MAIP: An Open-Source Tool to Enrich High-Throughput Screening Output and Identify Novel, Druglike Molecules with Antimalarial Activity.

Efforts to tackle malaria must continue for a disease that threatens half of the global population. Parasite resistance to current therapies requires new chemotypes that are able to demonstrate effectiveness and safety. Previously, we developed a machine-learning-based approach to predict compound antimalarial activity, which was trained on the compound collections of several organizations. The resulting prediction platform, MAIP, was made freely available to the scientific community and offers a solution to prioritize molecules of interest in virtual screening and hit-to-lead optimization. Here, we experimentally validate MAIP and demonstrate how the approach was used in combination with a robust compound selection workflow and a recently introduced innovative high-throughput screening (HTS) cascade to select and purchase compounds from a public library for subsequent experimental screening. We observed a 12-fold enrichment compared with a randomly selected set of molecules, and the eight hits we ultimately selected exhibit good potency and absorption, distribution, metabolism, and excretion (ADME) profiles.

Replenishing the malaria drug discovery pipeline: Screening and hit evaluation of the MMV Hit Generation Library 1 (HGL1) against asexual blood stage Plasmodium falciparum, using a nano luciferase reporter read-out.

A central challenge of antimalarial therapy is the emergence of resistance to the components of artemisinin-based combination therapies (ACTs) and the urgent need for new drugs acting through novel mechanism of action. Over the last decade, compounds identified in phenotypic high throughput screens (HTS) have provided the starting point for six candidate drugs currently in the Medicines for Malaria Venture (MMV) clinical development portfolio. However, the published screening data which provided much of the new chemical matter for malaria drug discovery projects have been extensively mined. Here we present a new screening and selection cascade for generation of hit compounds active against the blood stage of Plasmodium falciparum. In addition, we validate our approach by testing a library of 141,786 compounds not reported earlier as being tested against malaria. The Hit Generation Library 1 (HGL1) was designed to maximise the chemical diversity and novelty of compounds with physicochemical properties associated with potential for further development. A robust HTS cascade containing orthogonal efficacy and cytotoxicity assays, including a newly developed and validated nanoluciferase-based assay was used to profile the compounds. 75 compounds (Screening Active hit rate of 0.05%) were identified meeting our stringent selection criteria of potency in drug sensitive (NF54) and drug resistant (Dd2) parasite strains (IC50 ≤ 2 µM), rapid speed of action and cell viability in HepG2 cells (IC50 ≥ 10 µM). Following further profiling, 33 compounds were identified that meet the MMV Confirmed Active profile and are high quality starting points for new antimalarial drug discovery projects.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection.

OBJECTIVES: Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility. METHODS: Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA). RESULTS: A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector. CONCLUSIONS: The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.

ß-Arrestin-biased signaling of PTH analogs of the type 1 parathyroid hormone receptor.

Parathyroid hormone (PTH) is an anabolic agent that mediates bone formation through activation of the Gα(s)-, Gα(q)- and ß-arrestin-coupled parathyroid hormone receptor type 1 (PTH1R). Pharmacological evidence based on the effect of PTH(7-34), a PTH derivative that is said to preferentially activate ß-arrestin signaling through PTH1R, suggests that PTH1R-activated ß-arrestin signaling mediates anabolic effects on bone. Here, we performed a thorough evaluation of PTH(7-34) signaling behaviour using quantitative assays for ß-arrestin recruitment, Gα(s)- and Gα(q)-signaling. We found that PTH(7-34) inhibited PTH-induced cAMP accumulation, but was unable to induce ß-arrestin recruitment, PTH1R internalization and ERK1/2 phosphorylation in HEK293, CHO and U2OS cells. Thus, the ß-arrestin bias of PTH(7-34) is not apparent in every cell type examined, suggesting that correlating in vivo effects of PTH(7-34) to in vitro pharmacology should be done with caution.