The aquaporin protein family generally seems to be designed for the selective passage of water or glycerol. Charged molecules, metal ions and even protons are strictly excluded. Recently, particular aquaporin isoforms were reported to conduct unconventional permeants, i.e., the unpolar gases carbon dioxide and nitric oxide, the polar gas ammonia, the oxidative oxygen species hydrogen peroxide, and the metalloids antimonite, arsenite and silicic acid. Here, we summarize the available data on permeability properties and physiological settings of these aquaporins and we analyze which structural features might be connected to permeability for non-water, non-glycerol solutes.

Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water‐specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H+ ≫K+ >Rb+ >Na+ >Cs+ >Li+. Contrary to theoretical models, we found that electrostatic repulsion at the central asn‐pro‐ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali‐leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali‐tight but solute‐unselective NPA region as a feature of primordial channels and the proton‐tight and solute‐selective ar/R constriction variants as later adaptations within the AQP superfamily.

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis. Pentamidine is usable only in the early, non-cerebral stage of human African trypanosomiasis. An aquaglyceroporin, TbAQP2, is required for high-affinity pentamidine uptake. This study established the mechanism. Pentamidine does not pass TbAQP2 but blocks the unique selectivity filter via a single charged amidine and displacement of water from the channel, rendering it the first non-covalent, nanomolar aquaglyceroporin inhibitor. TbAQP2 does not act as a pentamidine transporter but a high-affinity receptor indicating drug uptake by endocytosis. The novel binding mode and proposed uptake mechanism open up possibilities for designing needed anti-trypanosomal small molecule drugs with regard to penetrating the blood-brain barrier for treatment of the severe, second disease stage.

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 fight against malaria, i.e. one of the three major infectious diseases and transmitted by mosquitos, is conducted at three levels: i. transmission control (by attacking the mosquito vector or biological processes of vector infection), ii. vaccination (by stimulating the immune system to produce antibodies against molecular parasite surface structures), and iii. antimalarial drugs (by developing and applying small molecules that interfere with vital biochemical pathways). Despite strong efforts on levels i. and ii., small molecule drugs remain an indispensible antimalarial means; however, emergence and spreading of resistant malaria parasites against all currently used drugs pose a growing threat to treatment success. Therefore, novel drug targets need to be identified and exploited. Here, we show that a recently discovered lactic acid transporter, PfFNT, is a novel valid drug target and we provide first compounds that potently block transport and kill malaria parasites. Lactic acid is the metabolic end product of the parasites’ energy generation metabolism and interfering with this biochemical pathway represents a new mode of action against malaria parasites.