Biology subjects

Pasaje, C. F. A.

Publications and source records attributed to Pasaje, C. F. A..

8 recordsLinked to original sources

bioRxiv2025

The Human Chk1 Inhibitor CHIR-124 Shows Multistage Activity Against Plasmodium falciparum via Dual Inhibition of PfArk1 and Hemozoin Formation

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 {beta}-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.

microbiology↗

bioRxiv2025

Targeting Aurora kinases as essential cell cycle regulators to deliver multi-stage antimalarials against Plasmodium falciparum

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 (<250 nM) against all proliferative stages of parasite development, including asexual blood stages, liver schizonts and male gametes. Among the most potent compounds, hesperadin and AT83 exhibit >1000x selectivity towards the parasite without concerns about mammalian cell toxicity. Importantly, we identified PfArk1 as the principal vulnerable Ark family member, with specific inhibition of PfArk1 as the primary target for hesperadin and the human anaplastic lymphoma kinase (ALK) inhibitor TAE684. Hesperadins whole-cell and protein activity validates it as a unique PfArk1 tool compound. Inhibition of PfArk1 results in the parasites inability to complete mitotic processes, presenting with unsegregated, multi-lobed nuclei caused by aberrant microtubule organization. This suggests that PfArk1 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 PfArk1.

molecular biology↗

bioRxiv2022

A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which in Toxoplasma gondii includes MIC8 and the microneme-associated CRMP complex. Here, we examine the function of the microneme protein CLAMP, uncovering its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP-deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. Nevertheless, SPATR, which appears to act as an accessory factor in Toxoplasma, is essential during Plasmodium falciparum blood stages. Our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

microbiology↗

bioRxiv2022

Identification of Antimalarial Compounds that Inhibit Apicomplexan AP2 Proteins

Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified compounds that interact with this domain. Four compounds were found to compete for DNA binding with AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P. falciparum trophozoites and results in a decrease in abundance of log2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti-Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that are targeted against the ApiAP2 family of proteins. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics. Author SummaryPlasmodium parasites are the causative agent of malaria, which resulted in over 600,000 deaths in 2021. Due to resistance arising for every antimalarial therapeutic deployed to date, new drug targets and druggable pathways must be explored. To address this concern, we used a molecular docking screen to predict competitors of DNA binding by the parasite specific family of Apicomplexan AP2 (ApiAP2) transcription factor proteins for testing in vitro and in vivo. We find that ApiAP2 competing compounds have antimalarial activity consistent with the disruption of gene regulation. This work will further our understanding of both the biological role and targetability of parasite transcriptional regulation.

molecular biology↗

bioRxiv2021

Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183

Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (ACS) inhibitor to enter preclinical development. Our studies demonstrated attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound showed exceptional in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocked P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identified ACS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. MMV693183 was well adsorbed after oral administration in mice, rats and dogs. Pharmacokinetic - pharmacodynamic modelling predicted that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. In conclusion, the ACS-targeting compound MMV693183 represents a promising addition to the portfolio of antimalarials in (pre)clinical development with a novel mode of action for the treatment of malaria and blocking transmission.

microbiology↗

bioRxiv2020

Selective expression of variant surface antigens enables Plasmodium falciparum to evade immune clearance in vivo

Plasmodium falciparum has developed extensive mechanisms to evade host immune clearance. Currently, most of our understanding is based on in vitro studies of individual parasite variant surface antigens and how this relates to the processes in vivo is not well-understood. Here, we have used a humanized mouse model to identify parasite factors important for in vivo growth. We show that upregulation of the specific PfEMP1, VAR2CSA and the RIFIN PF3D7_1254800 provides the parasite with protection from macrophage phagocytosis and natural killer cell mediated killing. Taken together, these findings reveal new insights on the molecular and cellular mechanisms that coordinate the immune escape process the parasite utilizes in vivo. As immune evasion may be particularly important during the establishment of the blood stage infection when parasite numbers are still relatively small, identification of specific parasite variant surface antigens provides targets for developing more effective vaccines by targeting parasite immune evasion.

microbiology↗

bioRxiv2020

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

Phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin, a first-line malaria treatment, and environmental factors. However, the functional role of PI(3)P in the Plasmodium stress response and a possible mechanism of protection were unknown. Here, we used multiple chemical probes including PI3K inhibitors and known antimalarial drugs to examine the importance of PI(3)P under thermal conditions that recapitulate malaria fever. Live cell microscopy using both chemical and genetic reporters revealed that PI(3)P stabilizes the acidic and proteolytic digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. These phenotypes are not observed with an inactive structural analog of the PI3K inhibitor. A chemoproteomic and biochemical approach identified PfHsp70-1 as a parasite PI(3)P-binding protein. Targeting PfHsp70-1 with a small molecule inhibitor phenocopied PI(3)P-deficient parasites under heat shock. Furthermore, tunable knockdown of PfHsp70-1 showed that PfHsp70-1 downregulation causes DV destabilization and hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1, and present a novel PI(3)P function in stabilizing the DV compartment during heat stress.

microbiology↗

bioRxiv2019

An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum

Establishing robust genome engineering methods in the malarial parasite, Plasmodium falciparum, has the potential to substantially improve the efficiency with which we gain understanding of this pathogens biology to propel treatment and elimination efforts. Methods for manipulating gene expression and engineering the P. falciparum genome have been validated. However, a significant barrier to fully leveraging these advances is the difficulty associated with assembling the extremely high AT content DNA constructs required for modifying the P. falciparum genome. These are frequently unstable in commonly-used circular plasmids. We address this bottleneck by devising a DNA assembly framework leveraging the improved reliability with which large AT-rich regions can be efficiently manipulated in linear plasmids. This framework integrates several key functional genetics outcomes via CRISPR/Cas9 and other methods from a common, validated framework. Overall, this molecular toolkit enables P. falciparum genetics broadly and facilitates deeper interrogation of parasite genes involved in diverse biological processes.

microbiology↗