Lyn-Marie Birkholtz

Introduction: The emergence of resistance to frontline antimalarial drugs underscores the urgent need for new chemotypes capable of targeting multiple P. falciparum life-cycle stages, including transmission stages. Methods: Building on the antimycobacterial scaffold BM212 , we generated and profiled a focused library of 1,3,5-trisubstituted pyrroles to define the structural features driving activity against asexual blood-stage (ABS) parasites and late-stage gametocytes (LGc) of P. falciparum. Restults and Discussion: Clear structure -activity relationship (SAR) trends emerged: the C3 cyclic amine was the dominant driver of life-cycle stage preference, with methylpiperazine substituent conferring potent gametocytocidal activity (compound 7, IC50 0.093 μM), whereas the dimethyl-azasilinane group enabled potent dual-stage profiles (compound 1 IC50 values 0.48 against LGc and 1.08 against ABS). At N1 and C5, electron-deficient aryl substituents (4-F-phenyl, 2-CF3-phenyl) consistently enhanced potency and improved physicochemical properties, while aliphatic rings (e.g., cyclobutyl) at N1 optimized hydrophobic fit and solubility. Specific combinations of these motifs yielded balanced dual-stage activity without compromising lipophilicity or metabolic stability. Several optimized analogues retained activity against the multidrug-resistant P. falciparum Dd2 strain, with IC50 values ranging from 1.01 to 1.54 μM and exhibited potent transmission-blocking activity, with compound 9 achieving 81% transmission-reducing activity in the standard membrane feeding assay (SMFA). Overall, this work positions BM212 -derived pyrroles as a promising and versatile multistage antiplasmodial chemotype, while delineating the key structural determinants required to achieve potency, selectivity, and transmission-blocking efficacy.

The high burden of malaria and growing resistance to frontline antimalarials demand new drug target combinations with reduced propensities for conferring parasite resistance. An attractive approach for circumventing antimalarial drug resistance is target repurposing, in which known drugs that act through protein targets of human origin that are also active against the human malaria parasite Plasmodium falciparum are exploited to identify novel antimalarial drug targets. Here, we show that the human checkpoint kinase 1 (Chk1) inhibitor CHIR-124 is active in vitro against both drug-sensitive and drug-resistant asexual blood stage parasites and competitively binds to several Plasmodium kinases. The compound also shows moderate activity against both the liver and gametocyte forms of the parasite. Further target investigation of CHIR-124 via conditional knockdown experiments confirmed that P. falciparum Aurora-related kinase 1 (PfArk1) is implicated in its parasiticidal activity. Notably, CHIR-124 also inhibits β-hematin (synthetic hemozoin) formation and causes a dose-dependent increase in free heme that correlates with inhibition of parasite growth. These findings suggest that polypharmacology is involved in the activity of CHIR-124 against P. falciparum via the dual inhibition of Plasmodium PfArk1 and hemozoin formation, both essential for parasite proliferation. This is further supported by in vitro drug combination experiments, morphological studies, and resistance generation attempts. This study validates the feasibility of dual Plasmodium kinase/hemozoin formation inhibitors active against resistant strains with decreased resistance risks in the fight against malaria.

The ongoing rise in antimalarial drug resistance underscores the urgent need for new drug candidates that specifically target novel mechanisms. Malaria parasites employ various epigenetic strategies to regulate gene expression throughout their complex life cycle, with histone lysine acetylation and methylation being well-studied and targeted by new antiplasmodials. By contrast, arginine methylation remains poorly explored. Plasmodium falciparum possesses three protein arginine methyltransferases (PRMTs) that maintain a unique and combinatorial histone arginine methylation landscape. Here, we present a chemical repositioning strategy to evaluate the efficacy of known PRMT inhibitors against malaria parasites. We identified a potent compound, TC-E 5003, which is active across multiple stages of parasite development. PfPRMT1 was proposed as the most likely target of TC-E 5003, with a distinct structure-activity relationship demonstrated by TC-E 5003 analogs from a hit expansion campaign. The chemotype exhibits a clear pharmacophore that elucidates the compound's mechanism of action. Overall, these findings open a new pathway for identifying multistage active antiplasmodial candidates targeting a novel protein family in P. falciparum.

A new class of benzoxaboroles with a phenylglycine appendage was found to display in vitro blood stage activity against the human malaria parasite Plasmodium falciparum (Pf). Structure-activity relationship studies of the starting hit compound 3 resulted in compounds active against PfNF54 drug-sensitive and PfK1 drug-resistant strains with an in vitro antiplasmodium IC₅₀ < 0.4 μM, selectivity over mammalian cell-lines (selectivity index > 47) and high aqueous solubility (160 to >200 μM). Selected compounds showed good in vitro metabolic stability when incubated with human, rat, and mouse liver microsomes and showed no cross-resistance against barcoded mutant lines. Two frontrunner compounds, 6 and 7, were dosed orally at 50 mg·kg^-1 using a standard quadrupole dosing regimen in a P. berghei mouse infection model and showed encouraging in vivo efficacy. This work identifies a promising new class of phenylglycine-based benzoxaboroles, which warrants further medicinal chemistry optimization.

Kinases play critical roles in the development and adaptation of Plasmodium falciparum and present novel opportunities for chemotherapeutic intervention. Mitotic kinases that regulate the proliferation of the parasites by controlling nuclear division, segregation, and cytokinesis. We evaluated the potential of human Aurora kinase (Aur) inhibitors to prevent P. falciparum development by targeting members of the Aurora‐related kinase (Ark) family in this parasite. Several human AurB inhibitors exhibited multistage potency (< 250 nM) against all proliferative stages of parasite development, including asexual blood stages, liver schizonts, and male gametes. The most potent compounds, hesperadin, TAE684, and AT83, exhibited > 1000x selectivity towards the parasite. Importantly, we identified Pf Ark1 as the principal vulnerable Ark family member, with specific inhibition of Pf Ark1 as the primary target for hesperadin. Hesperadin's whole‐cell and protein activity validates it as a unique Pf Ark1 tool compound. Inhibition of Pf Ark1 results in the parasite's inability to complete mitotic processes, presenting with unsegregated, multi‐lobed nuclei caused by aberrant microtubule organization. This suggests Pf Ark1 is the main Aur mitotic kinase in proliferative stages of Plasmodium , characterized by bifunctional AurA and B activity. This paves the way for drug‐discovery campaigns based on hesperadin targeting Pf Ark1.

Kinases play critical roles in the development and adaptation of Plasmodium falciparum and present novel opportunities for chemotherapeutic intervention. Mitotic kinases that regulate the proliferation of the parasites by controlling nuclear division, segregation, and cytokinesis. We evaluated the potential of human Aurora kinase (Aur) inhibitors to prevent P. falciparum development by targeting members of the Aurora‐related kinase (Ark) family in this parasite. Several human AurB inhibitors exhibited multistage potency (< 250 nM) against all proliferative stages of parasite development, including asexual blood stages, liver schizonts, and male gametes. The most potent compounds, hesperadin, TAE684, and AT83, exhibited > 1000x selectivity towards the parasite. Importantly, we identified Pf Ark1 as the principal vulnerable Ark family member, with specific inhibition of Pf Ark1 as the primary target for hesperadin. Hesperadin's whole‐cell and protein activity validates it as a unique Pf Ark1 tool compound. Inhibition of Pf Ark1 results in the parasite's inability to complete mitotic processes, presenting with unsegregated, multi‐lobed nuclei caused by aberrant microtubule organization. This suggests Pf Ark1 is the main Aur mitotic kinase in proliferative stages of Plasmodium , characterized by bifunctional AurA and B activity. This paves the way for drug‐discovery campaigns based on hesperadin targeting Pf Ark1.

Anticancer ATP-competitive inhibitors are a promising source of new starting points for antimalarial drug discovery. Herein, we present a novel antimalarial chemotype based on the anticancer human ataxia-telangiectasia-mutated (ATM) kinase inhibitor AZD0156. This class inhibits phosphatidylinositol 4-kinase IIIβ (PI4K) in the human malaria parasite Plasmodium, demonstrating remarkable activities against all stages of the Plasmodium falciparum life cycle. The current series exhibited a lower propensity for resistance and toxicity compared to previous Plasmodium PI4K inhibitors. The lead compound 18 was efficacious in a humanized NOD-scid IL-2Rγnull mouse model of P. falciparum malaria, with an ED90 value of 4.6 mg kg-1.

Kinases that play critical roles in the development and adaptation of Plasmodium falciparum present novel opportunities for chemotherapeutic intervention. Of particular interest are mitotic kinases that regulate the proliferation of the parasites by controlling nuclear division, segregation and cytokinesis. We evaluated the potential of human Aurora kinase (Aur) inhibitors to inhibit P. falciparum development by targeting members of the Aurora-related kinase (Ark) family in this parasite. Several human AurB inhibitors exhibited multistage potency (1000x selectivity towards the parasite without concerns about mammalian cell toxicity. Importantly, we identified Pf Ark1 as the principal vulnerable Ark family member, with specific inhibition of Pf Ark1 as the primary target for hesperadin and the human anaplastic lymphoma kinase (ALK) inhibitor TAE684. Hesperadin’s whole-cell and protein activity validates it as a unique Pf Ark1 tool compound. Inhibition of Pf Ark1 results in the parasite’s inability to complete mitotic processes, presenting with unsegregated, multi-lobed nuclei caused by aberrant microtubule organization. This suggests that Pf Ark1 is the main Aur mitotic kinase in proliferative stages of Plasmodium , characterized by bifunctional AurA and B activity. This paves the way for drug discovery campaigns based on hesperadin targeting Pf Ark1.