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Objectives: Rapid rate-of-kill (RoK) is a key parameter in the target candidate profile 1 (TCP1) for the next-generation antimalarial drugs for uncomplicated malaria, termed Single Encounter Radical Cure and Prophylaxis (SERCaP). TCP1 aims to rapidly eliminate the initial parasite burden, ideally as fast as artesunate, but minimally as fast as chloroquine. Here we explore whether the relative RoK of the Medicine for Malaria Venture (MMV) Malaria Box compounds is linked to their mode of action (MoA) and identify scaffolds of medicinal chemistry interest., Methods: We used a bioluminescence relative RoK (BRRoK) assay over 6 and 48 h, with exposure to equipotent IC50 concentrations, to compare the cytocidal effects of Malaria Box compounds with those of benchmark antimalarials., Results: BRRoK assay data demonstrate the following relative RoKs, from fast to slow: inhibitors of PfATP4>parasite haemoglobin catabolism>dihydrofolate reductase-thymidylate synthase (DHFR-TS)>dihydroorotate dehydrogenase (DHODH)>bc1 complex. Core-scaffold clustering analyses revealed intrinsic rapid cytocidal action for diamino-glycerols and 2-(aminomethyl)phenol, but slow action for 2-phenylbenz-imidazoles, 8-hydroxyquinolines and triazolopyrimidines., Conclusions: This study provides proof of principle that a compound's RoK is related to its MoA and that the target's intrinsic RoK is also modified by factors affecting a drug's access to it. Our findings highlight that as we use medicinal chemistry to improve potency, we can also improve the RoK for some scaffolds. Our BRRoK assay provides the necessary throughput for drug discovery and a critical decision-making tool to support development campaigns. Finally, two scaffolds, diamino-glycerols and 2-phenylbenzimidazoles, exhibit fast cytocidal action, inviting medicinal chemistry improvements towards TCP1 candidates., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies., (Copyright © 2018. Published by Elsevier Ltd.)

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.

Objectives: A future treatment for uncomplicated malaria will contain at least one component that exerts a rapid rate of kill. We describe here the validation and application of a simple, robust and rapid bioluminescence-based assay for the determination of the initial rate of kill in intra-erythrocytic asexual stages of Plasmodium falciparum ., Methods: A modification to the concentration-response bioluminescence [here termed bioluminescence relative rate of kill (BRRoK)] assay, utilizing exposure to fold-IC 50 concentrations (0.33× to  9×), was used to monitor the immediate cytocidal effect of 372 open-source compounds for antimalarial drug discovery available through the Medicines for Malaria Venture Malaria Box., Results: Antimalarial drugs that exert a rapid cytocidal effect produce a concentration-dependent loss of bioluminescence signal that correlates with available in vitro and in vivo estimates of parasite clearance time and parasite reduction ratio. Following the measurement of IC 50 for the Malaria Box compounds in Dd2 luc , the BRRoK assay was used to identify and rank 372 compounds for their initial cytocidal activity. Fifty-three compounds in the Malaria Box show an initial relative rate of kill greater than that of chloroquine, with 17 of these having an initial relative rate of kill greater than that of dihydroartemisinin., Conclusions: The BRRoK assay provides a rapid assay format for the estimation of a key pharmacodynamic property of antimalarial drug action. The simplicity and robustness of the assay suggests it would be readily scalable for high-throughput screening and a critical decision-making tool for antimalarial drug development., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

The methylerythritol phosphate (MEP) pathway is an essential metabolic pathway found in malaria parasites, but absent in mammals, making it a highly attractive target for the discovery of novel and selective antimalarial therapies. Using high-throughput screening, we have identified 2-phenyl benzo[d]isothiazol-3(2H)-ones as species-selective inhibitors of Plasmodium spp. 2-C-methyl- D -erythritol-4-phosphate cytidyltransferase (IspD), the third catalytic enzyme of the MEP pathway. 2-Phenyl benzo[d]isothiazol-3(2H)-ones display nanomolar inhibitory activity against P. falciparum and P. vivax IspD and prevent the growth of P. falciparum in culture, with EC 50 values below 400 nM. In silico modeling, along with enzymatic, genetic and crystallographic studies, have established a mechanism-of-action involving initial non-covalent recognition of inhibitors at the IspD binding site, followed by disulfide bond formation through attack of an active site cysteine residue on the benzo[d]isothiazol-3(2H)-one core. The species-selective inhibitory activity of these small molecules against Plasmodium spp. IspD and cultured parasites suggests they have potential as lead compounds in the pursuit of novel drugs to treat malaria.

Greater than the sum of its parts: Artemisinins are currently in phase I-II clinical trials against breast, colorectal and non-small-cell lung cancers. In an attempt to offer increased specificity, a series of hybrid artemisinin-polypyrrole minor groove binder conjugates are described. DNA binding/modelling studies and preliminary biological evaluation give insights into their mechanism of action and the potential of this strategy., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc(1). Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Malaria is responsible for approximately 1 million deaths annually; thus, continued efforts to discover new antimalarials are required. A HTS screen was established to identify novel inhibitors of the parasite's mitochondrial enzyme NADH:quinone oxidoreductase (PfNDH2). On the basis of only one known inhibitor of this enzyme, the challenge was to discover novel inhibitors of PfNDH2 with diverse chemical scaffolds. To this end, using a range of ligand-based chemoinformatics methods, ~17000 compounds were selected from a commercial library of ~750000 compounds. Forty-eight compounds were identified with PfNDH2 enzyme inhibition IC(50) values ranging from 100 nM to 40 μM and also displayed exciting whole cell antimalarial activity. These novel inhibitors were identified through sampling 16% of the available chemical space, while only screening 2% of the library. This study confirms the added value of using multiple ligand-based chemoinformatic approaches and has successfully identified novel distinct chemotypes primed for development as new agents against malaria., (© 2012 American Chemical Society)

Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite Plasmodium falciparum, hit to lead optimization led to identification of CK-2-68, a molecule suitable for further development. In order to reduce ClogP and improve solubility of CK-2-68 incorporation of a variety of heterocycles, within the side chain of the quinolone core, was carried out, and this approach led to a lead compound SL-2-25 (8b). 8b has IC(50)s in the nanomolar range versus both the enzyme and whole cell P. falciparum (IC(50) = 15 nM PfNDH2; IC(50) = 54 nM (3D7 strain of P. falciparum) with notable oral activity of ED(50)/ED(90) of 1.87/4.72 mg/kg versus Plasmodium berghei (NS Strain) in a murine model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the bc(1) complex of P. falciparum providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-pyridyl quinolones underlines the potential of this template for further lead optimization studies.

A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure-activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC(50) against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc(1), and studies to determine the potential advantage of this dual-targeting effect are in progress.

An abstract of the article "A novel drug for uncomplicated malaria: targeted high throughput screening (HTS) against the type II NADH:ubiquinone oxidoreductase (PfNdh2) of Plasmodium falciparum" that was presented at a conference titled "From Parasite to Prevention: Advances in the understanding of malaria" held in Edinburgh, Great Britain on October 20-22, 2010, is presented.