The complementation of yeast with human or Plasmodium falciparum Hsp90 confers differential inhibitor sensitivities

Abstract

Developing novel drugs against the unicellular parasite Plasmodium is complicated by the paucity of simple screening systems. Heat-shock proteins are an essential class of proteins for the parasite's cyclical life style between different cellular milieus and temperatures. The molecular chaperone Hsp90 assists a large variety of proteins, but its supporting functions for many proteins that are important for cancer have made it into a well-studied drug target. With a better understanding of the differences between Hsp90 of the malarial parasite and Hsp90 of its human host, new therapeutic options might become available. We have generated a set of isogenic strains of the budding yeast Saccharomyces cerevisiae where the essential yeast Hsp90 proteins have been replaced with either of the two human cytosolic isoforms Hsp90α or Hsp90β, or with Hsp90 from Plasmodium falciparum (Pf). All strains express large amounts of the Flag-tagged Hsp90 proteins and are viable. Even though the strain with Pf Hsp90 grows more poorly, it provides a tool to reconstitute additional aspects of the parasite Hsp90 complex and its interactions with substrates in yeast as a living test tube. Upon exposure of the set of Hsp90 test strains to the two Hsp90 inhibitors radicicol (Rd) and geldanamycin (GA), we found that the strain with Pf Hsp90 is relatively more sensitive to GA than to Rd compared to the strains with human Hsp90's. This indicates that this set of yeast strains could be used to screen for new Pf Hsp90 inhibitors with a wider therapeutic window

Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction

“…The inhibition of stage differentiation from bradyzoite to tachyzoite stage in T. gondii by GA is also evident [44]. Similarly, the inactivation of HSP90 by GA and RC with different sensitivities in E. tenella , L. donovani , P. falciparum , T. cruzi , T. brucei , and T. evansi has been proven [12,16-19,45]. Likewise, ATP-competitive inhibition of BoHSP90-A and BoHSP90-B may also help in investigating their roles in the invasion of parasite into the host cells and intracellular growth.…”

Differences in Conformational Dynamics between Plasmodium falciparum and Human Hsp90 Orthologues Enable the Structure-Based Discovery of Pathogen-Selective Inhibitors

“…The vectors expressing human Hsp90s have been previously described. 27 The mutation for PfHsp90 K313Q was generated by PCR site-directed mutagenesis with in subclone of PfHsp90 using a pair of complementary primers covering the m utation site: . TACTTCTCTCGTTCCTT; for human Hsp90α, GATCTGGG-GATCCATGCCTGAGGAAACCCAGAC and GGGAATTCTG-CAGCTACTCCACAAAAAGAGT).…”

“…Using this system, we demonstrated that already known Hsp90 inhibitors manifest a species selectivity arguing that it might be possible to identify or to design inhibitors with a more pronounced species selectivity. 27 An unbiased biochemical high-throughput screen of over 4000 compounds led to the identification of three additional molecules, including the natural compound harmine, with selective binding to PfHsp90 over human Hsp90. 28 Exploring Hsp90 from pathogens as a drug target is further encouraged by the finding that inhibiting Hsp90 may additionally suppress resistance to other drugs in both pathogenic fungi 29 and protozoans.…”

Characterization of Plasmodium falciparum co-chaperone p23: its intrinsic chaperone activity and interaction with Hsp90

“…Coupled with the lack of efficacious vaccines, there is a need to search for new anti-malarials and drug targets. Recent studies have recognized the potential of targeting PfHsp90 for malaria therapy as its inhibition effectively arrests the intra-erythrocytic development of P. falciparum [23,41,42]. Nevertheless, there are some considerations on the use of Hsp90-specific inhibitors as anti-malarials.…”

“…In a recent study, PfHsp90 was shown to be capable of supporting the survival of a yeast strain with both Hsp90 genes (Hsc82 and Hsp82) deleted, which is otherwise lethal [42]. This has led to the proposal of exploiting the viable yeast strain complemented with PfHsp90 to screen for inhibitors that are selective against the parasite Hsp90.…”