Heat shock protein 90 as a drug target against protozoan infections: biochemical characterization of HSP90 from Plasmodium falciparum and Trypanosoma evansi and evaluation of its inhibitor as a candidate drug.

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

Systems analysis of chaperone networks in the malarial parasite Plasmodium falciparum.

Molecular chaperones participate in the maintenance of cellular protein homeostasis, cell growth and differentiation, signal transduction, and development. Although a vast body of information is available regarding individual chaperones, few studies have attempted a systems level analysis of chaperone function. In this paper, we have constructed a chaperone interaction network for the malarial parasite, Plasmodium falciparum. P. falciparum is responsible for several million deaths every year, and understanding the biology of the parasite is a top priority. The parasite regularly experiences heat shock as part of its life cycle, and chaperones have often been implicated in parasite survival and growth. To better understand the participation of chaperones in cellular processes, we created a parasite chaperone network by combining experimental interactome data with in silico analysis. We used interolog mapping to predict protein-protein interactions for parasite chaperones based on the interactions of corresponding human chaperones. This data was then combined with information derived from existing high-throughput yeast two-hybrid assays. Analysis of the network reveals the broad range of functions regulated by chaperones. The network predicts involvement of chaperones in chromatin remodeling, protein trafficking, and cytoadherence. Importantly, it allows us to make predictions regarding the functions of hypothetical proteins based on their interactions. It allows us to make specific predictions about Hsp70-Hsp40 interactions in the parasite and assign functions to members of the Hsp90 and Hsp100 families. Analysis of the network provides a rational basis for the anti-malarial activity of geldanamycin, a well-known Hsp90 inhibitor. Finally, analysis of the network provides a theoretical basis for further experiments designed toward understanding the involvement of this important class of molecules in parasite biology.

Three-dimensional structure of heat shock protein 90 from Plasmodium falciparum: molecular modelling approach to rational drug design against malaria.

We have recently implicated heat shock protein 90 from Plasmodium falciparum (PfHsp90) as a potential drug target against malaria. Using inhibitors specific to the nucleotide binding domain of Hsp90, we have shown potent growth inhibitory effects on development of malarial parasite in human erythrocytes. To gain better understanding of the vital role played by PfHsp90 in parasite growth,we have modeled its three dimensional structure using recently described full length structure of yeast Hsp90.S equence similarity found between PfHsp90 and yeast Hsp90 allowed us to model the core structure with high confidence. The superimposition of the predicted structure with that of the template yeast Hsp90 structure reveals an RMSD of 3.31 Angstrom. The N-terminal and middle domains showed the least RMSD (1.76 Angstrom) while the more divergent C-terminus showed a greater RMSD (2.84 Angstrom) with respect to the template. The structure shows overall conservation of domains involved in nucleotide binding, ATPase activity, co-chaperone binding as well as inter-subunit interactions. Important co-chaperones known to modulate Hsp90 function in other eukaryotes are conserved in malarial parasite as well. An acidic stretch of amino acids found in the linker region, which is uniquely extended in PfHsp90 could not be modeled in this structure suggesting a flexible conformation. Our results provide a basis to compare the overall structure and functional pathways dependent on PfHsp90 in malarial parasite. Further analysis of differences found between human and parasite Hsp90 may make it possible to design inhibitors targeted specifically against malaria.

Recurrent fever promotes Plasmodium falciparum development in human erythrocytes.

The human malarial parasite Plasmodium falciparum (Pf) is exposed to wide temperature fluctuations during its life cycle, ranging from 25 degrees C in the mosquito vector and 37 degrees C in humans to 41 degrees C during febrile episodes in the patient. The repeated occurrence of fever at regular intervals is a characteristic of human malaria. We have examined the influence of repeated exposure to elevated temperatures encountered during fever on the intraerythrocytic development of the parasite. Using flow cytometry, we show that repeated exposure to temperatures mimicking febrile episodes promotes parasite development in human erythrocytes. Heat shock-mediated cytoprotection and growth promotion is dependent on the heat shock protein 90 (PfHsp90) multi-chaperone complex. Inhibition of PfHsp90 function using geldanamycin attenuates temperature-dependent progression from the ring to the trophozoite stage. Geldanamycin inhibits parasite development by disrupting the PfHsp90 complex consisting of PfHsp70, PfPP5, and tubulin, among other proteins. While explaining the contribution of febrile episodes to the pathogenesis of malaria, our results implicate temperature as an important environmental cue used by the parasite to coordinate its development in humans.

Heat shock protein 90 function is essential for Plasmodium falciparum growth in human erythrocytes.

Hsp90 is important for normal growth and development in eukaryotes. Together with Hsp70 and other accessory proteins, Hsp90 not only helps newly synthesized proteins to fold but also regulates activities of transcription factors and protein kinases. Although the gene coding for heat shock protein 90 from Plasmodium falciparum (PfHsp90) has been characterized previously, there is very little known regarding its function in the parasite. We have analyzed PfHsp90 complexes and addressed its role in parasite life cycle using Geldanamycin (GA), a drug known to interfere with Hsp90 function. Sedimentation analysis and size exclusion chromatography showed PfHsp90 to be in 11 s(20,(w)) complexes of approximately 300 kDa in size. Similar to the hetero-oligomeric complexes of Hsp90 in mammals, PfHsp70 was found to be present in PfHsp90 complexes. Homology modeling revealed a putative GA-binding pocket at the amino terminus of PfHsp90. The addition of GA inhibited parasite growth with LD(50) of 0.2 microm. GA inhibited parasite growth by arresting transition from Ring to trophozoite. Transition from trophozoite to schizonts and reinvasion of new erythrocytes were less significantly affected. While inducing the synthesis of PfHsp70 and PfHsp90, GA did not significantly alter the pattern of newly synthesized proteins. Pre-exposure to heat shock attenuated GA-mediated growth inhibition, suggesting the involvement of heat shock proteins. Specificity of GA action on PfHsp90 was evident from selective inhibition of PfHsp90 phosphorylation in GA-treated cultures. In addition to suggesting an essential role for PfHsp90 during parasite growth, our results highlight PfHsp90 as a potential drug target to control malaria.