High-throughput screening and meta-analysis for lead compounds in antimalarial drug discovery

(2025) 24:378 Van Truong et al. Malaria Journal https://doi.org/10.1186/s12936-025-05587-0 Malaria Journal Open Access RESEARCH High‑throughput screening and meta‑analysis for lead compounds in antimalarial drug discovery Nguyen Van Truong1, Seok‑Won Na1,2, Ji Hoon Park1,2, Tuyet‑Kha Nguyen1, Nguyen Sy Thau1,3, Thi‑Thanh Hang Chu1, Bazgha Sanaullah1, Ch Venkataramaiah1, Jin‑Hee Han1, Sung‑Hun Na4, Won‑Sun Park5, Wan‑Joo Chun6, Joo Hwan No2* and Eun‑Taek Han1* Abstract Background The discovery of novel antimalarial drugs against Plasmodium falciparum has become globally urgent due to the consistent increase in mortality, morbidity, and drug resistance in endemic areas. Methods Using an in-house library, novel antimalarial agents were identified through in vitro high-throughput screening (HTS) and meta-analysis. Hit compounds were selected from the primary HTS at 10 µM and confirmed in a dose-dependent manner to determine their IC₅₀ values. The identified hit molecules were further selected based on the following criteria: novelty, antimalarial activity (IC50), pharmacokinetic properties (Cmax and T1/2), mechanism of action, and safety (in vitro and in vivo) (CC50, SI, LD50, and MTD). In vitro and in vivo antimalarial activity against drug-sensitive and resistant strains (3D7, NF54 and K1, Dd2, Dd2-R539T (+), and CamWT-C580Y (+)) and the rodent Plasmodium berghei parasite-infected animal model, respectively, were subsequently used to validate hit compounds. Results Based on the top 3% threshold, 256 compounds were selected for dose‒response curve analysis from the HTS. Among them, 110 compounds without published research related to Plasmodium and 157 compounds with IC50 values < 1 µM were identified. Further analysis confirmed 69 compounds with median lethal doses, maximum tolerated doses or treated doses greater than 20 mg/kg, 48 compounds with FDA approval, 29 compounds characterized by Cmax > IC100 and T1/2 > 6 h, and 38 compounds with a potential mechanism in Plasmodium. Next, 19 candidates were further evaluated for in vitro inhibition of drug-resistant parasites and inhibition in a mouse model of P. berghei parasites. Notably, three potent inhibitors were identified, exhibiting 95.9% and 81.4% suppression via oral delivery at a dose of 50 mg/kg ONX-0914 and methotrexate, respectively, and 96.4% suppression via intraperitoneal delivery at a dose of 20 mg/kg of an antimony compound. In addition, strong in vitro antimalarial activity was demonstrated against CQ- and ART-sensitive and resistant strains (IC50 < 500 nM). Conclusions Combining HTS and meta-analysis provides a robust method for screening antimalarial candidate compounds and identifying new hits with in vivo activity as candidates to treat drug-resistant malarial strains. Keywords Malaria, High-throughput screening, Meta-analysis, Drug discovery *Correspondence: Joo Hwan No Eun‑Taek Han Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. Van Truong et al. Malaria Journal (2025) 24:378 Background Malaria, which is caused by Plasmodium parasites, remains a major global health burden, with an estimated 263 million cases and 597,000 deaths annually [1]. Along with increasing incidence, high rates of antimalarial drug resistance have been reported in Africa, the Americas, and Southeast Asia. Specifically, therapeutic efficacy studies (TESs) have shown treatment failure with both single or combination regimens, including with newer drugs such as dihydroartemisinin-piperaquine and artesunate-amodiaquine, primarily due to genetic mutations associated with drug resistance [2–5]. To achieve the goal of malaria eradication, there is an urgent need to expand antimalarial drug discovery efforts. However, drug discovery and development is a long, costly, and high-risk process, typically taking over a decade and costing an estimated over $1–2 billion for a drug before approval for human treatment [6, 7]. Among the advancements in technology that have accelerated drug discovery, high-throughput screening (HTS) has emerged as a powerful method for screening millions of compounds in pharmaceutical libraries. Over the past decade, HTS has significantly contributed to the antimalarial development pipeline, leading to the identification of new chemotypes that target the diseasecausing asexual stages of Plasmodium [8–10]. The pharmaceutical industry has developed HTS based on two primary approaches: phenotypic (whole-cell) screening and target-based screening. Phenotypic screening evaluates changes in parasites upon exposure to antimalarial compounds, whereas target-based screening assesses the effects of compounds on purified target proteins [11]. Recent analyses have shown that phenotypic approaches are more successful for small molecule inhibitors, largely due to advancements in high-resolution optical microscopy and improvements in image analysis software [12– 15]. In phenotypic HTS, parasite-infected red blood cells (RBCs) are stained with nucleic acid-conjugated fluorescence dyes, followed by the detection and classification of parasites at different developmental stages. Compared with the conventional SYBR Green I assay, which is often used as a reference method, phenotypic HTS has demonstrated enhanced accuracy in detecting antimalarial activity [11–14]. Following the HTS stage, an enormous workload remains for subsequent steps, including ‘hit’ confirmation, lead optimization, and preclinical evaluations before a candidate drug can proceed to clinical trials and receive approval from the U.S. Food and Drug Administration (FDA) [6, 16]. Drug discovery is an inherently challenging process, and the development of antimalarials presents additional difficulties due to several key factors, including the need for high tolerance and safety across diverse Page 2 of 19 endemic populations, ease of administration, short-term duration, availability of combination regimens to (...truncated)