Substrate-analogous inhibitors exert antimalarial action by targeting the Plasmodium lactate transporter PfFNT at nanomolar scale.

Resistance against all available antimalarial drugs calls for novel compounds that hit unexploited targets in the parasite. Here, we show that the recently discovered Plasmodium falciparum lactate/proton symporter, PfFNT, is a valid druggable target, and describe a new class of fluoroalkyl vinylogous acids that potently block PfFNT and kill cultured parasites. The original compound, MMV007839, is derived from the malaria box collection of potent antimalarials with unknown targets and contains a unique internal prodrug principle that reversibly switches between a lipophilic transport form and a polar, substrate-analogous active form. Resistance selection of cultured P. falciparum parasites with sub-lethal concentrations of MMV007839 produced a single nucleotide exchange in the PfFNT gene; this, and functional characterization of the resulting PfFNT G107S validated PfFNT as a novel antimalarial target. From quantitative structure function relations we established the compound binding mode and the pharmacophore. The pharmacophore largely circumvents the resistance mutation and provides the basis for a medicinal chemistry program that targets lactate and proton transport as a new mode of antimalarial action.

The amoeboidal Dictyostelium aquaporin AqpB is gated via Tyr216 and aqpB gene deletion affects random cell motility.

BACKGROUND INFORMATION: Regulation of aquaporin (AQPs) water channels mostly occurs on the transcriptional level or by intracellular trafficking. Direct AQP gating is comparatively rare and is described mainly for fungi and plants. Earlier, we identified a gated water-specific AQP in Dictyostelium discoideum, AqpB, which is opened by truncation of an extended intracellular loop D (AqpB Δ208-219). RESULTS: We show that Tyr216 of loop D is a key residue in the gating mechanism possibly involving phosphorylation. Mutation of Tyr216 to aspartate or glutamate initiated water permeability (Pf  = 69 µm/s) to the same extent as AqpB Δ208-219, whereas neither replacement of Tyr216 by a positive arginine (Y216R) nor introduction of a negative charge in a neighbouring position (T217D) opened the channel. We overexpressed AqpB wildtype and AqpB Δ208-219 as GFP fusion constructs in Dictyostelium amoebae and found that the truncated, permanently open AqpB yielded cells with reduced capability to cope with hypotonic stress. Genetic deletion of aqpB yielded a strain with significantly reduced speed of random motility (4.5 µm/min vs. 6.3 µm/min of wildtype cells). Yet, chemotaxis towards folate and cAMP was unaffected. In this context, we identified a second, so-far uncharacterised amoeboidal AQP, AqpD, whose role in directed cell locomotion needs to be established. CONCLUSION: Our data add to the completion of the Dictyostelium model for cell motility and show that knowledge on the expression, permeability properties and localisation of amoeboidal AQPs are crucial for further development of the system.

A widened substrate selectivity filter of eukaryotic formate-nitrite transporters enables high-level lactate conductance.

Bacterial formate-nitrite transporters (FNT) regulate the metabolic flow of small weak mono-acids derived from anaerobic mixed-acid fermentation, such as formate, and further transport nitrite and hydrosulfide. The eukaryotic Plasmodium falciparumFNT is vital for the malaria parasite by its ability to release the larger l-lactate substrate as the metabolic end product of anaerobic glycolysis in symport with protons preventing cytosolic acidification. However, the molecular basis for substrate discrimination by FNTs has remained unclear. Here, we identified a size-selective FNT substrate filter region around an invariant lysine at the bottom of the periplasmic/extracellular vestibule. The selectivity filter is reminiscent of the aromatic/arginine constriction of aquaporin water and solute channels regarding composition, location in the protein, and the size-selection principle. Bioinformatics support an adaptation of the eukaryotic FNT selectivity filter to accommodate larger physiologically relevant substrates. Mutations that affect the diameter at the filter site predictably modulated substrate selectivity. The shape of the vestibule immediately above the filter region further affects selectivity. This study indicates that eukaryotic FNTs evolved to transport larger mono-acid substrates, especially l-lactic acid as a product of energy metabolism.

Challenges and achievements in the therapeutic modulation of aquaporin functionality.

Aquaporin (AQP) water and solute channels have basic physiological functions throughout the human body. AQP-facilitated water permeability across cell membranes is required for rapid reabsorption of water from pre-urine in the kidneys and for sustained near isosmolar water fluxes e.g. in the brain, eyes, inner ear, and lungs. Cellular water permeability is further connected to cell motility. AQPs of the aquaglyceroporin subfamily are necessary for lipid degradation in adipocytes and glycerol uptake into the liver, as well as for skin moistening. Modulation of AQP function is desirable in several pathophysiological situations, such as nephrogenic diabetes insipidus, Sjögren's syndrome, Menière's disease, heart failure, or tumors to name a few. Attempts to design or to find effective small molecule AQP inhibitors have yielded only a few hits. Challenges reside in the high copy number of AQP proteins in the cell membranes, and spatial restrictions in the protein structure. This review gives an overview on selected physiological and pathophysiological conditions in which modulation of AQP functions appears beneficial and discusses first achievements in the search of drug-like AQP inhibitors.