The heat shock protein 90 of Plasmodium falciparum and antimalarial activity of its inhibitor, geldanamycin
© Kumar et al; licensee BioMed Central Ltd. 2003
Received: 16 July 2003
Accepted: 15 September 2003
Published: 15 September 2003
The naturally occurring benzoquinone ansamycin compound, geldanamycin (GA), is a specific inhibitor of heat shock protein 90 (Hsp90) and is a potential anticancer agent. Since Plasmodium falciparum has been reported to have an Hsp90 ortholog, we tested the possibility that GA might inhibit it and thereby display antiparasitic activity.
We provide direct recombinant DNA evidence for the Hsp90 protein of Plasmodium falciparum, the causative agent of fatal malaria. While the mRNA of Hsp90 was mainly expressed in ring and trophozoite stages, the protein was found in all stages, although schizonts contained relatively lower amounts. In vitro the parasitic Hsp90 exhibited an ATP-binding activity that could be specifically inhibited by GA. Plasmodium growth in human erythrocyte culture was strongly inhibited by GA with an IC50 of 20 nM, compared to the IC50 of 15 nM for chloroquine (CQ) under identical conditions. When used in combination, the two drugs acted synergistically. GA was equally effective against CQ-sensitive and CQ-resistant strains (3D7 and W2, respectively) and on all erythrocytic stages of the parasite.
Together, these results suggest that an active and essential Hsp90 chaperone cycle exists in Plasmodium and that the ansamycin antibiotics will be an important tool to dissect its role in the parasite. Additionally, the favorable pharmacology of GA, reported in human trials, makes it a promising antimalarial drug.
As the causative agent of malaria, Plasmodium sp. claims between one and two million human lives annually worldwide. Plasmodium falciparum is particularly lethal and causes cerebral malaria . A major area in malaria research is, therefore, focused on finding a potent and reliable anti-parasitic drug that would inhibit Plasmodium infection and growth. In nearly all the malaria-endemic populations, Plasmodium has developed resistance against the hallmark drug chloroquine and its derivatives [2–4]. It is thus appreciated that the new generation of drugs should use a rational strategy based on the structure and function of essential parasitic molecules. With this goal we have concentrated on understanding the signaling pathways of P. falciparum with special emphasis on protein phosphorylation. We and others have recently shown that P. falciparum contains a PP5 protein phosphatase containing a tetratricopeptide (TPR) domain [5, 6]. We also showed that PfPP5 interacts with a 90 kDa protein of the parasite that is antigenically similar to mammalian heat shock protein 90 (Hsp90) . Because of the enormous importance of PP5 and Hsp90 in cellular physiology and signaling [7–9], further studies of both Plasmodium proteins were warranted.
Hsp90 is the most abundant chaperone in cells and plays an essential role in the folding, and hence functioning, of a large number of proteins, especially those participating in cell cycle regulation and signal transduction [8, 9]. The list of the "client" proteins of Hsp90 is impressively long, and includes protein kinases such as Raf, Src, Lck, Wee1, MEK, Cdk4, Src, and CK2, and transcription factors such as steroid receptors and p53 [8, 9]. Because of this, Hsp90 has been used as a drug target in basic as well as clinical applications [10–15]. Recent studies have revealed a number of structural and functional aspects of Hsp90 that include the N-terminal ATP-binding domain and a sophisticated ATP-dependent conformational change in the protein [16–19]. At least two natural antibiotics – geldanamycin (GA) and radicicol – have been experimentally demonstrated to compete with ATP for binding to the N-terminal domain [16–20]. GA, in particular, is considered a highly specific inhibitor of Hsp90 and its derivative, 17-(allylamino)-17-Demethoxygeldanamycin (17AAG), is in Phase I trials as an antitumor agent [12–15]. Inhibition of Hsp90 by these antibiotics and others abolish Hsp90-dependent folding of immature client proteins and direct them to ubiquitin-mediated proteolytic degradation [21, 22]. The gene and cDNA sequence of PfHsp90 have been characterized, and the deduced protein sequence revealed its obvious similarity to Hsp90 from other species and its high conservation among P. falciparum isolates [23, 24]. The cDNA sequence was considered to correspond to this protein since a monoclonal antibody that reacted with the 90 kDa antigen was used to screen the cDNA library. Furthermore, the same antibody reacted with a 90 kDa Plasmodium protein that bound to ATP-agarose [23, 25]. Sera of humans, mice, and squirrel monkeys, exposed to Plasmodium, contained abundant amounts of antibody reactive to the 90 kDa protein [25–27], suggesting that it may have a major antigenic role in malaria.
Based on the foregoing, we conjectured that PfHsp90 might play a critical role in parasitic signaling and cell division, and by corollary, GA might inhibit P. falciparum growth. In this communication, we show that this is true and present detailed studies of the effect of GA on P. falciparum replication and morphology. Our evidence suggests that GA inhibits the ATP-binding activity of PfHsp90. This is likely to inhibit the Hsp90 chaperone cycle, thus providing a working hypothesis for the antiparasitic activity of GA.
Materials and Methods
Antibodies, reagents, parasite culture, and drug treatment
Monoclonal mouse antibody against Achlya Hsp90 was purchased from Sigma, and was found to react with PfHsp90 . Antibody to the T7-tag peptide was from Novagen (EMD Biosciences, Inc., Madison, WI, USA). Chloroquine (CQ) was purchased from Sigma, and [8-3H]-hypoxanthine, from Perkin Elmer. Geldanamycin was provided by NCI, and its stock solution and appropriate dilutions were made in DMSO.
P. falciparum (3D7 or W2) was grown on A-positive human erythrocytes at 5% hematocrit in the presence of homologous serum as described earlier . The parasite morphology and stage-specific development were evaluated by microscopic inspection of Giemsa-stained thin smears . To determine parasitemia, about 500 erythrocytes were examined and the number of infected erythrocytes was reported as percentage of the total. Stage-specific development was assessed by examining a minimum of 400 parasitized cells on each smear for differential counting of rings, trophozoites, schizonts, and pyknotic forms whose exact morphology could not be established. The fraction of each group was calculated as a percentage of the total number of parasitized cells.
When needed, the cultures were synchronized to ring stage by D-sorbitol treatment as described earlier [28, 30]. Four hours post-sorbitol treatment was taken as 0 h, and synchronized parasites were collected (or treated with drug) at various times afterwards, as described in the respective Figure legends. The different morphogenetic stages were timed as follows: early trophozoite (20 h); late trophozoite (26 h); early schizont (32 h); late schizont (40 h); ring (48 h). Synchrony persisted well through two cycles; the purity of individual stages was confirmed to be greater than 95% by light microscopic examination of the Geimsa-stained thin smears of the cultures .
Asynchronous or ring-stage synchronized culture at 4 h after sorbitol treatment was seeded in 12-well culture plates. When needed, the cultures were treated with the drugs (or DMSO control for GA and sterile de-ionized water control for CQ) for different time intervals as indicated in the figure legends. The 3H-hypoxanthine incorporation was carried out as described earlier  with minor modifications. All incorporations were measured in triplicate, and the average presented.
Cloning and expression of recombinant PfHsp90 cDNA
Interaction of PfHsp90 with ATP and GA
Characterization of Pf Hsp90 cDNA and stage-specific expression of the protein
To obtain direct evidence for the identity of PfHsp90 and to initiate studies of its biochemistry, we cloned its cDNA and expressed the recombinant protein in bacteria. Results (Fig. 1) show that the T7-tagged recombinant protein reacts with anti-T7 tag as well as human Hsp90 antibodies, thus confirming the cDNA sequence. The observed Mr of the protein (about 92 k) is in agreement with the predicted molecular weight of PfHsp90 (86.2 kDa) plus approximately 2 kDa for the T7-tag. The protein also has a predicted acidic pI of 4.94 http://us.expasy.org/tools/pi_tool.html, which should retard its mobility on SDS-PAGE. The stage-specific expression pattern of the Hsp90 mRNA (Fig. 1B) confirmed previous findings  that the mRNA is abundant in the ring and early trophozoite stages, but extremely low in schizont, indicating potential regulation at the level of transcription or mRNA stability. Our data closely matched the recent results of Bozdech et al  obtained by microarray analysis of the PfHsp90 transcript throughout the intraerythrocytic developmental cycle (Fig. 1C). Measurement of Hsp90 protein by immunoblot analysis (Fig. 1D) revealed comparable amounts in all stages except the late schizont, where only low amounts are present. Comparing the RNA and protein levels we conjecture that Hsp90 is translated mainly in the ring and trophozoite stages; however, it is a relatively stable protein that continues to persist through much of the schizont stage, starting to disappear only in the late schizonts.
Interaction of PfHsp90 with ATP and GA
Crystallographic as well as biochemical studies have documented that Hsp90 possesses an ATP-binding activity, and that GA, by virtue of its structural similarity to ATP competes for binding to the N-terminal ATP-binding pocket [8, 16, 17, 19, 20]. In vitro, purified Hsp90 was shown to bind to immobilized ATP linked to the matrix via the γ-phosphate, and this could be abolished by pre-incubation of the Hsp90 with GA . Binding was also inhibited by free ATP and ADP, but not by GTP. Since the biochemistry of parasitic Hsp90 has not been studied, we carried out preliminary experiments in an attempt to understand GA action. As described under Discussion, p23 is a co-chaperone important to the function of Hsp90, and previous work showed that the formation of the p23-Hsp90 complex requires ATP and Mg+2 [36, 37]. We have, therefore, adopted similar conditions for binding of native parasitic Hsp90 to ATP-Sepharose. As shown in Fig. 2, upon incubation of P. falciparum extracts with ATP-Sepharose, PfHsp90 was indeed detected in the bound material by immunoblot assay. In the control experiment, ovalbumin did not bind to the column , demonstrating specificity of binding (data not shown). The association of PfHsp90 was abolished in the presence of EDTA, confirming a role of divalent cations . ATP and ADP also inhibited binding in a concentration-dependent manner whereas GTP failed to do so.
Antimalarial effect of GA is independent of CQ-resistance
Since CQ-resistance is a major problem in malaria therapy, and multi-drug resistance is a common phenomenon in Plasmodium and other parasites [4, 40], we tested if CQ-resistant parasites were simultaneously GA-resistant. Fig. 3 shows that GA is equally effective against the CQ-sensitive strain 3D7 and the CQ-resistant strain W2.
Geldanamycin and chloroquine are synergistic inhibitors of Plasmodium growth
GA-mediated inhibition is rapid
GA causes death and disintegration of all parasitic stages
Our in vitro results presented here demonstrate that GA can function as an effective antimalarial, at least in erythrocytic culture, and that it is effective against chloroquine-resistant strains as well. The impressive reduction (5–10 fold) of IC50 of both drugs when used together (Fig. 4) suggests that GA can be used in combination with CQ. As mentioned before, GA and its derivatives are already FDA-approved for Phase I clinical trials in cancer patients, particularly those with advanced solid malignancies of the breast [11–15]. When treated with 17-(allylamino)-17-demethoxygeldanamycin (17AAG), derivative of GA, breast cancer cells were arrested in G1, underwent subsequent mammary differentiation, and then apoptosis. Interestingly, despite the spectrum of important proteins that are degraded in response to these drugs, they showed antitumor activity in animals at doses that are not particularly toxic. In human cancer patients, micromolar peak concentrations were achieved without significant toxicity, which suggested a favorable pharmacology [12–14]. Thus, it appears that GA and 17AAG are worth testing in animal and human Plasmodium infections.
It is important to note that pairs of antimalarial drugs interact with various degrees of cooperativity or antagonism which must be experimentally determined. For example, while mefloquine and clotrimazole are synergistic, CQ and clotrimazole are antagonistic . The demonstration that CQ and GA act synergistically (Fig. 4) makes antimalarial therapy with a combination of these two drugs a viable option. It is also gratifying to find that development of CQ-resistance did not simultaneously lead to a resistance to GA (Fig. 3). Together, these results reinforce the facts that the CQ-resistant strains do not cause an efflux of GA and that the target of GA and CQ are indeed different, i.e., while GA most likely inhibits Hsp90, the principle target of CQ is the parasitic digestive vacuole and requires PfCRT, a membrane transporter .
Besides GA, there are a number of other compounds that also inhibit Hsp90 and interfere with its chaperone function. These include members of the ansamycin antibiotic family, namely herbimycin, and macbecin I and II, which are structurally similar to GA and bind to the nucleotide-binding pocket of Hsp90, and coumarin-type antibiotics, exemplified by novobiocin, originally discovered as an inhibitor of bacterial DNA gyrase B [42–44]. Radicicol, a macrocyclic antifungal structurally unrelated to GA, also specifically binds to and inhibits Hsp90. As expected, many of these drugs inhibit the ATPase activity of the Hsp90 complex, and all of them promote proteolytic degradation of Hsp90 client proteins. Based on the high degree of sequence similarity between PfHsp90 and mammalian Hsp90, we predict that all of these compounds may also act as antimalarials.
There is now mounting evidence, albeit indirect, supporting the existence of a highly active protein chaperone system in Plasmodia. First, our results and previous publications have led to the characterization of a Plasmodium ortholog of Hsp90, an ATP-utilizing molecular chaperone conserved across evolution, and sequence analysis suggested that there might be others (Fig. 8). Consistent with its greater need in conditions that lead to protein misfolding, PfHsp90 transcript levels increased three to four fold when erythrocytic parasite culture was shifted from 37°C to 41°C .
Second, as mentioned before, a large variety of important cell cycle proteins, kinases, and transcription factors depend on Hsp90 for proper folding and stability [8, 9]. Consistent with this task, Hsp90 is known to be essential in all eukaryotes. The mechanism of Hsp90 action is complex and many of its aspects are still being elucidated. In its chaperone cycle, Hsp90 forms transient complexes with a number of participating proteins that include Hsp70, Hip (Hsp70 interacting protein), Hop (p60), p23, and immunophilins [8, 9]. The orthologs of all of these proteins also seem to exist in Plasmodium. Multiple Hsp70-like genes and a grp78 (glucose-regulated protein), another stress protein of the same family, have been described in Plasmodium [45, 46]. Like PfHsp90, these proteins are also highly immunogenic, and the transcription of these genes is elevated upon heat shock. A 58 kDa Plasmodium protein (called heat shock-related protein, Hrp) has been identified that has significant similarity to Hip and contains TPR motifs . A homology search of the P. falciparum genome sequence also revealed sequences with significant homology to Hop (chr14_1.gen_156) and p23 (chr14_1.gen_248) [, our unpublished data]. The Plasmodium Hop and Hsp70 proteins also contain TPR motifs, and Hsp90 is known to have a propensity to interact with TPR-domain proteins [8, 9]. We have already shown that PfHsp90 binds to the TPR-phosphatase, PfPP5 . Taken together, these findings not only suggest the existence of an active chaperone pathway in Plasmodium, but also point to the possibility that many members of the pathway are regulated by reversible phosphorylation. Clearly, further characterization of this process will lead to important directions in the regulation of parasitic gene expression.
Folding and stability of proteins are intimately connected: the vast majority of improperly folded proteins are generally ligated to ubiquitin and degraded by the proteasome machinery . Thus, by blocking Hsp90 function, GA promotes the degradation of the proteins that depend on Hsp90 for optimal folding [21, 22, 44]. Recent reports have, in fact, described a proteasome S4 ATPase homolog  and ubiquitin in P. falciparum  and, interestingly, polyubiquitin expression was regulated during parasite development and by heat shock. It is thus tempting to speculate that Plasmodium has an ATP-ubiquitin-proteasome pathway that is functionally similar to higher eukaryotes and mediates non-lysosomal degradation of cytosolic proteins of the parasite. Our preliminary results (not shown) indicated that a specific subset of P. falciparum proteins is indeed degraded in GA-treated parasites, suggesting that this may make additional contributions to the antimalarial mechanism of GA.
After our manuscript was submitted, a recent publication by Banumathy et al  came to our attention that described inhibition of P. falciparum by GA, thus vindicating the basic conclusion of our paper. Unfortunately, however, use of different procedures and lack of appropriate controls made evaluation of some of their results difficult. For example, these authors reported an IC50 (termed LD50) of 200 nM for GA against erythrocytic cultures of P. falciparum 3D7, which is ten-fold higher than our IC50 value of 20 nM. We believe the difference is due to the fact that Banumathy et al exposed the parasite culture to GA for 24 hr only, whereas we exposed for 48 hr. Initially, we used a 24 hr exposure protocol, and also obtained an IC50 value of 200 nM; however, under the same conditions (i.e., 24 hr exposure), CQ exhibited an IC50 of 250 nM, which is more than ten times the accepted value in literature [38, 39]. Thus, we optimized the drug exposure time to 48 hr, which resulted in the IC50 values of 20 nM and 15 nM, respectively for GA and CQ (Fig. 3). Banumathy et al did not use any known antimalarial drug as positive control. These authors also performed density gradient and pull-down experiments to determine the binding partners of PfHsp90; however, lack of quantitation and specific detection of the associated proteins left their identities uncertain. Based solely on GA's ability to inhibit Plasmodium growth, Banumathy et al concluded that Hsp90 is essential in the parasitic life cycle. We believe the conclusion is premature as it is still unknown whether Hsp90 is the exclusive parasitic target of GA. Isolation of spontaneous GA-resistant mutants of P. falciparum and mapping of the mutations to specific PfHsp90 gene(s), currently in progress in our laboratory, should shed light on the relative roles and essentiality of Hsp90 in the parasite.
The heat shock protein 90 (Hsp90) of P. falciparum is highly similar to its orthologs in other species in both sequence and biochemical activities that are relevant to its chaperone function. The inhibitory effect of geldanamycin on the ATP-binding activity of Plasmodium Hsp90 offers a potential biochemical mechanism for the antiparasitic effect of geldanamycin. Further characterization of the parasitic Hsp90 chaperone pathway and its client proteins may provide important targets for novel antiparasitic drugs. The relatively low IC50 (20 nM) of GA against Plasmodium and the substantially higher concentration (in the micromolar range) achieved in human serum without overt toxicity make it a potential candidate for further development as an antimalarial.
S. B. provided overall guidance, performed the ATP-binding experiments, Northern analyses, and sequence alignment, and wrote the manuscript; A. M. cloned and expressed the recombinant PfHsp90; and R.K. did the all the rest. Preliminary results describing the antimalarial activity of geldanamycin were reported in the Molecular Parasitology Meeting XIII, Marine Biological Laboratory, Woods Hole, MA, USA (September 22–26, 2002; Abstract # 280A). S. B. was a recipient of a Burroughs Wellcome New Initiatives in Malaria Research Award in the initial stages of this study. This research was also supported by NIH grant AI45803 from the National Institute of Allergy and Infectious Diseases (to S. B.). Thanks are due to: Dr. Debopam Chakrabarti (University of Central Florida), Dr. Donald J. Krogstad (Tulane University), and the ATCC / MR4 reagent bank for the P. falciparum strains; Drs. Robert J. Schultz and Ven L. Narayanan (National Cancer Institute, NIH) for the kind gift of geldanamycin; Anja Oldenburg for expert technical assistance; Nicolle E. Garmon for help with word processing; Drs. John Foster (Department of Microbiology and Immunology) and Colin Ohrt (Walter Reed Army Institute of Research, MD, USA) for guidance in the isobologram plot; the ExPASy web site http://us.expasy.org/ for free sequence analysis software; PlasmoDB http://plasmodb.org/ for sequence resources; and Dr. Joseph DeRisi and PLoS  for the adoption of the microarray data in Fig. 1C.
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