Throughout the latter half of this century, the development and spread of resistance to most front-line antimalarial compounds used in the prevention and treatment of the most severe form of human malaria has given cause for grave clinical concern. Polymorphisms in pfmdr1, the gene encoding the P-glycoprotein homologue 1 (Pgh1) protein of Plasmodium falciparum, have been linked to chloroquine resistance; Pgh1 has also been implicated in resistance to mefloquine and halofantrine. However, conclusive evidence of a direct causal association between pfmdr1 and resistance to these antimalarials has remained elusive, and a single genetic cross has suggested that Pgh1 is not involved in resistance to chloroquine and mefloquine. Here we provide direct proof that mutations in Pgh1 can confer resistance to mefloquine, quinine and halofantrine. The same mutations influence parasite resistance towards chloroquine in a strain-specific manner and the level of sensitivity to the structurally unrelated compound, artemisinin. This has important implications for the development and efficacy of future antimalarial agents.

Pantothenic acid, a precursor of coenzyme A (CoA), is essential for the growth of pathogenic microorganisms. Since the structure of pantothenic acid was determined, many analogues of this essential metabolite have been prepared. Several have been demonstrated to exert an antimicrobial effect against a range of microorganisms by inhibiting the utilization of pantothenic acid, validating pantothenic acid utilization as a potential novel antimicrobial drug target. This review commences with an overview of the mechanisms by which various microorganisms acquire the pantothenic acid they require for growth, and the universal CoA biosynthesis pathway by which pantothenic acid is converted into CoA. A detailed survey of studies that have investigated the inhibitory activity of analogues of pantothenic acid and other precursors of CoA follows. The potential of inhibitors of both pantothenic acid utilization and biosynthesis as novel antibacterial, antifungal and antimalarial agents is discussed, focusing on inhibitors and substrates of pantothenate kinase, the enzyme catalysing the rate-limiting step of CoA biosynthesis in many organisms. The best strategies are considered for identifying inhibitors of pantothenic acid utilization and biosynthesis that are potent and selective inhibitors of microbial growth and that may be suitable for use as chemotherapeutic agents in humans.

The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H ؉ ions and thereby regulates its cytosolic pH (pH i ), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH i and to recover from an imposed intracellular acidification in the absence of extracellular Na ؉ , thus ruling out the involvement of a Na ؉ /H ؉ exchanger in both processes. Both phenomena were ATPdependent. Amiloride and the related compound ethylisopropylamiloride caused a substantial reduction in the resting pH i of the parasite, whereas EMD 96785, a potent and allegedly selective inhibitor of Na ؉ /H ؉ exchange, had relatively little effect. The resting pH i of the parasite was also reduced by the sulfhydryl reagent N-ethylmaleimide, by the carboxyl group blocker N,N-dicyclohexylcarbodiimide, and by bafilomycin A 1 , a potent inhibitor of V-type H ؉ -ATPases. Bafilomycin A 1 blocked pH i recovery in parasites subjected to an intracellular acidification and reduced the rate of acidification of a weakly buffered solution by parasites under resting conditions. The data are consistent with the hypothesis that the malaria parasite, like other parasitic protozoa, has in its plasma membrane a V-type H ؉ -ATPase, which serves as the major route for the efflux of H ؉ ions.Malaria, one of the most important infectious diseases in the world today, is caused by parasitic protozoa of the genus Plasmodium. These are unicellular, eukaryotic organisms, which, during the course of their complex lifecycle, invade the red blood cells of their vertebrate host. Having entered a red cell, the invading parasite lies dormant for some hours (the ring stage), after which it begins a period of rapid growth (the trophozoite stage) followed by division (schizogony), resulting in the generation of 20 -30 new parasites.The metabolic and biosynthetic activity of the malaria trophozoite is intense. The parasite is wholly reliant on glycolysis as its energy source, and it consumes glucose and produces lactic acid at a rate some 100 times higher than does a normal, uninfected erythrocyte (1, 2). The high metabolic activity of the parasite generates a substantial intracellular acid load. In addition, in the in vivo situation, malaria infection commonly gives rise to a pronounced extracellular acidosis (3). For the parasite to remain viable, it must therefore have an effective means of protecting its intracellular pH (pH i ) 1 from both intraand extracellular acid loads.Eukaryotic cells extrude H ϩ via a variety of different mechanisms. Plant cells, yeast, various protozoa, and a number of invertebrate and vertebrate cell types have in their plasma membrane H ϩ -ATPases that utilize energy derived from the hydrolysis of ATP to pump H ϩ ions from the cell cytosol (4). These are either P-type ATPases (so-called because they form an acyl-phosphate intermediate during their reaction cycle) or V-type ATPases (so-called because they were first described on t...

The growth of the human malaria parasite, Plasmodium falciparum, within its host erythrocyte is reliant on the uptake of a number of essential nutrients from the extracellular medium. One of these is pantothenic acid, a water-soluble vitamin that is a precursor of coenzyme A. In this study we show that normal uninfected erythrocytes are impermeable to pantothenate but that the vitamin is taken up rapidly into malaria-infected cells via a transport pathway that has the characteristics (furosemide sensitivity, nonsaturability) of previously characterized, broad specificity permeation pathways induced by the intracellular parasite in the host cell membrane. The transport of pantothenate therefore constitutes a critical physiological role for these pathways. Inside the parasitized cell pantothenate undergoes phosphorylation, the first step in its conversion to coenzyme A. Parasites within saponin-permeabilized erythrocytes were shown to take up and phosphorylate pantothenate, consistent with the intracellular parasite having both a pantothenate transporter and a pantothenate kinase. Comparisons of the rate of phosphorylation of pantothenate by lysates prepared from uninfected and infected erythrocytes revealed that the pantothenate kinase activity of the P. falciparum trophozoite is some 10-fold higher than that of its host cell and that most, if not all, of the phosphorylation of pantothenate within the malaria-infected cell occurs within the intracellular parasite. These results contrast with those of previous studies in which it was proposed that the avian malaria parasite Plasmodium lophurae lacks pantothenate kinase (as well as the other enzymes for the synthesis of coenzyme A) and is reliant upon the uptake of preformed coenzyme A from the host cell cytosol.Malaria is caused by a protozoan parasite (genus Plasmodium) which, during the course of its life cycle, invades the red blood cells of its vertebrate host. This strategy serves to protect the parasite from attack by the immune system of the host; however, it poses significant challenges to the protozoan with regard to obtaining essential nutrients from the plasma.One such compound is pantothenic acid, a water-soluble vitamin (M r 219; Fig. 1) that serves as a precursor of the enzyme cofactor coenzyme A. Early evidence for the importance of exogenous pantothenate in supporting the growth of the intracellular malaria parasite came from the observation that in chickens infected with the avian parasite Plasmodium gallinaceum a dietary deficiency of pantothenate resulted in a marked reduction in the parasitemia (1). Forty years later it was demonstrated in in vitro growth assays that the sustained growth of the human malaria parasite Plasmodium falciparum within human erythrocytes is dependent upon there being pantothenate in the extracellular medium (2). The requirement of the intracellular malaria parasite for an exogenous supply of pantothenate implies that there is a mechanism by which this compound can gain entry into the infected erythrocyte. However, thi...

Growth of the virulent human malaria parasite Plasmodium falciparum is dependent on an extracellular supply of pantothenate (vitamin B5) and is susceptible to inhibition by pantothenate analogues that hinder pantothenate utilization. In this study, on the hunt for pantothenate analogues with increased potency relative to those reported previously, we screened a series of pantothenamides (amide analogues of pantothenate) against P. falciparum and show for the first time that analogues of this type possess antiplasmodial activity. Although the active pantothenamides in this series exhibit only modest potency under standard in vitro culture conditions, we show that the potency of pantothenamides is selectively enhanced when the parasite culture medium is pre-incubated at 37°C for a prolonged period. We present evidence that this finding is linked to the presence in Albumax II (a serum-substitute routinely used for in vitro cultivation of P. falciparum) of pantetheinase activity: the activity of an enzyme that hydrolyzes the pantothenate metabolite pantetheine, for which pantothenamides also serve as substrates. Pantetheinase activity, and thereby pantothenamide degradation, is reduced following incubation of Albumax II-containing culture medium for a prolonged period at 37°C, revealing the true, sub-micromolar potency of pantothenamides. Importantly we show that the potent antiplasmodial effect of pantothenamides is attenuated with pantothenate, consistent with the compounds inhibiting parasite proliferation specifically by inhibiting pantothenate and/or CoA utilization. Additionally, we show that the pantothenamides interact with P. falciparum pantothenate kinase, the first enzyme involved in converting pantothenate to coenzyme A. This is the first demonstration of on-target antiplasmodial pantothenate analogues with sub-micromolar potency, and highlights the potential of pantetheinase-resistant pantothenamides as antimalarial agents.