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

Abstract

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.

FIGURE 1.

PfHsp90 is a hyperactive ATPase. A, determination of binding affinity of ATP to PfHsp90 using tryptophan fluorescence. The change in fluorescence (ΔF) was plotted against ATP concentrations. B, rate of ATP hydrolysis was measured by the direct conversion of radiolabeled ATP to ADP. A Michaelis-Menten plot shows the fractional cleavage of γ-³²P-labeled ATP plotted against ATP concentration. C, a comparison of ATP hydrolysis of Hsp90 from chicken (Gallus gallus), human (Homo sapiens), yeast, and Plasmodium. Plasmodium Hsp90 has higher ATPase activity as compared with human and chicken and is similar to yeast Hsp90. A.U., arbitrary units.

FIGURE 2.

GA potently inhibits PfHsp90 ATPase activity. A and B, determination of binding affinity of GA toward PfHsp90 and hHsp90 using tryptophan fluorescence. The change in fluorescence (ΔF) was plotted against GA concentrations for PfHsp90 (A) and hHsp90 (B). C and D, the IC_50(ATPase) of PfHsp90 (F) and hHsp90 (G) ATPase activity was determined by plotting percent activity remaining versus GA concentration in logarithmic scale. A.U., arbitrary units.

FIGURE 3.

GA specifically binds to PfHsp90. Parasite lysate was incubated with GA-coupled beads or control beads. Bound (lanes 2 and 6) and unbound proteins (lanes 3 and 7) were analyzed by SDS-PAGE followed by Coomassie staining (left side) or immunoblotting using anti-PfHsp90 (right side). WB, Western blot.

FIGURE 4.

Acetylation disrupts PfHsp90 complex. PfHsp90 acetylation status was determined by treating parasite-infected erythrocytes using histone deacetylase inhibitor TSA. A, domain distribution of acetylation sites in PfHsp90. CR, charged linker region; TPR, tetratricopeptide repeat. B, P. falciparum-infected erythrocytes were treated/untreated with histone deacetylase inhibitors. Parasite lysate was prepared as described under “Experimental Procedures” and analyzed using a Superdex 200 gel filtration column. 500-μl fractions were collected and immunoblotted for PfHsp90 (top panel). The bottom panel shows quantitation of the profile. Error bars have been represented to indicate standard error from three independent experiments.

FIGURE 5.

Hsp90 inhibitors potently inhibit parasite growth. A and B, IC₅₀ values for GA and 17AAG in the P. falciparum 3D7 strain were determined using a [³H]hypoxanthine incorporation assay. IC₅₀ values were determined by plotting the percentage of incorporation of [³H]hypoxanthine against different concentrations of drug in logarithmic scale. A, percentage of [³H]hypoxanthine incorporation upon GA treatment for 48 h. B, percentage of [³H]hypoxanthine incorporation upon 17AAG treatment for 48 h. C, comparison of the IC₅₀ values among different organisms. Error bars have been represented to indicate standard error from three independent experiments.

FIGURE 6.

Efficacy of 17AAG, a GA derivative, in rodent model of malaria. A, representative Giemsa-stained tail smears of 17AAG-untreated (left) and -treated (right) P. berghei-infected mice after 6 days postinfection. B, plot of percentage of parasitemia against the number of days postinfection. (C) Percentage viability of 17AAG treated/untreated mice plotted against time. Error bars have been represented to indicate standard error from three independent experiments.

FIGURE 7.

Multiple sequence alignment of Hsp90s from various protozoan parasites. Hsp90 sequences for P. falciparum (PfHsp90; GenBank^TM accession number CAA82765.1), Plasmodium vivax (PvHsp90; GenBank accession number EDL43724.1), Eimeria tenella (EtHsp90; GenBank accession number AAB97088.1), Toxoplasma gondii (TgHsp90; GenBank accession number AAP44977.1), Cryptosporidium parvum Iowa II (CpHsp90; GenBank accession number EAK89246.1), Babesia bovis (BbHsp90; RefSeq accession number XP_001611554.1), L. donovani infantum (LdHsp90; Protein Information Resource accession number S57415), Leishmania major (LmHsp90; GenBank accession number CAJ05959.1), T. brucei (TbHsp90; Protein Information Resource accession number A44983), T. cruzi strain CL Brener (TcHsp90; RefSeq accession number XP_814892.1), Entamoeba histolytica (EhHsp90; RefSeq accession number XP_653132.1), and Trichomonas vaginalis (Tvhsp90; RefSeq accession number XP_001583398.1) were obtained from NCBI. The Tehsp90 sequence was obtained from the sequence of the cloned gene obtained in this study. All the residues required for GA binding are conserved across protozoan parasites. The residues that interact with GA are indicated (●) and marked (♦, hydrogen bond; ■, van der Waals; ♢, with H₂O).

FIGURE 8.

Hsp90 inhibitor binds to TeHsp90 and cures infection in mice. A, determination of binding affinity of GA toward TeHsp90 by tryptophan fluorescence. The change in fluorescence (ΔF) was plotted against GA concentration for TeHsp90. B, GA-immobilized beads specifically pull down TeHsp90 from T. evansi lysate. The GA pulldown fraction was analyzed by SDS-PAGE (upper panel) and two-dimensional electrophoresis (lower panel). C and D, in vivo efficacy of Hsp90 inhibitors on the survivability of T. evansi-infected mice. C, plot of number of parasites against the number of days postinfection. D, percentage of viability of 17AAG-treated/untreated mice plotted against time. A.U., arbitrary units. Error bars have been represented to indicate standard error from three independent experiments.