Paul Horrocks

Background/Objectives: The discovery of antimalarial compounds with novel mechanisms of action and distinct rates of kill (RoK) is essential to address emerging drug resistance in Plasmodium falciparum. Natural product libraries represent a valuable and chemically diverse source of potential new antiplasmodial scaffolds. This study aimed (i) to evaluate a modified bioluminescence relative rate-of-kill (mBRRoK) assay as a rapid triage platform for screening large compound libraries with previously unknown antiplasmodial activity, enabling simultaneous assessment of potency and RoK, and (ii) to identify novel compounds with potent and selective in vitro erythrocytic activity. Methods: A fixed two-concentration mBRRoK screen was applied to 1165 compounds from the PhytoQuest natural product library. Antiplasmodial activity and RoK profiles were assessed over 48 h using two genetically distinct luciferase-expressing P. falciparum strains (Dd2^luc and NF54^luc) with distinct drug resistance backgrounds. Reproducibility was evaluated across biological replicates. Selected hits underwent secondary profiling, including EC₅₀ determination and HepG2 cytotoxicity assessment to establish potency and selectivity. Results: The primary screen identified 36 lead compounds demonstrating potent activity within 48 h, encompassing both fast- and slow-acting phenotypes. Activity was reproducible and largely strain-independent across both parasite lines. Secondary profiling prioritised four compounds (100657, 101158, 101160, and 101173) with nanomolar-to-micromolar antiplasmodial potency and favourable selectivity indices relative to mammalian cell cytotoxicity. Conclusions: The mBRRoK assay provides a robust and scalable framework for integrating potency and pharmacodynamic assessment in early antimalarial discovery. This strategy enabled efficient prioritisation of selective natural product-derived leads with distinct killing profiles, supporting their progression toward further optimisation and preclinical development.

The use of plant-derived natural products for the treatment of tropical parasitic diseases often has ethnopharmacological origins. As such, plants grown in temperate regions remain largely untested for novel anti-parasitic activities. We describe here a screen of the PhytoQuest Phytopure library, a novel source comprising over 600 purified compounds from temperate zone plants, against in vitro culture systems for Plasmodium falciparum, Leishmania mexicana, Trypanosoma evansi and T. brucei. Initial screen revealed 6, 65, 15 and 18 compounds, respectively, that decreased each parasite’s growth by at least 50% at 1–2 µM concentration. These initial hits were validated in concentration-response assays against the parasite and the human HepG2 cell line, identifying hits with EC₅₀ < 1 μM and a selectivity index of >10. Two sesquiterpene glycosides were identified against P. falciparum, four sterols against L. mexicana, and five compounds of various scaffolds against T. brucei and T. evansi. An L. mexicana resistant line was generated for the sterol 700022, which was found to have cross-resistance to the anti-leishmanial drug miltefosine as well as to the other leishmanicidal sterols. This study highlights the potential of a temperate plant secondary metabolites as a novel source of natural products against tropical parasitic diseases.

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. 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. 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. 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.

Cylicodiscus gabunensis Harms (Family Leguminosae) (CG) is an African medicinal plant used as a treatment of various ailments including malaria, liver diseases, and gastrointestinal disturbances. Its extracts showed potent in vitro antibacterial activity. However, the antibacterial components are unknown. In this study, the stem bark of the CG plant was extracted and its antibacterial property against a panel of Gram-negative and Gram-positive bacterial strains assessed using the disk diffusion assay method. Bioassay-guided fractionation of the bioactive extracts was employed to identify bioactive constituents using both gas and liquid chromatography mass spectrometry. Chemical synthesis was used to make the analogues of gallic acid. Microplate dilution assays and scanning electron microscopy (SEM) were used to evaluate the antibacterial properties and mechanism of action of the active fractions and pure compounds. The most bioactive sub-fractions derived from CG comprised of ethyl gallate, gallic acid and polyphenols. Five alkyl/alkenyl gallates were synthesized. A preliminary structure-activity relationship of gallic acid derivatives was obtained using the synthetic analogues and a series of commercially available phenolic compounds. Increasing the length of alkyl chains generally increases the potency of the alkyl gallates. Introducing a double bond with restricted conformations of the C-5 side chain has little effect on the antibacterial property. SEM analysis of the effect of alkyl gallates on Staphylococcus aureus indicates that they appear to interrupt S. aureus bacterial cell wall integrity. The results of this research rationalise the ethnobotanical use of C. gabunensis and suggest that gallate derivatives may serve as promising antibacterial agents for the treatment of infectious diseases.

The protozoan parasite Leishmania causes leishmaniasis; a spectrum of diseases of which there are an estimated 1 million new cases each year. Current treatments are toxic, expensive, difficult to administer, and resistance to them is emerging. New therapeutics are urgently needed, however, screening the infective amastigote form of the parasite is challenging. Only certain species can be differentiated into axenic amastigotes, and compound activity against these does not always correlate with efficacy against the parasite in its intracellular niche. Methods used to assess compound efficacy on intracellular amastigotes often rely on microscopy-based assays. These are laborious, require specialist equipment and can only determine parasite burden, not parasite viability. We have addressed this clear need in the anti-leishmanial drug discovery process by producing a transgenic L. mexicana cell line that expresses the luciferase NanoLuc-PEST. We tested the sensitivity and versatility of this transgenic strain, in comparison with strains expressing NanoLuc and the red-shifted firefly luciferase. We then compared the NanoLuc-PEST luciferase to the current methods in both axenic and intramacrophage amastigotes following treatment with a supralethal dose of Amphotericin B. NanoLuc-PEST was a more dynamic indicator of cell viability due to its high turnover rate and high signal:background ratio. This, coupled with its sensitivity in the intramacrophage assay, led us to validate the NanoLuc-PEST expressing cell line using the MMV Pathogen Box in a two-step process: i) identify hits against axenic amastigotes, ii) screen these hits using our bioluminescence-based intramacrophage assay. The data obtained from this highlights the potential of compounds active against M. tuberculosis to be re-purposed for use against Leishmania. Our transgenic L. mexicana cell line is therefore a highly sensitive and dynamic system suitable for Leishmania drug discovery in axenic and intramacrophage amastigote models. The protozoan parasite Leishmania causes a spectrum of diseases collectively known as leishmaniasis. The parasite is transmitted to humans by the bite of its vector, the sand fly, following which the parasite invades host white blood cells, particularly macrophages. Leishmaniasis is classified as a neglected tropical disease, and is endemic in 97 countries. Symptoms of the disease depend on the species of Leishmania. These include skin lesions, destruction of the mucosal membranes, and the visceral form which is usually fatal if untreated. Current therapeutic options for leishmaniasis have a number of associated problems that include toxicity, the development of drug resistance and poor patient compliance due to lengthy and painful treatment regimens. New therapeutics are therefore urgently needed. The ability to screen potential drug candidates requires robust screening assays. Currently, screening the intracellular parasite relies on microscopy-based techniques that require expensive equipment, are time consuming and only detect parasite burden, not viability. By using a transgenic cell line that expresses the NanoLuc-PEST luciferase, we show that we have a parasite-specific viability marker that can be used to measure the efficacy of compounds against the intracellular parasite. We validate the potential of this cell line by screening the MMV Pathogen Box.

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-IC₅₀ 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 IC₅₀ 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.