The stakes are considerable. According to the World Health Organisation, malaria caused an estimated 282 million cases and roughly 610,000 deaths in 2024, an increase of nine million cases over the previous year, driven in part by the spread of artemisinin-resistant parasites. Resistance to chloroquine and sulphadoxine is also well documented. As conventional targets falter, researchers are turning to proteins the parasite simply cannot live without, and molecular chaperones have emerged as prime candidates.

Hsp90 is the master regulator of proteostasis in eukaryotic cells, shepherding client proteins into their functional shapes and, in Plasmodium, even chaperoning the 26S proteasome. The parasite’s main cytosolic isoform, PfHsp90α, is a validated antimalarial target: the ansamycin antibiotic geldanamycin blocks its ATPase activity, and derivatives such as 17-AAG prolong survival of infected mice. But there is a catch. PfHsp90α and its human orthologue are so similar in sequence that selective inhibition has proved elusive, and 17-AAG lacked the selectivity needed for malaria therapy.

That is where the parasite’s second cytosolic Hsp90 comes in. The gene, PF3D7_1443900, encodes a protein that shares only 27.6 percent sequence identity with PfHsp90α and roughly 28 percent with human Hsp90α and β. A piggyBac transposon mutagenesis screen had already flagged the gene as essential for parasite survival, yet its divergence raised doubts about whether it was a true, functional Hsp90 at all. The new study answers that question with a combination of evolutionary, structural and genetic evidence.

Phylogenetic analysis of 69 Hsp90 sequences, aligned with MAFFT and trimmed to 584 informative columns, placed the Plasmodium putative isoforms in their own well-separated clade, distinct from canonical cytosolic Hsp90s, the mitochondrial paralogue TRAP1 and the endoplasmic reticulum-resident GRP94. The team therefore renamed the protein PfHsp90β. Structural modelling with AlphaFold confirmed the canonical three-domain architecture, with an N-terminal Bergerat-fold ATP-binding pocket flanked by the conserved B1, B2 and B3 motifs, and a middle domain retaining the asparagine, arginine and glutamine residues that position ATP’s γ-phosphate during hydrolysis.

The deviations, however, are what make PfHsp90β interesting. It lacks the charged linker that modulates N-domain conformation in canonical Hsp90s, and it is missing the C-terminal MEEVD motif through which TPR-domain cochaperones such as Hop/Sti1 dock. It also carries an extended 91-amino-acid disordered region at its C-terminus. Together these features imply a chaperone with a different conformational landscape and, potentially, a different set of cochaperone dependencies, perhaps more like the bacterial HtpG, which associates directly with Hsp70 without a MEEVD tether.

Functionally, the evidence is compelling but nuanced. Semiquantitative RT-PCR showed PfHsp90β expression peaking at the metabolically active trophozoite stage, ninefold higher than at the ring stage. In yeast, the protein could not fully replace Hsp82 in a plasmid shuffling assay, presumably because it cannot engage yeast cochaperones. Yet when expressed in a temperature-sensitive iGD170hsp82 strain, it rescued growth at the restrictive temperature: after four hours at 37 degrees Celsius, the PfHsp90β-expressing strain survived at 37 percent, far above the 11 percent of the empty-vector control, though below the 69 percent supported by yeast Hsp82 itself. Partial complementation, but genuine chaperone activity nonetheless.

The team then turned the yeast system into a drug screen. Using isogenic Δpdr5 strains, in which the major drug export pump is deleted to boost inhibitor uptake, each carrying either yeast Hsp82, PfHsp90α or PfHsp90β, they tested six bona fide Hsp90 inhibitors. Molecular docking with AutoDock Vina and Smina, validated by redocking crystallised ligands to within two angstroms, ranked NVP-AUY922, CCT018159, ganetespib and MPC-3100 as the strongest binders to PfHsp90β. In the survival assays, only CCT018159, a resorcinol-class diaryl pyrazole, selectively impaired the PfHsp90β strain, cutting survival by about 40 percent, while leaving strains carrying PfHsp90α or yeast Hsp82 largely unharmed. Docking suggested why: the compound’s resorcinol ring hydrogen-bonds with Gly254 and Thr233, its benzodioxane oxygen contacts Asn224, and Lys140 stacks over an aromatic ring in a π-cation interaction.

Crucially, CCT018159 also kills the parasite itself. In cultured P. falciparum, the compound showed half-maximal inhibitory concentrations of 1.6 micromolar against the drug-sensitive 3D7 strain and 2.8 micromolar against the multidrug-resistant Dd2 strain at the ring stage, with similar values at the trophozoite stage. Activity against Dd2 matters, because resistance mechanisms often undermine otherwise promising compounds. The authors caution that selectivity rests so far on docking and yeast surrogate data; direct biochemical confirmation against purified PfHsp90β remains to be done, and full-length protein expression has proved difficult, possibly because a long disordered N-terminal region is toxic to bacterial hosts.

Even with those caveats, the study delivers a rare commodity in antimalarial research: a parasite protein that is essential, structurally divergent from its human counterpart, and demonstrably druggable with an existing small molecule. With a negative mutant fitness score of minus 3.033 and an ATP pocket whose lining differs substantially from both PfHsp90α and human Hsp90, PfHsp90β now stands as a legitimate target for structure-guided drug development. As artemisinin resistance spreads, the parasite’s most idiosyncratic chaperone may prove to be one of its most exploitable vulnerabilities.

Subject of Research: Functional characterisation and selective inhibition of the putative Hsp90 paralogue PfHsp90β in Plasmodium falciparum

Article Title: Plasmodium falciparum putative Hsp90 functionally complements yeast Hsp90 and is inhibited in an isoform-specific manner

Article References: Khamitkar, S. R., Bag, M., Kini, V. R., Singh, M., Sethi, B. K., & Bhattacharyya, S. (2026). Plasmodium falciparum putative Hsp90 functionally complements yeast Hsp90 and is inhibited in an isoform-specific manner. Frontiers in Parasitology, 5, Article 1944050. https://doi.org/10.3389/fpara.2026.1944050

Image Credits: AI Generated

DOI: 10.3389/fpara.2026.1944050

Keywords: Plasmodium falciparum, Hsp90, PfHsp90β, molecular chaperones, antimalarial drug discovery, CCT018159, yeast complementation, molecular docking, drug resistance, malaria, protein homeostasis, Frontiers in Parasitology