Biology subjects

Okombo, J.

Publications and source records attributed to Okombo, J..

3 recordsLinked to original sources

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↗

bioRxiv2024

Identification of the drug/metabolite transporter 1 as a marker of quinine resistance in a NF54xCam3.II P. falciparum genetic cross.

The genetic basis of Plasmodium falciparum resistance to quinine (QN), a drug used to treat severe malaria, has long been enigmatic. To gain further insight, we used FRG-NOD human liver-chimeric mice to conduct a P. falciparum genetic cross between QN-resistant (Cam3.II) and QN-sensitive (NF54) parasites, which also differ in their susceptibility to chloroquine (CQ). By applying different selective conditions to progeny pools prior to cloning, we recovered 120 unique recombinant progeny. Drug profiling and quantitative trait loci analyses of the progeny revealed predominant peaks on chromosomes 7 and 12 associated with CQ and QN resistance, that is consistent with a multifactorial mechanism of resistance for these compounds. CQ and monodesethyl-CQ (md-CQ) resistance mapped to a chromosome 7 region harboring pfcrt as expected. However, for QN, resistance mapped to a dominant chromosome 7 peak centered 295 kb downstream of pfcrt, with pfcrt showing a smaller peak. We identified the drug/metabolite transporter 1 (DMT1) as the top chromosome 7 candidate due to its structural similarity to PfCRT and proximity to the peak. Deleting DMT1 in QN-resistant Cam3.II parasites significantly sensitized the parasite to QN but not to the other drugs tested, suggesting that DMT1 mediates QN response specifically. We localized DMT1 to structures associated with vesicular trafficking, as well as the parasitophorous vacuolar membrane, lipid bodies, and the digestive vacuole. We also observed that mutant DMT1 transports more QN than the wild-type isoform in vitro. Gene editing confirmed an additional role for mutant PfCRT in mediating QN resistance. In addition, we identified an ATP-dependent zinc metalloprotease (FtsH1) as one of the top candidates in the chromosome 12 locus and confirmed its role as a potential mediator of QN resistance and a modulator of md-CQ resistance using CRISPR/Cas9 SNP-edited lines. Interestingly, this chromosome 12 region mapped to resistance to both CQ and QN and was preferentially co-inherited with pfcrt. Our study demonstrates that DMT1 is a novel marker of QN resistance and that a new chromosome 12 locus associates with CQ and QN response, with ftsh1 as a potential candidate, suggesting these genes in addition to pfcrt should be genotyped in surveillance and clinical settings.

genetics↗

bioRxiv2019

Genetic screens reveal a central role for heme biosynthesis in artemisinin susceptibility

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria, however, resistance threatens to undermine global control efforts. To explore artemisinin resistance in apicomplexan parasites broadly, we used genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncovered the putative porphyrin transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provided evidence that mitochondrial metabolism can modulate resistance. We show that disruption of a top candidate from the screens, the mitochondrial protease DegP2, lowered levels of free heme and decreased DHA susceptibility, without significantly altering fitness in culture. Deletion of the homologous gene in P. falciparum, PfDegP, similarly lowered heme levels and DHA susceptibility. These results expose the vulnerability of the heme biosynthetic pathway for genetic perturbations that can lead to survival in the presence of DHA. We go on to show that chemically reducing heme biosynthesis can decrease the sensitivity of both T. gondii and P. falciparum to DHA, suggesting guidelines for developing combination therapies.

microbiology↗