The parasite faces relentless genotoxic pressure during its life inside human red blood cells. As it digests host hemoglobin, free heme is released and oxidized from the ferrous to the ferric state, generating reactive oxygen species that inflict a range of DNA lesions, including double-strand breaks. Compounding this vulnerability, Plasmodium species apparently lack the classical nonhomologous end joining pathway, leaving homologous recombination as the primary, and likely exclusive, route for repairing broken chromosomes. That singular reliance makes the recombination machinery an attractive target for antimalarial drugs, because the parasite cannot fall back on an alternative repair system.

PfBlm sits at the heart of this machinery. Previous work has shown that the helicase is upregulated in response to DNA damage, interacts directly with key recombination proteins such as PfRad51 and PfMre11, and that overexpression of the wild-type protein enhances parasite survival under genotoxic stress while a helicase-dead mutant fails to rescue repair defects. Parasites lacking Pfblm show telomere lengthening, relaxed subtelomeric chromatin, and upregulation of subtelomeric var genes, pointing to additional roles in telomere maintenance and in suppressing uncontrolled diversification of the virulence genes that help the parasite evade the immune system. Earlier studies also demonstrated that the small-molecule inhibitor ML216, which blocks PfBlm’s helicase activity, sensitizes parasites to DNA-damaging agents and synergizes with frontline antimalarials.

The Hyderabad team, led by Mrinal Kanti Bhattacharyya, asked whether the molecular chaperone PfHsp90 governs PfBlm’s stability and function. Hsp90 is an ATP-dependent chaperone whose client proteins include many stress-response and genome-maintenance factors. In P. falciparum, Hsp90 is essential for parasite growth, and the geldanamycin analog 17-AAG has been validated as a specific inhibitor of PfHsp90 activity in vitro. The group had previously established that PfRad51 and the sirtuin histone deacetylase PfSir2A are bona fide PfHsp90 clients, and that combining a PfHsp90 inhibitor with a PfRad51 inhibitor produces strong synergistic antimalarial effects in both drug-sensitive 3D7 and multidrug-resistant Dd2 parasite strains.

To test whether PfBlm also depends on PfHsp90, the researchers first probed for a physical interaction. A yeast two-hybrid assay, in which PfHsp90 was fused to a GAL4 DNA-binding domain and PfBlm to a GAL4 activation domain, showed reporter gene expression consistent with an interaction, although the lack of growth on more stringent adenine-selection plates indicated the association is relatively weak. Critically, co-immunoprecipitation from P. falciparum 3D7 parasite lysates using an anti-PfHsp90 antibody pulled down PfBlm, while preimmune IgG did not, confirming that the two proteins associate within the parasite itself. Notably, PfBlm was also detected in the unbound supernatant fraction, suggesting that only a portion of the cellular PfBlm pool is engaged with PfHsp90 at any given moment.

The team then examined what happens to PfBlm when chaperone function is lost. They expressed PfBlm in a temperature-sensitive yeast strain in which both endogenous HSP90 genes are deleted and viability is maintained by a temperature-sensitive hsp82 allele. At the permissive temperature of 25 degrees Celsius, PfBlm remained stable, but after four hours at the restrictive temperature of 37 degrees Celsius, PfBlm levels dropped by roughly 75 percent, as quantified from western blots of three independent experiments. No such temperature-dependent decline occurred in a wild-type yeast strain, confirming that the depletion resulted specifically from the loss of Hsp90 chaperone activity rather than heat stress itself.

Parallel experiments in the parasite told a consistent story. Treating synchronized ring-stage cultures with 1.7 micromolar 17-AAG for twelve hours significantly reduced PfBlm abundance at the late trophozoite stage. When the proteasome inhibitor MG132 was added alongside 17-AAG, PfBlm levels were restored, and MG132 alone actually raised steady-state PfBlm, indicating that the helicase normally turns over through the proteasome and that PfHsp90 is required to protect it from proteasomal degradation. Semi-quantitative RT-PCR showed that PfBLM transcript levels were not downregulated by 17-AAG; in fact they increased, implying transcriptional upregulation in response to chaperone inhibition. The protein loss, therefore, is a post-transcriptional effect driven by degradation, not reduced gene expression.

The decisive question was whether this chaperone-client relationship matters for DNA repair. Because PfHsp90 inhibition also depletes PfRad51, blocking the chaperone alone cannot selectively reveal PfBlm-dependent repair defects. The researchers therefore designed a drug-combination experiment using a sensitive PCR-based repair assay. Synchronized parasites were exposed to ultraviolet irradiation at 100 joules per square meter to induce genome-wide double-strand breaks, and repair kinetics were tracked by amplifying a long 7,200-base-pair genomic fragment, normalized against a short 269-base-pair fragment, at time points before damage, immediately after, and at 12 and 24 hours of recovery. The team first identified doses of each drug that were individually harmless to repair: 510 nanomolar 17-AAG left repair fully intact, 250 nanomolar ML216 caused no inhibition, and 500 nanomolar ML216 inhibited repair by only about 16 percent.

The combination, however, was dramatically more damaging than either drug alone. Co-treatment with 510 nanomolar 17-AAG and 500 nanomolar ML216 produced roughly 60 percent inhibition of double-strand break repair, far exceeding the approximately 16 percent expected from a purely additive effect. This statistically significant excess indicates a synergistic interaction, consistent with PfHsp90 activity contributing to PfBlm abundance and function. The growth-inhibition assays reinforced the point: in the presence of the IC50 concentration of 17-AAG, the IC50 of ML216 fell from 1.579 micromolar to 0.041 micromolar, a more than 38-fold potentiation of the helicase inhibitor’s activity. The interpretation is that 17-AAG shrinks the pool of functional PfBlm by destabilizing it, so a much lower dose of ML216 is sufficient to disable the residual active protein, pushing cellular PfBlm activity below the threshold needed for repair.

The findings extend the known clientele of PfHsp90 among recombination factors and suggest a broader regulatory role for the chaperone in parasite genome biology. PfHsp90 stabilizes the pro-recombinogenic PfRad51 while also supporting the anti-recombinogenic PfBlm, which restrains var gene diversification and preserves subtelomeric chromatin, and it additionally modulates var silencing through PfSir2A. This balancing act allows the parasite to maintain core genome integrity while permitting the controlled recombination that underlies antigenic variation, a key virulence strategy. Intriguingly, a similar physical association between human Hsp90 and the Bloom helicase has been reported in immortalized human cells, where the chaperone modulates Blm’s helicase activity on telomeric DNA substrates in a substrate-dependent manner, hinting at an evolutionarily conserved chaperone-helicase partnership. Whether such context-dependent regulation operates in the malaria parasite remains to be determined. The authors propose that the PfHsp90-PfBlm axis could be exploited to develop new chemotherapeutic strategies against malaria, with simultaneous targeting of chaperone and helicase offering a way to block DNA repair and limit parasite survival, particularly valuable at a time when resistance to existing antimalarials continues to spread.

Subject of Research: Chaperone-dependent stabilization of the PfBlm DNA repair helicase and dual-inhibitor antimalarial strategy in Plasmodium falciparum

Article Title: Inhibition of Plasmodium DNA double strand repair by the combined action of 17-AAG and ML216

Article References: Inhibition of Plasmodium DNA double strand repair by the combined action of 17-AAG and ML216. (n.d.). https://doi.org/10.3389/fpara.2026.1896831

Image Credits: AI Generated

DOI: 10.3389/fpara.2026.1896831

Keywords: Plasmodium falciparum, malaria, PfBlm helicase, PfHsp90, DNA double-strand break repair, homologous recombination, 17-AAG, ML216, molecular chaperone, RecQ helicase, antimalarial drug targets, proteasomal degradation