Because the mammary glands are significantly much larger length of development at night lymph node was assessed

Serendipitously, however, we discovered that the antiplasmodial potency of pantothenamides is enhanced considerably when the parasite culture medium used for growth assays is pre-incubated at 37uC for a prolonged period. Consequently, sub-micromolar concentrations of pantothenamides that have no effect in freshly prepared medium inhibit parasite growth effectively in the preincubated medium. We present evidence that links this finding to the presence in parasite culture medium of pantetheinase activity, the activity of an enzyme that catalyzes the hydrolysis of pantetheine to pantothenate and cysteamine. In animals, pantetheinase activity is typically linked with the Vanin proteins, soluble or membrane bound proteins that belong to the nitrilase superfamily, the members of which share an invariant Glu-Lys-Cys catalytic triad. We show, using an in vitro primary amine detection assay, that a pantothenamide selected from the series tested here is hydrolyzed in the presence of Albumax II, demonstrating Albumax II to be a source of pantetheinase activity. Furthermore, we show that recombinant human pantetheinase reduces the antiplasmodial potency of the pantothenamide in the pre-incubated medium in vitro, and, that the attenuating effect of the pantetheinase is alleviated by incubation of the pantetheinase-supplemented medium at 37uC. Together these data are consistent with pantetheinase-mediated pantothenamide degradation occurring in medium freshly supplemented with Albumax II or serum under in vitro culture conditions, lowering the effective pantothenamide concentration, and thereby masking the sub-micromolar antiplasmodial potency of pantothenamides. Importantly, we Wortmannin PI3K inhibitor demonstrate that the potent antiplasmodial effect of the pantothenamides in the medium pre-incubated at 37uC is alleviated with pantothenate, and therefore results specifically from inhibition of pantothenate and/or CoA utilization. We also show that all of the pantothenamides in this series inhibit P. falciparum PanK-catalysed pantothenate phosphorylation. The data presented here provide additional validation of pantothenate and CoA utilization as potential antiplasmodial drug targets. The data presented thus far are consistent with there being a heat-labile component in Albumax-complete RPMI that antagonizes the activity of pantothenamides. In an attempt to identify such a component, the activity of a selected pantothenamide in aged Albumax-complete RPMI supplemented immediately prior to the assay with various components of Albumax-complete RPMI, was investigated. A component of the medium that was found to antagonize the activity of pantothenamides was Albumax II, the lipid-rich bovine serum albumin preparation used as a serum substitute. As shown in Figure 5A, the addition of Albumax II to aged Albumax-complete medium reduced the activity of compound 12. In the presence of the additional Albumax II, compound 12 had little-to-no effect on parasite growth even at a concentration of 200 mM, a concentration that inhibits parasite growth completely in aged Albumax-complete RPMI without additional Albumax. Furthermore, the activity of pantothenol was unaffected by supplementation with the additional Albumax II, consistent with Albumax II specifically influencing the potency of pantothenamides. To investigate whether the attenuating effect of Albumax II could be linked to the increased potency of pantothenamides in Albumax-complete RPMI incubated for a prolonged period at 37uC, we determined whether the attenuating effect of the additional Albumax II could be alleviated by incubating the aged medium to which additional Albumax II had been added for a further 40 h at 37uC. The pantothenamide was indeed more potent when tested in aged Albumax-complete RPMI supplemented with Albumax II and aged a second time. The possibility that this increased potency was due to further inactivation of an independent component of the aged medium was investigated by testing the pantothenamide in aged medium incubated for the same total length of time as the aged Albumax-complete RPMI supplemented with Albumax II and then incubated. Compound 12 was slightly more active in the Albumax-complete RPMI subjected to two 40 h incubations at 37uC than in the Albumaxcomplete RPMI subjected to a single 40 h incubation. This demonstrated that a component of the Albumax-complete RPMI had not been fully depleted/inactivated after the initial 40 h and hence that the pantothenamide had not reached a maximum potency in this medium after the initial 40 h incubation. Nonetheless, the increase in potency was far less than the increase in potency observed following incubation of the aged medium supplemented with additional Albumax II, consistent with the Albumax II being sensitive to heat treatment.

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