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Mikrotubuli sind filamentöse intrazelluläre Polymere, die als grundlegende Bestandteile subzellulärer Strukturen in Eukaryoten dienen. Diese Studie verwendet einen vergleichenden Ansatz, um zu untersuchen, wie sich die intrinsischen dynamischen und biochemischen Eigenschaften von Tubulin zwischen verschiedenen Spezies unterscheiden, und zeigt ihre Konsequenzen in zwei verschiedenen physiologischen Kontexten: 1) Bestimmung der Spindelgröße bei Fröschen der Gattung Xenopus und 2) Spezifität von Mikrotubuli-Inhibitoren für Plasmodium falciparum-Mikrotubuli über denen ihres menschlichen Wirts. In den Eiern der Froschgattung Xenopus wird die Länge der meiotischen Spindel biochemisch festgelegt und erreicht unabhängig von räumlichen Einschränkungen eine Obergrenze. Messungen der Dynamik von Xenopus-Mikrotubuli zeigen, dass X. laevis-Mikrotubuli sowohl schneller wachsen als auch länger leben als die von X. tropicalis. Darüber hinaus spielt die Quantifizierung der Länge und Massenverteilung der Xenopus-Mikrotubuli zusammen mit den Reaktionen der Eiextrakt-Spindelanordnung eine Rolle für die intrinsische Dynamik der Mikrotubuli bei der Modulation der Spindellänge. Mikrotubuli sind auch Wirkstofftargets bei Pilz- und parasitären Helmintheninfektionen und haben in den letzten Jahrzehnten die Aufmerksamkeit als potenzielles Wirkstoffziel beim Malariaparasiten Plasmodium falciparum auf sich gezogen. Um die Dynamik und Medikamentspezifität von Mikrotubuli von P. falciparum zu charakterisieren, haben wir Tubulin direkt von den Parasiten gereinigt. Zum ersten Mal wurden hier dynamische P. falciparum-Mikrotubuli in vitro rekonstituiert und eine parasitenspezifische Unterdrückung der Dynamik von Mikrotubuli durch Oryzalin und Amiprofos-Methyl direkt nachgewiesen. Diese Studie legt einen experimentellen Rahmen fest, um direkt auf parasitenspezifische Hemmung von Mikrotubuli zu testen, die bisher unter Verwendung bestehender in-vitro-Ansätze nicht beobachtet wurden., Microtubules are filamentous intracellular polymers that are fundamental components of subcellular structures including the spindle, the cytoskeleton, and flagella in eukaryotes. This study uses a comparative approach to investigate how the intrinsic dynamic and biochemical characteristics of tubulin vary between species and demonstrates their consequences in two different physiological contexts: 1) Spindle size control in Xenopus frogs, and 2) The specificity of microtubule inhibitors for Plasmodium falciparum microtubules over those of their human host. In Xenopus frog eggs, the length of the spindle is biochemically controlled and reaches an upper limit independent of spatial constraints. In this study, in vitro measurements of Xenopus microtubule dynamics show that X. laevis microtubules are both faster-growing and longer-lived X. tropicalis, independent of the influence of microtubule-associated proteins. Furthermore, quantification of Xenopus microtubule length and mass distributions, combined with egg extract spindle assembly reactions, establishes a role for intrinsic microtubule dynamics in modulating spindle length. Microtubules are also established drug targets in fungal and parasitic helminth infections and have in the past decades drawn attention as a potential drug target in the malaria parasite Plasmodium falciparum. In order to characterize P. falciparum microtubule dynamics, structure, and drug specificity, we have used an affinity chromatography-based approach to purify tubulin directly from blood-stage parasites. For the first time, dynamic P. falciparum microtubules have been reconstituted in vitro and parasite-specific suppression of microtubule dynamics by oryzalin and amiprofos methyl has been directly demonstrated. This study establishes an experimental framework to directly test for parasite-specific microtubule inhibition, microtubule structure, and interactions with MAPs that previously have not observed using existing in vitro approaches.

Malaria-causing Plasmodium parasites are developing resistance to antimalarial drugs, providing the impetus for new antiplasmodials. Although pantothenamides show potent antiplasmodial activity, hydrolysis by pantetheinases/vanins present in blood rapidly inactivates them. We herein report the facile synthesis and biological activity of a small library of pantothenamide analogues in which the labile amide group is replaced with a heteroaromatic ring. Several of these analogues display nanomolar antiplasmodial activity against Plasmodium falciparum and/or Plasmodium knowlesi, and are stable in the presence of pantetheinase. Both a known triazole and a novel isoxazole derivative were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although they fail to suppress Plasmodium berghei proliferation in vivo, the pharmacokinetic and contact time data presented provide a benchmark for the compound profile likely required to achieve antiplasmodial activity in mice and should facilitate lead optimization.

The biosynthesis of the essential metabolic cofactor coenzyme A (CoA) has been receiving increasing attention as a new target that shows potential to counter the rising resistance to established antimicrobials. In particular, phosphopantothenoylcysteine synthetase (PPCS)—the second CoA biosynthesis enzyme that is found as part of the bifunctional CoaBC protein in bacteria, but is monofunctional in eukaryotes—has been validated as a target through extensive genetic knockdown studies in Mycobacterium tuberculosis. Moreover, it has been identified as the molecular target of the fungal natural product CJ-15,801 that shows selective activity against Staphylococcus aureus and the malaria parasite Plasmodium falciparum. As such, CJ-15,801 and 4′-phospho-CJ-15,801 (its metabolically active form) are excellent tool compounds for use in the development of new antimicrobial PPCS inhibitors. Unfortunately, further study and analysis of CJ15,801 is currently being hampered by several unique challenges posed by its synthesis. In this study we describe how these challenges were overcome by using a robust palladium-catalyzed coupling to form the key N-acyl vinylogous carbamate moiety with retention of stereochemistry, and by extensive investigation of protecting groups suited to the labile functional group combinations contained in this molecule. We also demonstrate that using TBAF for deprotection causes undesired off-target effects related to the presence of residual tertiary ammonium salts. Finally, we provide a new method for the chemoenzymatic preparation of 4′-phospho-CJ-15,801 on multi-milligram scale, after showing that chemical synthesis of the molecule is not practical. Taken together, the results of this study advances our pursuit to discover new antimicrobials that specifically target CoA biosynthesis and/or utilization.