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

Abstract

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 uM), rapid speed of action and cell viability in HepG2 cells (IC50 ≥ 10 uM). 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.

A small-molecule myosin inhibitor as a targeted multi-stage antimalarial

“…Parasitemia and distribution of parasite stages was determined by microscopic examination of Giemsa-stained thin smears. Luminescence measurements were performed as described previously [32].…”

“…P. falciparum NF54-nanoGlo parasites [32] were seeded at a density of 10 8 parasites in 10 mL culture medium at 5% hematocrit and cultured in a semi-automated culture system [34] under pressure of KNX-115 at 3xIC 50 . Compound containing medium was refreshed twice per day and parasitemia was followed over time by virtue of Nanoluciferase activity.…”

“…P. falciparum NF54-nanoGlo parasites [32] were synchronized by sorbitol treatment as described previously [33] and seeded at a density of 2% parasitemia in 5% hematocrit in RPMI1640 medium supplemented with 10% human type A serum, 367 µM hypoxanthine, 25 mM Hepes and 25 mM sodium bicarbonate and cultured in a semi-automated suspension culture system as described before [34]. Cultures were treated with saturating amount of test compound (5 µM KNX-115), control compound dihydroartemisinin (DHA, 1 µM) or vehicle controls (0.1% DMSO) and samples were taken for thin smears and luminescence measurements at regular intervals.…”