Inhibition of the Plasmodium falciparum formate-nitrite transporter (PfFNT) has emerged as a promising strategy for antimalarial drug development. PfFNT inhibitors display high potency against blood-stage parasites and have shown efficacy in vivo. Here, we demonstrate activity against liver-stage parasites of the front-running compound, (Z)-4,4,5,5,5-pentafluoro-3-hydroxy-1-(pyridin-3-yl)pent-2-en-1-one (BH267m), highlighting the potential for chemopreventive applications. Consequently, we investigated the formation of metal complexes of PfFNT inhibitors as a strategy to generate long-acting depot formulations enabling sustained drug release. Complexes with Mg2+, Ca2+, and Zn2+ were prepared via chelation through the pharmacophoric vinylogous acid moiety and characterized by spectroscopic methods. The resulting solids formed amorphous particles in the low micrometer range with metal-dependent aqueous solubilities. In vitro release studies using a dialysis assay revealed sustained, near zero-order ligand release, with zinc-based complexes displaying particularly low solubility and slow release kinetics. Release rates varied by approximately one order of magnitude depending on metal ion and ligand structure. Simulated plasma concentrations suggested sustained exposure of one- to tenfold of the respective in vitro EC50 values within the current ligand portfolio. These findings indicate that coordination of vinylogous acid motifs with metal ions may provide a basis for developing long-acting depot formulations of chelating anti-infective agents.

Cryptosporin, a fungal metabolite, exhibited potent antimalarial activity against both asexual blood stage Plasmodium falciparum and liver-stage Plasmodium berghei with minimal human HepG2 toxicity. Unlike atovaquone, cryptosporin's mechanism is independent of mitochondrial electron transport. Minimum inoculum of resistance showed a low risk of resistance development. RNA-Seq analysis revealed the upregulation of genes associated with sexual development including many canonical markers such as Pfs25, and PfCCp3, suggesting a stress response that is also seen when parasites are treated with artemisinin. In vitro evolution and whole genome sequencing analysis identified a mutation (F138Y) in PfAQP (PF3D7_1132800) and duplications of the two superoxide dismutase genes, PfSOD-1 (PF3D7_0814900) and PfSOD-2 (PF3D7_0623500). CRISPR/Cas9 editing confirmed that the F138Y mutation in PfAQP was sufficient to confer resistance to cryptosporin. Alignment of the P. falciparum structure with that of HsAQP3 suggests the mutation may impact transport of hydrogen peroxide and the transition between open and closed conformations. Indeed, studies with BY4742 Δfps1 yeast expressing PfAQP showed that the permeability of PfAQP was not affected by cryptosporin and that it is likely not a direct target. Taken together, this study highlights the role of PfAQP in the resistance development of cryptosporin. In addition, cryptosporin likely induces high levels of oxidative stress which results in the duplications of oxidative dismutase genes as part of the parasite's defense response. These findings highlight the role of PfAQP in mediating drug resistance, the mechanism of which warrants further research.

Transmembrane facilitation of substrates by channels and secondary active transporters results in a defined steady-state concentration ratio across the membrane. Evidence is accumulating that asymmetry in the structural build of the transporters, or interaction with asymmetric partner proteins, can shift the position of the transmembrane equilibrium by biased transport directionality. For instance, the bacterial lactose transporter, LacY, and two amino acid transporters, i.e., the human excitatory amino acid carrier, EAAC1, and the yeast lysine permease, Lyp1, were reported to exhibit distinct transport kinetics in the inward and outward direction by protein-intrinsic properties. A recent example is transport modulation of human monocarboxylate transporters, MCT, by shedding of the extracellular domain of an ancillary protein, basigin. Loss of the domain selectively increases export of lactate from lung cancer cells by a factor of four, contributing to the Warburg effect and malignancy. Further, intrinsic properties of monocarboxylate transporters involving asymmetric affinities of substrate binding, or biased open probabilities were shown to generate preference for one transport direction. Here, we discuss molecular mechanisms and physiological contexts of asymmetric secondary active transmembrane transport. Focus is laid on experimentally established cases, and examples are given in which putative bias in transport directionality may have been overlooked.

Aim Human monocarboxylate transporters 1-4, MCT, are key for the lactate/H+ exchange between glycolytic and oxidative cancer cells, white and red muscle fibers, or in the astrocyte-neuron shuttle. The common MCT transport mechanism involves three conserved residues, that is, a substrate-attracting Lys and a conformation-locking Asp/Arg salt bridge (positions 38 and 309/313 in MCT1). Yet, it remained unclear which sites define isoform-specific substrate affinity and preferred transport directionality.Methods Here, we analyzed structural differences in the binding sites of MCT1-4 and determined their impact on the biophysical transport properties and inhibitor binding using radiolabeled transport assays in yeast.Results We found differences in amino acid positions with sidechain hydroxyl groups. While the higher affinity MCT1 and MCT2 carry Phe/Ser-OH (pos. 367/371 in MCT1), the lower affinity MCT3 and MCT4 have Tyr-OH/Gly. Mutation of Phe/Ser-OH in MCT1 to Tyr-OH/Gly markedly decreased the affinity for lactate and pyruvate, while it did not change the affinity for propionate. The maximal transport velocity increased with decreasing affinity, and the preferred transport directionality shifted toward export. Likewise, replacing Met151 by Ala shifted transport bias possibly by eliminating conformation-stabilizing sulfur-aromatic interactions. Moreover, these mutations lowered the activity of the clinical candidate MCT1 inhibitor AZD3965 by three orders of magnitude providing insight into the molecular drug binding mode and explaining the strong preference for MCT1 over MCT4.Conclusions Together, subtle changes in the arrangement of sidechains in the MCT binding site determine basic monocarboxylate/H+ transport properties that impact lactate-related physiology, namely cellular metabolism, reprogramming, and signaling.

The sodium/proton-exchanging ATPase of Plasmodium falciparum malaria parasites, PfATP4, is an emerging drug target. Inhibition results in detrimental cell swelling due to cytosolic accumulation of sodium and alkalization. PfATP4 is a sodium-releasing type II P-type ATPase restricted to apicomplexan parasites. Experimental data on structure-function relationships of the isolated protein are absent. Here, we produced and purified the soluble catalytic domain of PfATP4 and evaluated kinetic properties by in vitro phosphate colorimetry. The protein exhibited Mg2+-dependent ATPase activity at the same order of magnitude as the native cellular PfATP4 and was insensitive to the presence of sodium. AlphaFold 3-based structure and ATP/Mg2+ interaction predictions identified key residues of the nucleotide binding domain (Lys619, Lys652, Arg703). Replacement of the lysines by methionine decreased the enzymatic activity to one quarter. Individual mutation of the putative Mg2+-coordinating Asp865 of the phosphorylation domain was tolerated, while a joint replacement with Asp869 decreased ATPase again to one quarter. Mutation of the putative γ-phosphate receiving Asp451 maintained the rate of Pi release. Our data attribute typical functional roles for P-type ATPases to the basic and acidic residues of the soluble PfATP4 catalytic domain and show that its ATP hydrolysis is independent of phosphorylation of Asp451.

Metabolomics provides snapshots of states of metabolites under specific conditions, with nuclear magnetic resonance (NMR) being one of the few noninvasive techniques. However, when applied to intact cells (e.g., yeast or mammalian cells) or tissues, traditional 1H NMR often suffers from overlapping signals from numerous metabolites and intracellular macromolecules such as proteins. To address this, we employed deuterium-labeled tracers that do not suffer from background interference and streamline targeted flux analysis. Deuterium magnetic resonance spectroscopy (DMRS) enables rapid, noninvasive measurement of metabolic flux without specialized equipment. In our study, we first measured T1, T2, and chemical shifts for 26 deuterium-labeled compounds in phosphate-buffered saline: parameters functional for optimal DMRS settings. Among the 26 deuterated compounds tested with food-grade baker's yeast (Saccharomyces cerevisiae) as an easily accessible model solution, we observed and tracked the real-time consumption of pyruvate, glucose, fumarate, acetone, and nicotinamide. We redirected yeast metabolism by (i) varying concentrations of added pyruvate and (ii) osmotic pressure by changing buffer density. This study underscores DMRS's potential as a robust, versatile tool for dissecting metabolic transformations exemplified here with the convenient yeast cell systems active for hundreds of minutes under typical NMR observation conditions.

Inhibition of the lactate transporter PfFNT is a valid novel mode of action against malaria parasites. Current pyridine-substituted pentafluoro-3-hydroxy-pent-2-en-1-ones act as substrate analogs with submicromolar EC50 in vitro, and >99.7% activity in mice. The recently solved structure of a PfFNT-inhibitor complex visualized the binding mode. Here, we extended the inhibitor layout by series of amine- and anilide-linked pyridine p-substituents to generate additional interactions in the cytoplasmic vestibule. Virtual docking indicated hydrogen bonding to Tyr31 and Ser102. Fluorescence cross-correlation spectroscopy yielded respectively enhanced target affinity. Strikingly, the in vitro activity increased by 1 order of magnitude to 14.8 nM at negligible cytotoxicity. While p-amine substitutions were rapidly metabolized, the more stable p-acetanilide cleared 99.7% of parasites at 4 × 50 mg kg-1 in a mouse malaria model. Future stabilization of the p-substitution against metabolism may translate the gain in in vitro potency to the in vivo situation.

Inhibitors of ʟ-lactate transport are in development as a novel mode of action in antitumor therapy and malaria. Previously, we used radiolabeled ʟ-lactate to assay transport via the human monocarboxylate transporter 1, MCT1, and the structurally unrelated malaria parasite's transporter, PfFNT. We encountered a sensitivity limit at IC50 around 100 nM possibly resulting from the required high cell number per sample. Here, we describe a sensitive background-free high-throughput assay in yeast based on fluorescent iLACCO biosensors. We used iLACCO for co-expression and fusions with the transporter protein. Uptake of ʟ-lactate produced strong intensiometric fluorescent responses that could be monitored in cell suspensions using a fluorometer and in individual cells by fluorescence microscopy. The signal decreased dose-dependently in the presence of specific MCT1 and PfFNT inhibitors. Re-evaluation of 36 PfFNT inhibitors yielded IC50 values below 100 nM now matching previous data on Ki compound affinity to isolated transporter protein.

Various issues including the overuse of antibiotics has led to the development of threatening multidrug-resistant bacterial strains urging development of novel anti-infectives. One quarter of current clinical phase III antibiotic drug candidates address ribosomal protein translation as a target. Here, we describe an effective cell-free in vitro screening system for inhibitors of bacterial ribosome activity with direct fluorescence read-out. Using ribosomal S30 extracts from Escherichia coli, Salmonella enterica, and Pseudomonas putida, the validity of this system is demonstrated by concentration-dependent inhibition of translation by a set of different classes of translation-targeting drugs. The single-compartment cell-free translation reaction is compatible with multi-well formats. Fluorophore formation of green fluorescent protein or monomeric NeonGreen occurs in an hour time frame without the need of adding reagents for secondary enzymatic detection saving handling time, and prohibiting false positives. As label-free readout, the dose response further allows for IC50 determination in the same setup. Together, we show that cell-free production of fluorescent proteins for the discovery of ribosome-targeting an-tibiotics is feasible and amenable to high-throughput applications.

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 x 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. 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.

Human monocarboxylate/H+ transporters, MCT, facilitate the transmembrane translocation of vital weak acid metabolites, mainly L-lactate. Tumors exhibiting a Warburg effect rely on MCT activity for L-lactate release. Recently, high-resolution MCT structures revealed binding sites for anticancer drug candidates and the substrate. Three charged residues, Lys 38, Asp 309, and Arg 313 (MCT1 numbering) are essential for substrate binding and initiation of the alternating access conformational change. However, the mechanism by which the proton cosubstrate binds and traverses MCTs remained elusive. Here, we report that substitution of Lys 38 by neutral residues maintained MCT functionality in principle, yet required strongly acidic pH conditions for wildtype-like transport velocity. We determined pH-dependent biophysical transport properties, Michaelis-Menten kinetics, and heavy water effects for MCT1 wildtype and Lys 38 mutants. Our experimental data provide evidence for the bound substrate itself to accept and shuttle a proton from Lys 38 to Asp 309 initiating transport. We have shown before that substrate protonation is a pivotal step in the mechanisms of other MCT-unrelated weak acid translocating proteins. In connection with this study, we conclude that utilization of the proton binding and transfer capabilities of the transporter-bound substrate is probably a universal theme for weak acid anion/H+ cotransport.

(1) Background: Several members of the ubiquitous aquaporin family, AQP, of water and neutral solute channels carry a cysteine residue in the selectivity filter region. Traditionally, toxic mercury-containing compounds are used to bind to the cysteine as covalent AQP inhibitors for physiological studies or analysis of structure–function relationships. (2) Methods: We tested thiol-reactive methylthiosulfonate reagents, MTS, as alternative Cys modifiers for AQP inhibition. Three MTS reagents transferring S-alkyl moieties of increasing size, i.e., S-methyl, S-n-propyl, and S-benzyl, were used with yeast-expressed water-selective AQP1 and the aquaglyceroporin AQP9. Respective Cys-to-Ala variants and mouse erythrocytes that naturally express AQP1 and AQP9 served as controls. (3) Results: Both wildtype AQP isoforms were inhibited by the Cys modifiers in a size-dependent manner, whereas the Cys-to-Ala-variants exhibited resistance. Sub-millimolar concentrations and incubation times in the minute range were sufficient. The modifications were reversible by treatment with the thiol reagents acetylcysteine, ACC, and dithiothreitol, DTT. (4) Conclusions: MTS reagents represent a valid alternative of low toxicity for the inhibition of mercurial-sensitive AQPs.

(1) Background: Human aquaporin-9 (AQP9) conducts several small uncharged metabolites, such as glycerol, urea, and lactic acid. Certain brain tumors were shown to upregulate AQP9 expression, and the putative increase in lactic acid permeability was assigned to severity. (2) Methods: We expressed AQP9 and human monocarboxylate transporter 1 (MCT1) in yeast to determine the uptake rates and accumulation of radiolabeled l-lactate/l-lactic acid in different external pH conditions. (3) Results: The AQP9-mediated uptake of l-lactic acid was slow compared to MCT1 at neutral and slightly acidic pH, due to low concentrations of the neutral substrate species. At a pH corresponding to the pK(a) of l-lactic acid, uptake via AQP9 was faster than via MCT1. Substrate accumulation was fundamentally different between AQP9 and MCT1. With MCT1, an equilibrium was reached, at which the intracellular and extracellular l-lactate/H+ concentrations were balanced. Uptake via AQP9 was linear, theoretically yielding orders of magnitude of higher substrate accumulation than MCT1. (4) Conclusions: The selectivity of AQP9 for neutral l-lactic acid establishes an ion trap for l-lactate after dissociation. This may be physiologically relevant if the transmembrane proton gradient is steep, and AQP9 acts as the sole uptake path on at least one side of a polarized cell.

The transmembrane transport of weak acid and base metabolites depends on the local pH conditions that affect the protonation status of the substrates and the availability of co-substrates, typically protons. Different protein designs ensure the attraction of substrates and co-substrates to the transporter entry sites. These include electrostatic surface charges on the transport proteins and complexation with seemingly transport-unrelated proteins that provide substrate and/or proton antenna, or enzymatically generate substrates in place. Such protein assemblies affect transport rates and directionality. The lipid membrane surface also collects and transfers protons. The complexity in the various systems enables adjustability and regulation in a given physiological or pathophysiological situation. This review describes experimentally shown principles in the attraction and facilitation of weak acid and base transport substrates, including monocarboxylates, ammonium, bicarbonate, and arsenite, plus protons as a co-substrate.

Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10−8 M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function.

Monocarboxylate transporter isoforms 1-4, MCT, of the solute carrier SLC16A family facilitate proton-coupled transport of l-lactate. Growth of tumors that exhibit the Warburg effect, that is, high rates of anaerobic glycolysis despite availability of oxygen, relies on swift l-lactate export, whereas oxygenic cancer cells import circulating l-lactate as a fuel. Currently, MCTs are viewed as promising anticancer targets. Small-molecule inhibitors have been found, and, recently, high-resolution protein structures have been obtained. Key questions, however, regarding the exact binding sites of cysteine-modifying inhibitors and the substrate translocation cycle lack a conclusive experimental basis. Here, we report Cys159 of the ubiquitous human MCT1 to reside in a critical hinge region of the alternating access-type transporter. We identified Cys159 as the binding site of the organomercurial pCMBS. The inhibitory effect of pCMBS was proposed to be indirect via modification of the chaperone basigin. We provide evidence that pCMBS locks MCT1 in its outward open conformation in a wedge-like fashion. We corroborated this finding using smaller cysteine-modifying reagents that size-dependently inhibited l-lactate transport. The smallest modifiers targeted additional cysteines as shown by a C159S mutant. We found a Cys399/Cys400 pair to constitute the second hinge of the transporter that tolerated only individual replacement by serine. The hinge cysteines, in particular the selectively addressable Cys159, provide natural anchors for placing probes into MCTs to report, for instance, on the electrostatics or hydration upon binding of the transported l-lactate substrate and the proton cosubstrate.

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay.

The protozoan parasite Plasmodium falciparum causes the most severe and prevailing form of malaria in sub-Saharan Africa. Previously, we identified the plasmodial lactate transporter, PfFNT, a member of the microbial formate-nitrite transporter family, as a novel antimalarial drug target. With the pentafluoro-3-hydroxy-pent-2-en-1-ones, we discovered PfFNT inhibitors that potently kill P. falciparum parasites in vitro. Four additional human-pathogenic Plasmodium species require attention, that is, P. vivax, most prevalent outside of Africa, and the regional P. malariae, P. ovale and P. knowlesi. Herein, we show that the plasmodial FNT variants are highly similar in terms of protein sequence and functionality. The FNTs from all human-pathogenic plasmodia and the rodent malaria parasite were efficiently inhibited by pentafluoro-3-hydroxy-pent-2-en-1-ones. We further established a phenotypic yeast-based FNT inhibitor screen, and found very low compound cytotoxicity and monocarboxylate transporter 1 off-target activity on human cells, particularly of the most potent FNT inhibitor BH267.meta, allowing these compounds to proceed towards animal model malaria studies.