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Malaria parasites activate a broad-selectivity ion channel on their host erythrocyte membrane to obtain essential nutrients from the bloodstream. This conserved channel, known as the plasmodial surface anion channel (PSAC), has been linked to parasite clag3 genes in P. falciparum, but epigenetic switching between the two copies of this gene hinders clear understanding of how the encoded protein determines PSAC activity. Here, we used linkage analysis in a P. falciparum cross where one parent carries a single clag3 gene to overcome the effects of switching and confirm a primary role of the clag3 product with high confidence. Despite Mendelian inheritance, CLAG3 conditional knockdown revealed remarkably preserved nutrient and solute uptake. Even more surprisingly, transport remained sensitive to a CLAG3 isoform-specific inhibitor despite quantitative knockdown, indicating that low doses of the CLAG3 transgene are sufficient to confer block. We then produced a complete CLAG3 knockout line and found it exhibits an incomplete loss of transport activity, in contrast to rhoph2 and rhoph3, two PSAC-associated genes that cannot be disrupted because nutrient uptake is abolished in their absence. Although the CLAG3 knockout did not incur a fitness cost under standard nutrient-rich culture conditions, this parasite could not be propagated in a modified medium that more closely resembles human plasma. These studies implicate oligomerization of CLAG paralogs encoded by various chromosomes in channel formation. They also reveal that CLAG3 is dispensable under standard in vitro conditions but required for propagation under physiological conditions. Author summary: Malaria, a globally important infectious disease, is caused by parasites that invade and grow in circulating red blood cells to avoid host immune attack. Infected red blood cells have increased uptake of diverse nutrients, fueling parasite growth; this uptake is mediated by an ion channel that transports essential nutrients across the red blood cell membrane. Three proteins made by the parasite have been linked to this channel, but how they increase uptake is unknown. Here, we used mapping in a genetic cross of two strains of the virulent human malaria parasite to confirm a primary role of one protein known as CLAG3. We then used gene editing to produce a parasite that has reduced CLAG3 levels when a stabilizing chemical is removed; surprisingly, solute transport was minimally changed despite a 90% reduction in CLAG3. Gene editing was also used to make a parasite without any CLAG3. This knockout parasite had reduced nutrient uptake, but it still grew normally in media with high nutrient levels; it was unable to grow when nutrient levels were lowered to levels like those in the human bloodstream. The complex effects of channel inhibitors on these genetically modified parasites suggests that CLAG3 and the two other proteins interact with each other to form large protein clusters in the red blood cell membrane; these clusters may form the nutrient uptake pore. Our studies indicate that CLAG3 is required for parasite survival and growth in the bloodstream and that the channel it produces can be targeted to make new antimalarial drugs. [ABSTRACT FROM AUTHOR]

Malaria parasites increase their host erythrocyte’s permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel’s structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing. [ABSTRACT FROM AUTHOR]

Malaria parasites grow within vertebrate erythrocytes and increase host cell permeability to access nutrients from plasma. This increase is mediated by the plasmodial surface anion channel (PSAC), an unusual ion channel linked to the conserved clag gene family. Although PSAC recognizes and transports a broad range of uncharged and charged solutes, it must efficiently exclude the small Na+ ion to maintain infected cell osmotic stability. Here, we examine possible mechanisms for this remarkable solute selectivity. We identify guanidiniumas an organic cation with high permeability into human erythrocytes infected with Plasmodium falciparum, but negligible uptake by uninfected cells. Transport characteristics and pharmacology indicate that this uptake is specifically mediated by PSAC. The rank order of organic and inorganic cation permeabilities suggests cation dehydration as the rate-limiting step in transport through the channel. The high guanidinium permeability of infected cells also allows rapid and stringent synchronization of parasite cultures, as required formolecular and cellular studies of this pathogen. These studies provide important insights into how nutrients and ions are transported via PSAC, an established target for antimalarial drug development. [ABSTRACT FROM AUTHOR]

A series of novel tetra substituted thiophenes were synthesized, characterized, and evaluated for their anti-inflammatory activity in carrageenin induced rat paw edema model—an acute in vivo model. Compounds V1, V3, V11, V12, V17, and V18 showed good anti-inflammatory activity, indicating the importance of oxime moiety in modulating the activity. The structure–activity relationship studies explore “the aliphatic oxime esters” attached via a ketone bridge to fifth position of the thiophene, and indicate that this feature may enhance the anti-inflammatory activity as compared to aromatic oximes. Since free radicals are implicated in various inflammatory disorders, the free radical scavenging activity of some of the synthesized candidates was assessed using 1,1-diphenyl-2-picryl hydrazyl assay. The oxime containing analogs exhibited weak to moderate activity as free radical scavengers in DPPH assay. A plausible reasoning for its free radical scavenging ability is discussed. All the compounds were also screened in nitro blue tetrazolium model, to assess them as superoxide anion radical scavengers. A direct correlation between anti-inflammatory activity and free radical scavenging activity was not seen. The results disclose a new class of anti-inflammatory agents designed and synthesized for the first time wherein the utility of aliphatic oxime esters in modulating the anti-inflammatory activity profile is apparent. This will give us potential anti-inflammatory leads. [Copyright &y& Elsevier]

Compounds incorporating thiophene moiety, a pi excess five membered heterocycle, have attracted a great deal of research interest, owing to the therapeutic utility of the template as useful drug molecular scaffolding. We report the synthesis and pharmacological evaluation of thiophenes substituted with 4-methanesulfonyl benzoyl moiety at the fifth position of the ring, as possible anti-inflammatory lead candidates. The aryl sulfonyl methyl thiophene analogs AP29, AP82, and AP37, when screened for anti-inflammatory activity in carrageenin induced rat paw edema, an acute in vivo model, exhibited moderate to good activity at a dose level of 100mg/kg body weight P.o compared to Ibuprofen. In a five day formalin induced rat paw edema, a chronic in vivo anti-inflammatory model, candidates AP29, AP82, and AP37 inhibited the disease progression by 53%, 34%, and 65%, respectively on the fifth day, at a dose level of 100 mg/kg body weight P.o compared to Rofecoxib, Ibuprofen, and Dexamethasone at therapeutic doses which gave a protection of 53.8%, 81.5%, and 81.5%, respectively. The replacement of the 4-methanesulfonyl benzoyl moiety in AP82 with the pyridine template, 3,5-dimethyl-4-methoxy-2-pyridyl function, gave rise to AP84, which was less active in the acute model, but gave 54% and 75% protection both during the first day and fifth day, respectively, in the chronic model. A dual mechanism of action is proposed for AP84, a non-steroidal drug which has exhibited remarkable activity when compared to the steroid dexamethasone. These results open up new avenues in designing novel anti-inflammatory drugs as dual inhibitors with the incorporation of a pyridine template as part of the pharmacophore. [Copyright &y& Elsevier]

Summary: Development of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound''s affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes. PaperFlick: Display Omitted [Copyright &y& Elsevier]

Abstract: Erythrocytes infected with malaria parasites have increased permeability to various solutes. These changes may be mediated by an unusual small conductance ion channel known as the plasmodial surface anion channel (PSAC). While channel activity benefits the parasite by permitting nutrient acquisition, it can also be detrimental because water-soluble antimalarials may more readily access their parasite targets via this channel. Recently, two such toxins, blasticidin S and leupeptin, were used to select mutant parasites with altered PSAC activities, suggesting acquired resistance via reduced channel-mediated toxin uptake. Surprisingly, although these toxins have similar structures and charge, we now show that reduced permeability of one does not protect the intracellular parasite from the other. Leupeptin accumulation in the blasticidin S-resistant mutant was relatively preserved, consistent with retained in vitro susceptibility to leupeptin. Subsequent in vitro selection with both toxins generated a double mutant parasite having additional changes in PSAC, implicating an antimalarial resistance mechanism for water-soluble drugs requiring channel-mediated uptake at the erythrocyte membrane. Characterization of these mutants revealed a single conserved channel on each mutant, albeit with distinct gating properties. These findings are consistent with a shared channel that mediates uptake of ions, nutrients and toxins. This channel''s gating and selectivity properties can be modified in response to in vitro selective pressure. [Copyright &y& Elsevier]