Biology subjects

Boonyalai, N.

Publications and source records attributed to Boonyalai, N..

6 recordsLinked to original sources

bioRxiv2026

Potent reaction hijacking inhibitors of Plasmodium falciparum asparagine tRNA synthetase

Malaria remains one of the major threats to human health. Breakthrough drugs with high potency and low resistance risk are needed to combat the ever-increasing resistance to currently deployed antimalarials. Here, we explore a series of 4-amino-quinazoline-based sulfonamides, with drug-like physicochemical parameters and a synthetically accessible scaffold. Exemplars exhibit nanomolar potency against blood stage Plasmodium cultures, with up to 300-fold selectivity compared with a mammalian cell line. The compounds are also active against transmissible stages of P. falciparum and are refractory to resistance development. Targeted mass spectrometry reveals that the compounds act as reaction hijacking inhibitors targeting P. falciparum aminoacyl tRNA synthetases (aaRSs). Subtle changes to the chemical structure switch the main target from cytoplasmic tRNA threonine synthetase (PfThrRS) to cytoplasmic asparagine synthetase (PfAsnRS), a change that is associated with increased potency and selectivity. The target preference was confirmed by selective knock-down of different P. falciparum aaRSs and by tolerance selection in a mutator line. Consistent with aaRS targets, exemplar compounds activate the amino acid starvation response. Recombinant enzyme inhibition and thermal stabilisation assays confirm the susceptibility of PfAsnRS to reaction hijacking and show that human AsnRS is less susceptible. A molecular model of Asn-tRNA-bound PfAsnRS reveals that a potent hijacker adopts a pose similar to adenosine 5-monophosphate (AMP). An AlphaFold model of the native PfAsnRS dimer helps explain the tolerance-conferring effect of a mutation at the dimer interface.

biochemistry↗

bioRxiv2025

Shared Binding Site but Divergent Resistance Profiles Uncover Novel Resistance Mechanisms in Plasmodium HSP90 Inhibitors

Drug resistance is a widespread problem across therapeutic areas including malaria, but what accounts for resistance propensity remains poorly understood. Here, we reveal that two HSP90 inhibitors targeting the identical ATP-binding site exhibit dramatically different resistance profiles in P. falciparum. Geldanamycin readily selected 10 distinct resistance mutations conferring up to 22-fold resistance, while AUY-922 required 44 weeks to yield a single A41S mutation with only 2-fold resistance to AUY-922 but not to geldanamycin. Resistance mapping in parasites and yeast revealed geldanamycin resistance mutations distributed throughout the binding pocket whereas AUY-922 resistance mutations localized close to the ATP-binding site. Unexpectedly, the A41S mutation enhanced AUY-922 binding affinity without changing geldanamycin binding. In silico analysis suggested this enhancement occurs through additional hydrogen bonding, yet stronger binding correlated with resistance. In yeast, A41S had opposite effects, hypersensitizing cells to all HSP90 inhibitors tested. Additionally, conditional HSP90 knockdown increased geldanamycin sensitivity but left AUY-922 activity unaffected, indicating different target dependencies despite shared binding sites. Based on these data, we propose a multi-target hypothesis where AUY-922s lower resistance risk stems from engaging multiple HSP90 family members. Our findings reveal how enhanced drug-target binding can paradoxically correlate with resistance and demonstrate that resistance risk cannot be predicted from binding site identity alone, providing insights for developing more durable drugs across therapeutic areas.

microbiology↗

bioRxiv2025

Mechanistic Insights into Dual-Active Liver and Blood-Stage Antiplasmodials

The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG) respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), and TCMDC-141334 and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modelled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration.

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

The Parasite Intraerythrocytic Cycle and Human Circadian Cycle are Coupled During Malaria Infection

During infections with malaria parasites P. vivax, patients exhibit rhythmic fevers every 48 hours. These fever cycles correspond with the time parasites take to traverse the Intraerythrocytic Cycle (IEC) and may be guided by a parasite-intrinsic clock. Different species of Plasmodia have cycle times that are multiples of 24 hours, suggesting they may be coordinated with the host circadian clock. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC were correlated across multiple patients, suggesting that the cycles are coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable anti-malarial therapies that disrupt this coupling.

systems biology↗

bioRxiv2018

Function and essentiality of Plasmodium falciparum Plasmepsin V

The malaria parasite replicates within erythrocytes. The pathogenesis of clinical malaria is in large part due to the capacity of the parasite to remodel its host cell. To do this, intraerythrocytic stages of Plasmodium falciparum export more than 300 proteins that dramatically alter the morphology of the infected erythrocyte as well as its mechanical and adhesive properties. P. falciparum plasmepsin V (PfPMV) is an aspartic protease that processes proteins for export into the host erythrocyte and is thought to play a key role in parasite virulence and survival. However, although standard techniques for gene disruption as well as conditional protein knockdown have been previously attempted with the pfpmv gene, complete gene removal or knockdown was not achieved so direct genetic proof that PMV is an essential protein has not yet been established. Here we have used a conditional gene excision approach combining CRISPR-Cas9 gene editing and DiCre-mediated recombination to functionally inactivate the pfpmv gene. The resulting mutant parasites displayed a severe growth defect. Detailed phenotypic analysis showed that development of the mutant parasites was arrested at the ring-to-trophozoite transition in the erythrocytic cycle following gene excision, likely due to a defect in protein export. Our findings are the first to elucidate the effects of PMV gene disruption, showing that it is essential for parasite viability in asexual blood stages. The mutant parasites can now be used as a platform to further dissect the Plasmodium protein export pathway.

molecular biology↗