mediated by hMCT1 and 4. Finally, we investigated the conserved aspartic acids of hMCT2 and 4, and revealed that these residues were essential for l-lactate transport. These findings suggested that the extracellular aspartic acids conserved in hMCT1, 2, and 4 played important roles in transport activity and pH dependency, and can function as a first step of substrate and H+ recognition and transport from the extracellular to the intracellular region. These findings contributed to enhance our understanding of the transport process of hMCT1, 2, and 4.

Introduction of Scaffold Nitrogen Atoms Renders Inhibitors of the Malarial l-Lactate Transporter, PfFNT, Effective against the Gly107Ser Resistance Mutation.

The spreading of malaria parasites, Plasmodium falciparum, with resistance to all known drugs calls for novel classes of inhibitors with new modes of action. Recently, we discovered and validated the plasmodial l-lactate transporter, PfFNT, as a novel antimalarial drug target. However, treatment of parasites with a screening hit from the malaria box compound collection, MMV007839, gave rise to a PfFNT Gly107Ser resistance mutation decreasing inhibitor affinity by 2 orders of magnitude. Here, we show that newly introduced nitrogen atoms into the inhibitor scaffold can act as hydrogen bond acceptor sites to the serine hydroxyl. The gain in affinity led to almost equal inhibition of wildtype PfFNT and the Gly107Ser mutation. The most potent inhibitor of this work, BH267.meta, killed cultured P. falciparum parasites with nanomolar efficacy and did not give rise to new resistance formation in vitro. Its deduced pharmacokinetic properties appear suitable for further drug development.

Characteristic Analysis of Homo- and Heterodimeric Complexes of Human Mitochondrial Pyruvate Carrier Related to Metabolic Diseases.

Human mitochondrial pyruvate carriers (hMPCs), which are required for the uptake of pyruvate into mitochondria, are associated with several metabolic diseases, including type 2 diabetes and various cancers. Yeast MPC was recently demonstrated to form a functional unit of heterodimers. However, human MPC-1 (hMPC-1) and MPC-2 (hMPC-2) have not yet been individually isolated for their detailed characterization, in particular in terms of their structural and functional properties, namely, whether they exist as homo- or heterodimers. In this study, hMPC-1 and hMPC-2 were successfully isolated in micelles and they formed stable homodimers. However, the heterodimer state was found to be dominant when both hMPC-1 and hMPC-2 were present. In addition, as heterodimers, the molecules exhibited a higher binding capacity to both substrates and inhibitors, together with a larger structural stability than when they existed as homodimers. Taken together, our results demonstrated that the hetero-dimerization of hMPCs is the main functional unit of the pyruvate metabolism, providing a structural insight into the transport mechanisms of hMPCs.

Monocarboxylate Transporters (SLC16): Function, Regulation, and Role in Health and Disease.

The solute carrier family 16 (SLC16) is comprised of 14 members of the monocarboxylate transporter (MCT) family that play an essential role in the transport of important cell nutrients and for cellular metabolism and pH regulation. MCTs 1-4 have been extensively studied and are involved in the proton-dependent transport of L-lactate, pyruvate, short-chain fatty acids, and monocarboxylate drugs in a wide variety of tissues. MCTs 1 and 4 are overexpressed in a number of cancers, and current investigations have focused on transporter inhibition as a novel therapeutic strategy in cancers. MCT1 has also been used in strategies aimed at enhancing drug absorption due to its high expression in the intestine. Other MCT isoforms are less well characterized, but ongoing studies indicate that MCT6 transports xenobiotics such as bumetanide, nateglinide, and probenecid, whereas MCT7 has been characterized as a transporter of ketone bodies. MCT8 and MCT10 transport thyroid hormones, and recently, MCT9 has been characterized as a carnitine efflux transporter and MCT12 as a creatine transporter. Expressed at the blood brain barrier, MCT8 mutations have been associated with an X-linked intellectual disability, known as Allan-Herndon-Dudley syndrome. Many MCT isoforms are associated with hormone, lipid, and glucose homeostasis, and recent research has focused on their potential roles in disease, with MCTs representing promising novel therapeutic targets. This review will provide a summary of the current literature focusing on the characterization, function, and regulation of the MCT family isoforms and on their roles in drug disposition and in health and disease. SIGNIFICANCE STATEMENT: The 14-member solute carrier family 16 of monocarboxylate transporters (MCTs) plays a fundamental role in maintaining intracellular concentrations of a broad range of important endogenous molecules in health and disease. MCTs 1, 2, and 4 (L-lactate transporters) are overexpressed in cancers and represent a novel therapeutic target in cancer. Recent studies have highlighted the importance of MCTs in glucose, lipid, and hormone homeostasis, including MCT8 in thyroid hormone brain uptake, MCT12 in carnitine transport, and MCT11 in type 2 diabetes.

Whole-Exome Sequencing Reveals a Rare Missense Variant in SLC16A9 in a Pedigree with Early-Onset Gout.

Gout is a common inflammatory arthritis triggered by monosodium urate deposition after longstanding hyperuricemia. In the general community, the disease is largely polygenic in genetic architecture, with many polymorphisms having been identified in gout or urate-associated traits. In a small proportion of cases, rare high penetrant mutations associated with monogenic segregation of the disease in families have been demonstrated to be disease causative. In this study, we recruited a two-generation pedigree with early-onset gout. To elucidate the genetic predisposition, whole-exome sequencing (WES) was performed. After comprehensive variant analyses and cosegregation testing, we identified a missense variant (c.277C>A, p.L93M) in SLC16A9, an extremely rare variant in genetic databases. Moreover, in silico assessments showed strong pathogenicity. This variant cosegregated with the disease phenotype perfectly in the family and is located in a highly conserved functional domain. A few studies supported our results of the association between SLC16A9 and gout and serum urate levels. In conclusion, we provide the first evidence for the association of rare missense in SLC16A9 with early-onset gout. These findings not only expand our current understanding of gout but also may have further implications for the treatment and prevention of gout.

Monocarboxylate Transporter 1 in Brain Diseases and Cancers.

BACKGROUND: Monocarboxylate Transporter 1 (MCT1), an important membrane transport protein, mediates the translocation of monocarboxylates together with protons across biological membranes. Due to its pathological significance, MCT1 plays an important role in the progression of some diseases, such as brain diseases and cancers. METHODS: We summarize the general description of MCT1 and provide a comprehensive understanding of the role of MCT1 in brain diseases and cancers. Furthermore, this review discusses the opportunities and challenges of MCT1- targeting drug-delivery systems in the treatment of brain diseases and cancers. RESULTS: In the brain, loss of MCT1 function is associated with pathologies of degeneration and injury of the nervous system. In tumors, MCT1 regulates the activity of signaling pathways and controls the exchange of monocarboxylates in aerobic glycolysis to affect tumor metabolism, proliferation and invasion. Meanwhile, MCT1 also acts as a good biomarker for the prediction and diagnosis of cancer progressions. CONCLUSION: MCT1 is an attractive transporter in brain diseases and cancers. Moreover, the development of MCT1- based small molecule drugs and MCT1 inhibitors in the clinic is promising. This review systematically summarizes the basic characteristics of MCT1 and its role in brain diseases and cancers, laying the foundation for further research on MCT1.

Mechanistic basis of L-lactate transport in the SLC16 solute carrier family.

In human and other mammalian cells, transport of L-lactate across plasma membranes is mainly catalyzed by monocarboxylate transporters (MCTs) of the SLC16 solute carrier family. MCTs play an important role in cancer metabolism and are promising targets for tumor treatment. Here, we report the crystal structures of an SLC16 family homologue with two different bound ligands at 2.54 and 2.69 Å resolution. The structures show the transporter in the pharmacologically relevant outward-open conformation. Structural information together with a detailed structure-based analysis of the transport function provide important insights into the molecular working mechanisms of ligand binding and L-lactate transport.

Homology modeling and site-directed mutagenesis identify amino acid residues underlying the substrate selection mechanism of human monocarboxylate transporters 1 (hMCT1) and 4 (hMCT4).

Human monocarboxylate transporters (hMCTs/SLC16As) mediate the transport of monocarboxylic compounds across plasma membranes. Among the hMCTs, hMCT1 and hMCT4 are expressed in various tissues, and transport substrates involved in energy metabolism. Both transporters mediate L-lactate transport, but, although hMCT1 also transports L-5-oxoproline (L-OPro), this compound is minimally transported by hMCT4. Thus, we were interested in the molecular mechanism responsible for the difference in substrate specificity between hMCT1 and hMCT4. Therefore, we generated 3D structure models of hMCT1 and hMCT4 to identify amino acid residues involved in the substrate specificity of these transporters. We found that the substrate specificity of hMCT1 was regulated by residues involved in turnover number (M69) and substrate affinity (F367), and these residues were responsible for recognizing (directly or indirectly) the -NH- moiety of L-OPro. Furthermore, our homology model of hMCT1 predicted that M69 and F367 participate in hydrophobic interactions with another region of hMCT1, emphasizing its potentially important role in the binding and translocation cycle of L-OPro. Mutagenesis experiments supported this model, showing that efficient L-OPro transport required a hydrophobic, long linear structure at position 69 and a hydrophobic, γ-branched structure at position 367. Our work demonstrated that the amino acid residues, M69 and F367, are key molecular elements for the transport of L-OPro by hMCT1. These two residues may be involved in substrate recognition and/or substrate-induced conformational changes.

The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state.

The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae In contrast to earlier hypotheses, we demonstrate that MPC is a hetero-dimer, not a multimeric complex. When not engaged in hetero-dimers, the yeast Mpc proteins can also form homo-dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero-dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition.

Molecular docking of anti-inflammatory drug diclofenac with metabolic targets: Potential applications in cancer therapeutics.

Diclofenac is a potent NSAID of clinical choice, which is widely used for containing inflammation. Moreover, recent experimental evidences overwhelmingly substantiate its antineoplastic potential. However, the precise molecular mechanisms of diclofenac's anticancer activity remain poorly understood. Neoplastic cells display reprogrammed metabolic features, which are manifested and regulated by a complex networking of molecular pathways. However, the effect of diclofenac on tumor cell metabolism are not yet clearly deciphered. Hence, the present investigation was carried out to identify and characterize key diclofenac targets of tumor metabolism, cell survival and chemoresistance. The interactions of diclofenac with such targets was analysed by PatchDock and YASARA (Yet Another Scientific Artificial Reality Application). The docking ability of diclofenac with its targets was based on analysis of dissociation constant (Kd), geometric shape complementarity score (GSC score), approximate interface area (AI area) and binding energy. The findings of this investigation reveal that diclofenac is capable of interacting with all of the selected molecular targets. Prominent interactions were observed with GLUT1, MCT4, LDH A, COX1, COX2, BCRP/ABCG2, HDM2/MDM2 and MRP1 compared to other targets. Interactions were of noncovalent nature involving ionic, hydrophobic interactions, Van der Waals forces and H-bonds, which varied depending on targets. This study for the first time, characterizes the nature of molecular interactions of diclofenac with selected targets involved in cancer cell metabolism, pH homeostasis, chemosensitivity, cell signalling and inflammation. Hence, these findings will be highly beneficial in optimizing the utility of diclofenac in development of novel cancer therapeutics.

Transport of 2,4-dichloro phenoxyacetic acid by human Na+-coupled monocarboxylate transporter 1 (hSMCT1, SLC5A8).

Using X. laevis oocyte expression system, we investigated whether human Na+-coupled monocarboxylate transporter 1 (SLC5A8, hSMCT1) is involved in 2,4-dichlorophenoxyacetate (2,4-D) uptake by the renal tubular epithelial cells. 2,4-D is a herbicide that causes nephrotoxicity. Heterologous expression of hSMCT1 in X. laevis oocytes conferred the ability to take up 2,4-D; the induced uptake process was Na+-dependent and electrogenic. The Na+-dependent uptake of 2,4-D was inhibited not only by known hSMCT1 substrates, but also by many structural analogs of 2,4-D. The currents induced by 2,4-D, 4-chlorophenoxyacetate (4-CPA) and 2-methyl-4-chlorophenoxyacetate (MCPA) were saturable: the rank order of the maximal induced current and the affinity for hSMCT1was 2,4-D > 4-CPA > MCPA. The relationship between the structures of the derivatives and their transport activity implied specific structural features in a compound for recognition as a substrate by hSMCT1. Furthermore, we have demonstrated using purified rabbit renal brush-border membrane vesicles that 2,4-D potently inhibited the Na+-dependent uptake of pyroglutamate, a typical substrate for Smct1, and that 2,4-D uptake process was Na+-dependent, saturable and inhibitable by a potent blocker, ibuprofen. We conclude that hSMCT1 is involved partially in the renal reabsorption of 2,4-D and its derivatives and their nephrotoxicity.

The intracellular parasite Toxoplasma gondii harbors three druggable FNT-type formate and l-lactate transporters in the plasma membrane.

Toxoplasma gondii is a globally prevalent parasitic protist. It is well-known for its ability to infect almost all nucleated vertebrate cells, which is reflected by its unique metabolic architecture. Its fast-growing tachyzoite stage catabolizes glucose via glycolysis to yield l-lactate as a major by-product that must be exported from the cell to prevent toxicity; the underlying mechanism remains to be elucidated, however. Herein, we report three formate-nitrite transporter (FNT)-type monocarboxylate/proton symporters located in the plasma membrane of the T. gondii tachyzoite stage. We observed that all three proteins transport both l-lactate and formate in a pH-dependent manner and are inhibited by 2-hydroxy-chromanones (a class of small synthetic molecules). We also show that these compounds pharmacologically inhibit T. gondii growth. Using a chemical biology approach, we identified the critical residues in the substrate-selectivity region of the parasite transporters that determine differential specificity and sensitivity toward both substrates and inhibitors. Our findings further indicate that substrate specificity in FNT family proteins from T. gondii has evolved such that a functional repurposing of prokaryotic-type transporters helps fulfill a critical metabolic role in a clinically important parasitic protist. In summary, we have identified and characterized the lactate transporters of T. gondii and have shown that compounds blocking the FNTs in this parasite can inhibit its growth, suggesting that these transporters could have utility as potential drug targets.

Succinate-acetate permease from Citrobacter koseri is an anion channel that unidirectionally translocates acetate.

Acetate is an important metabolite in metabolism and cell signaling. Succinate-Acetate Permease (SatP) superfamily proteins are known to be responsible for acetate transport across membranes, but the nature of this transport remains unknown. Here, we show that the SatP homolog from Citrobacter koseri (SatP_Ck) is an anion channel that can unidirectionally translocate acetate at rates of the order of ~107 ions/s. Crystal structures of SatP_Ck in complex with multiple acetates at 1.8 Å reveal that the acetate pathway consists of four acetate-binding sites aligned in a single file that are interrupted by three hydrophobic constrictions. The bound acetates at the four sites are each orientated differently. The acetate at the cytoplasmic vestibule is partially dehydrated, whereas those in the main pore body are fully dehydrated. Aromatic residues within the substrate pathway may coordinate translocation of acetates via anion-π interactions. SatP_Ck reveals a new type of selective anion channel and provides a structural and functional template for understanding organic anion transport.