Sheptide A: an antimalarial cyclic pentapeptide from a fungal strain in the Herpotrichiellaceae.

As part of ongoing efforts to isolate biologically active fungal metabolites, a cyclic pentapeptide, sheptide A (1), was discovered from strain MSX53339 (Herpotrichiellaceae). The structure and sequence of 1 were determined primarily by analysis of 2D NMR and HRMS/MS data, while the absolute configuration was assigned using a modified version of Marfey's method. In an in vitro assay for antimalarial potency, 1 displayed a pEC50 value of 5.75 ± 0.49 against malaria-causing Plasmodium falciparum. Compound 1 was also tested in a counter screen for general cytotoxicity against human hepatocellular carcinoma (HepG2), yielding a pCC50 value of 5.01 ± 0.45 and indicating a selectivity factor of ~6. This makes 1 the third known cyclic pentapeptide biosynthesized by fungi with antimalarial activity.

Alkyne modified purines for assessment of activation of Plasmodium vivax hypnozoites and growth of pre-erythrocytic and erythrocytic stages in Plasmodium spp.

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite's ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesised that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalysed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.

Aminoalkoxycarbonyloxymethyl Ether Prodrugs with a pH-Triggered Release Mechanism: A Case Study Improving the Solubility, Bioavailability, and Efficacy of Antimalarial 4(1H)-Quinolones with Single Dose Cures.

Preclinical and clinical development of numerous small molecules is prevented by their poor aqueous solubility, limited absorption, and oral bioavailability. Herein, we disclose a general prodrug approach that converts promising lead compounds into aminoalkoxycarbonyloxymethyl (amino AOCOM) ether-substituted analogues that display significantly improved aqueous solubility and enhanced oral bioavailability, restoring key requirements typical for drug candidate profiles. The prodrug is completely independent of biotransformations and animal-independent because it becomes an active compound via a pH-triggered intramolecular cyclization-elimination reaction. As a proof-of-concept, the utility of this novel amino AOCOM ether prodrug approach was demonstrated on an antimalarial compound series representing a variety of antimalarial 4(1H)-quinolones, which entered and failed preclinical development over the last decade. With the amino AOCOM ether prodrug moiety, the 3-aryl-4(1H)-quinolone preclinical candidate was shown to provide single-dose cures in a rodent malaria model at an oral dose of 3 mg/kg, without the use of an advanced formulation technique.

Open-source discovery of chemical leads for next-generation chemoprotective antimalarials.

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

Microphysical space of a liver sinusoid device enables simplified long-term maintenance of chimeric mouse-expanded human hepatocytes.

While many advanced liver models support hepatic phenotypes necessary for drug and disease studies, these models are characterized by intricate features such as co-culture with one of more supporting cell types or advanced media perfusion systems. These systems have helped elucidate some of the critical biophysical features missing from standard well-plate based hepatocyte culture, but their advanced designs add to their complexity. Additionally, regardless of the culture system, primary hepatocyte culture systems suffer from reproducibility issues due to phenotypic variation and expensive, limited supplies of donor lots. Here we describe a microfluidic bilayer device that sustains primary human hepatocyte phenotypes, including albumin production, factor IX production, cytochrome P450 3A4 drug metabolism and bile canaliculi formation for at least 14 days in a simple monoculture format with static media. Using a variety of channel architectures, we describe how primary cell phenotype is promoted by spatial confinement within the microfluidic channel, without the need for perfusion or co-culture. By sourcing human hepatocytes expanded in the Fah, Rag2, and Il2rg-knockout (FRG™-KO) humanized mouse model, utilizing a few hundred hepatocytes within each channel, and maintaining hepatocyte function for weeks in vitro within a relatively simple model, we demonstrate a basic primary human hepatocyte culture system that addresses many of the major hurdles in human hepatocyte culture research.