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The Malaria Drug Accelerator (MalDA) is a consortium of 15 leading scientific laboratories. The aim of MalDA is to improve and accelerate the early antimalarial drug discovery process by identifying new, essential, druggable targets. In addition, it seeks to produce early lead inhibitors that may be advanced into drug candidates suitable for preclinical development and subsequent clinical testing in humans. By sharing resources, including expertise, knowledge, materials, and reagents, the consortium strives to eliminate the structural barriers often encountered in the drug discovery process. Here we discuss the mission of the consortium and its scientific achievements, including the identification of new chemically and biologically validated targets, as well as future scientific directions., Competing Interests: Declaration of Interests K.J.D. holds stock in TropIQ Health Sciences. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Most phenotypic screens aiming to discover new antimalarial chemotypes begin with low cost, high-throughput tests against the asexual blood stage (ABS) of the malaria parasite life cycle. Compounds active against the ABS are then sequentially tested in more difficult assays that predict whether a compound has other beneficial attributes. Although applying this strategy to new chemical libraries may yield new leads, repeated iterations may lead to diminishing returns and the rediscovery of chemotypes hitting well-known targets. Here, we adopted a different strategy to find starting points, testing ∼70,000 open source small molecules from the Global Health Chemical Diversity Library for activity against the liver stage, mature sexual stage, and asexual blood stage malaria parasites in parallel. In addition, instead of using an asexual assay that measures accumulated parasite DNA in the presence of compound (SYBR green), a real time luciferase-dependent parasite viability assay was used that distinguishes slow-acting (delayed death) from fast-acting compounds. Among 382 scaffolds with the activity confirmed by dose response (<10 μM), we discovered 68 novel delayed-death, 84 liver stage, and 68 stage V gametocyte inhibitors as well. Although 89% of the evaluated compounds had activity in only a single life cycle stage, we discovered six potent (half-maximal inhibitory concentration of <1 μM) multistage scaffolds, including a novel cytochrome bc1 chemotype. Our data further show the luciferase-based assays have higher sensitivity. Chemoinformatic analysis of positive and negative compounds identified scaffold families with a strong enrichment for activity against specific or multiple stages.

High-content screening (HCS) is a cell-based type of phenotypic screening that combines multiple simultaneous readouts with a high level of throughput. A particular benefit of this form of screening for drug discovery is the ability to perform the interrogation in a biologically relevant system. This approach has greatly advanced the field of drug discovery for cryptosporidiosis, a diarrheal disease caused by protozoan parasites of Cryptosporidium spp. These parasites are obligate intracellular parasites and cannot be cultured in vitro without the support of a host cell, limiting the options for potential assay readout. Here we describe an established 384- or 1536-well format high-content imaging (HCI) assay of Cryptosporidium-infected HCT-8 human ileocecal adenocarcinoma cells. This HCS assay is a powerful tool to assess large numbers of compounds to power drug discovery, as well as to phenotypically characterize known Cryptosporidium-active compounds.

Visceral leishmaniasis (VL) and Chagas disease (CD) are caused by kinetoplastid parasites that affect millions of people worldwide and impart a heavy burden against human health. Due to the partial efficacy and toxicity-related limitations of the existing treatments, there is an urgent need to develop novel therapies with superior efficacy and safety profiles to successfully treat these diseases. Herein we report the application of whole-cell phenotypic assays to screen a set of 150,000 compounds against Leishmania donovani, a causative agent of VL, and Trypanosoma cruzi, the causative agent of CD, with the objective of finding new starting points to develop novel drugs to effectively treat and control these diseases. The screening campaign, conducted with the purpose of global open access, identified twelve novel chemotypes with low to sub-micromolar activity against T. cruzi and/or L. donovani. We disclose these hit structures and associated activity with the goal to contribute to the drug discovery community by providing unique chemical tools to probe kinetoplastid biology and as hit-to-lead candidates for drug discovery., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lymphatic filariasis and onchocerciasis diseases caused by filarial parasite infections can lead to profound disability and affect millions of people worldwide. Standard mass drug administration campaigns require repetitive delivery of anthelmintics for years to temporarily block parasite transmission but do not cure infection because long-lived adult worms survive the treatment. Depletion of the endosymbiont Wolbachia, present in most filarial nematode species, results in death of adult worms and therefore represents a promising target for the treatment of filariasis. Here, we used a high-content imaging assay to screen the pure compounds collection of the natural products library at The Scripps Research Institute for anti- Wolbachia activity, leading to the identification of kirromycin B (1) as a lead candidate. Two additional congeners, kirromycin (2) and kirromycin C (3), were isolated and characterized from the same producing strain Streptomyces sp. CB00686. All three kirromycin congeners depleted Wolbachia in LDW1 Drosophila cells in vitro with half-maximal inhibitory concentrations (IC 50 ) in nanomolar range, while doxycycline, a registered drug with anti- Wolbachia activity, showed lower activity with an IC 50 of 152 ± 55 nM. Furthermore, 1-3 eliminated the Wolbachia endosymbiont in Brugia pahangi ovaries ex vivo with higher efficiency (65%-90%) at 1 μM than that of doxycycline (50%). No cytotoxicity against HEK293T and HepG2 mammalian cells was observed with 1-3 at the highest concentration (40 μM) used in the assay. These results suggest kirromycin is an effective lead scaffold, further exploration of which could potentially lead to the development of novel treatments for filarial nematode infections.

Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets Wolbachia , a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in L. sigmodontis , Brugia malayi , and Onchocerca ochengi in vivo preclinical models of filarial disease and in vitro selectivity against Loa loa (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (∼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against Cryptosporidium spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of Cryptosporidium infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.