The process of discovering compounds with biological activity is a significant challenge, demanding the application of robust and predictive methodologies to ensure reliable outcomes. Bioinformatics and cheminformatics analyses serve as foundational tools in the search for novel antimalarial agents. Their effectiveness is evident through validation and research efforts led by esteemed organizations such as MalDA, MMV, the Bill and Melinda Gates Foundation, and the pharmaceutical industry33.
A crucial aspect of antimalarial drug discovery involves the identification of novel targets essential to the parasite’s life cycle and conserved among various Plasmodium species. This strategy aims to encompass species previously overlooked as harmful to humans34. Consequently, the integration of computational strategies plays a crucial role in the discovery process, enabling the exploration of innovative mechanisms of action for potential new antimalarials.
The NMT enzyme was validated as a target for antimalarial drugs in 201430 with many inhibitors initially repurposed from other organisms21,35–37 and through High Throughput Screening (HTS) campaigns38,39. The latest NMT inhibitors, highlighted by Rodríguez-Hernández and colleagues, particularly compounds 12b and 30a from the series of hybrids of DDD8564630 and IMP-100240 PvNMT inhibitors studies, demonstrated significant potency in vitro against PvNMT, with IC50 of 0.0368 µM and 0.089 µM, respectively. However, their efficacy against hypnozoites and schizonts was observed to be in the low micromolar range31.
In our study, we initially conducted an analysis of structural conformations within a collection of crystal structures containing the new PvNMT inhibitor. This analysis allowed us to discern the various binding modes employed by the inhibitors. In parallel, a gathered set of compounds from the literature that had been tested against PvNMT, rigorously validating our shape-based and docking computational models, in accordance with established good practices41,42. Subsequently, a virtual screening campaign was conducted. Our approach involved utilizing a predicted ΔG difference between Plasmodium and human proteins as a filter to reevaluate the docking scores. This strategy enabled us to prioritize compounds that showed potential selectivity for the parasite. As a result, we identified and prioritized 23 compounds with promising characteristics for further investigation.
During the experimental validation phase, seven compounds exhibited a phenotypic effect against the modified yeast NMT strains. Next, the compounds were assessed for their efficacy against P. falciparum strains, considering its significant identity (> 80%) of NMT between the species43. Notably, in P. falciparum strains assays, four of these compounds demonstrated significant activity against three parasite strains, all at a single concentration of 5 µM, without any indications of cross-resistance and no signs of cytotoxicity (< 50 µM) or hemolysis, which are indicative of a favorable chemical safety profile. It's important to consider that the observed micromolar activity against the parasites may be influenced by the compounds' permeability to erythrocytic cells. Moreover, various efflux pumps, such as mdr1 and mrp1 in Plasmodium, known for their role in resistance mechanisms44–46, could affect the activity of these compounds. In yeast cells, the primary efflux pump for drugs, the gene PDR5, was deleted in the strains we used47. This deletion leads to an increase in the concentration of compounds inside the cells, potentially explaining the divergent results observed for some compounds. One standout compound from these assays, LabMol-395, displayed an antimalarial efficacy below 0.17 µM against P. falciparum strains examined. However, this particular compound did not exhibit a yeast phenotype against NMT, indicating that its antimalarial activity likely involves a different target.
On enzymatic NMT assay, LabMol-394 exhibited modest activity, resulting in a 52% decrease in the activity of PvNMT at a concentration of 20 µM, displaying selectivity towards vivax over the human isoform. Molecular dynamics simulations revealed that LabMol-394 demonstrated interactions comparable to PvNMT inhibitors, including those used in the shape-based modeling. However, it lacked interaction with Ser319, previously suggested as a crucial residue in the proposed mechanism of NMT inhibition30. Throughout our simulations, LabMol-394 did not engage with a crucial residue, Leu410. However, it sustained significant π-stacking interactions with Tyr211 and Phe105 residues, while also forming a hydrogen bond with Ser387. Although Ser387 is not currently associated with inhibition in the literature, we hypothesize it as a potential mechanism.
Chemically, LabMol-394 features a quinoline scaffold extensively documented for its diverse biological activities48. This scaffold is particularly renowned for its well-established antimalarial effects, primarily attributed to its inhibition of β-hematin and the formation of an irreversible complex with the heme group49. These actions disrupt the development of the parasite in both liver and red blood cells. Notably, recent reports unveiled a novel quinoline inhibitor targeting the translation elongation factor 2 (PfEF2), demonstrating multistage antimalarial properties and currently progressing through clinical stages50–53. This discovery underscores the significance of heterocycles as crucial sources of chemical activities and introduces new mechanisms of inhibition aimed at pivotal targets in the Plasmodium life cycle.
The prioritized candidates in this study were experimentally validated and the results demonstrated a correlation between antimalarial activity and the absence of cytotoxicity. LabMol-395 emerged as promising candidate for target identification, due to its favorable antimalarial profile. Furthermore, enzymatic assays revealed LabMol-394 as the most promising PvNMT inhibitor, exhibiting selectivity against the parasite. Remarkably, there was a good correlation between the results obtained from the yeast system and enzymatic assays in NMT. This study makes a significant contribution to the exploration of the quinazoline scaffold, showcasing activity against Plasmodium vivax NMT and offering prospects for further optimization and development.