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.

Pantothenamides are secondary or tertiary amides of pantothenic acid, the vitamin precursor of the essential cofactor and universal acyl carrier coenzyme A. A recent study has demonstrated that pantothenamides inhibit the growth of blood-stage Plasmodium falciparum with submicromolar potency by exerting an effect on pantothenic acid utilization, but only when the pantetheinase present in the growth medium has been inactivated. Here, we demonstrate that small modifications of the pantothenamide core structure are sufficient to counteract pantetheinase-mediated degradation and that the resulting pantothenamide analogues still inhibit the in vitro proliferation of P. falciparum by targeting a pantothenic acid-dependent process (or processes). Finally, we investigated the toxicity of the most potent analogues to human cells and show that the selectivity ratio exceeds 100 in one case. Taken together, these results provide further support for pantothenic acid utilization being a viable target for antimalarial drug discovery.

cPantothenamides inhibit blood-stage Plasmodium falciparum with potencies (50% inhibitory concentration [IC 50 ], ϳ20 nM) similar to that of chloroquine. They target processes dependent on pantothenate, a precursor of the essential metabolic cofactor coenzyme A. However, their antiplasmodial activity is reduced due to degradation by serum pantetheinase. Minor modification of the pantothenamide structure led to the identification of ␣-methyl-N-phenethyl-pantothenamide, a pantothenamide resistant to degradation, with excellent antiplasmodial activity (IC 50 , 52 ؎ 6 nM), target specificity, and low toxicity.O ne-half of the world's population (ϳ3.4 billion people) is at risk of contracting malaria, with pregnant women and children Ͻ5 years of age being especially vulnerable. In 2013, the WHO estimated that malaria caused ϳ584,000 deaths globally, with the majority occurring in Africa (1). Although efforts to control and to eliminate malaria in the past 15 years have saved an estimated 3.3 million lives (1), drug-resistant parasites continue to emerge (2). This places the progress in the fight against the disease under pressure, especially since there is no effective vaccine against malaria (3). Several new drug targets have been identified in recent years (4); however, these targets now need to be exploited through the development of directed treatments.We are interested in targeting the biosynthesis of the essential cofactor coenzyme A (CoA) from the water-soluble vitamin B 5 (pantothenate, compound 1 in Fig. 1) for antimalarial drug development (5, 6). It has been shown that extracellular pantothenate is essential for intracellular malaria parasites (7), which indicates that Plasmodium falciparum does not utilize exogenous CoA but must synthesize CoA de novo (8).Pantothenate analogues interfere with the ability of P. falciparum to utilize the vitamin, with many analogues being characterized as growth inhibitors of the blood-stage parasites (9-11). Furthermore, a recent study showed that CoA biosynthesis can be targeted by a chemically diverse set of inhibitors that do not resemble pantothenate, the most potent of which had a 50% inhibitory concentration (IC 50 ; the concentration that inhibits parasite proliferation by 50%) of 120 nM against blood-stage parasites (12). These studies support pantothenate utilization (and therefore CoA biosynthesis and CoA-dependent processes) as an antiplasmodial target.Recently we showed that N-substituted pantothenamides (PanAms), a specific class of pantothenate analogues, have excellent antiplasmodial activity. Among these, N-phenethyl-pantothenamide (N-PE-PanAm) (compound 2 in Fig. 1) exhibited an IC 50 of 20 nM (13); this potency is comparable to that of chloroquine (14, 15). In practice, however, the antiplasmodial activity of the PanAms is decreased since they are degraded by pantetheinase (13), a ubiquitous enzyme of the Vanin protein family that is present in serum (16,17). Pantetheinase normally catalyzes the hydrolysis of pantetheine (a CoA-derived metabolite) to for...

N-Substituted pantothenamides (PanAms) are pantothenate analogues with up to nanomolar potency against blood-stage Plasmodium falciparum (the most virulent species responsible for malaria). Although these compounds are known to target coenzyme A (CoA) biosynthesis and/or utilization, their exact mode of action (MoA) is still unknown. Importantly, PanAms that retain the natural β-alanine moiety are more potent than other variants, consistent with the involvement of processes that are selective for pantothenate (the precursor of CoA) or its derivatives. The transport of pantothenate and its phosphorylation by P. falciparum pantothenate kinase (PfPanK, the first enzyme of CoA biosynthesis) are two such processes previously highlighted as potential targets for the PanAms' antiplasmodial action. In this study, we investigated the effect of PanAms on these processes using their radiolabeled versions (synthesized here for the first time), which made possible the direct measurement of PanAm uptake by isolated blood-stage parasites and PanAm phosphorylation by PfPanK present in parasite lysates. We found that the MoA of PanAms does not involve interference with pantothenate transport and that inhibition of PfPanK-mediated pantothenate phosphorylation does not correlate with PanAm antiplasmodial activity. Instead, PanAms that retain the β-alanine moiety were found to be metabolically activated by PfPanK in a selective manner, forming phosphorylated products that likely inhibit other steps in CoA biosynthesis or are transformed into CoA antimetabolites that can interfere with CoA utilization. These findings provide direction for the ongoing development of CoA-targeted inhibitors as antiplasmodial agents with clinical potential.

Resistance to trypanocidal drugs has been detected in various African countries and is a serious impediment to the control of livestock trypanosomosis. To determine whether drug resistant trypanosome strains are present in the Zambézia Province of Mozambique a study was initiated. To assess the effect of the farming system and the drug-use regimen on the development of drug resistance, trypanosome isolates were collected from cattle from subsistence and commercial livestock production systems. The susceptibility of seven isolates against isometamidium chloride, diminazene aceturate and homidium chloride was tested in mice using a multiple-dose test. In four of the seven isolates high levels of drug resistance to diminazene aceturate and isometamidium chloride were detected. In most cases the observed levels of drug resistance correlated with the drug-use practices in the particular livestock production system.

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Eggs represent one of the most important low-cost sources of high-quality animal protein and minerals. However, the occurrence of contamination with antimicrobial drug residues and antimicrobial-resistant microorganisms has been increasing and represents a serious food safety risk. This study was carried out to assess the microbial and antimicrobial drug residue contamination of chicken eggs commercialized in selected neighbourhoods in Morogoro municipality, Tanzania. A total of two hundred and fifty-five eggs were sourced from forty-four stores in ten neighbourhoods and were examined for bacterial and antimicrobial drug residue contamination using standard microbiological assays, and the Bacillus subtilis ATCC 3491 as the test organism. Bacterial contamination was detected in 53.3% (136/255) of the egg yolk samples, with significant differences between sampling sites (p = 0.001). Bacterial total viable counts in the contaminated samples ranged from 102 to 109 CFU/mL, with a mean value of 6.42×108 CFU/mL (CI 95% = 1.45×108 – 1.60×108 CFU/mL). The most prevalent bacteria were Bacillus spp. (39.7%), followed by the Coagulase-negative Staphylococcus (16.9%), Escherichia coli (8.8%), Streptococcus spp. (5.9%), Staphylococcus aureus (4.4%), Enterobacter spp. (3.7%), Salmonella spp. (3.0%) and Pseudomonas spp. (1.5%). Antimicrobial drug residues were detected in 40.8% (104/255) of the samples in egg white and nil (0%) in the yolk. The contamination with antimicrobial residues showed significant differences between sample collection sites (p = 0.000), but not with bacterial counts (p = 0.862) and isolated bacteria in each sample collection site (p = 0.497). The presence of various bacteria and drug residues above permissible maximum residue levels in eggs predisposes consumers to poison and antimicrobial-resistant bacteria.