A review of recent patents on the protozoan parasite HSP90 as a drug target.

Diseases caused by protozoan parasites are still an important health problem. These parasites can cause a wide spectrum of diseases, some of which are severe and have high morbidity or mortality if untreated. Since they are still uncontrolled, it is important to find novel drug targets and develop new therapies to decrease their remarkable social and economic impact on human societies. In the past years, human HSP90 has become an interesting drug target that has led to a large number of investigations both at state organizations and pharmaceutical companies, followed by clinical trials. The finding that HSP90 has important biological roles in some protozoan parasites like Plasmodium spp, Toxoplasma gondii and trypanosomatids has allowed the expansion of the results obtained in human cancer to these infections. This review summarizes the latest important findings showing protozoan HSP90 as a drug target and presents three patents targeting T. gondii, P. falciparum and trypanosomatids HSP90.

Disruption of the expression of a non-coding RNA significantly impairs cellular differentiation in Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. Asexual replication of T. gondii in humans and intermediate hosts is characterized by two forms: rapidly growing "tachyzoites" and latent "bradyzoite" tissue cysts. Tachyzoites are responsible for acute illness and congenital neurological birth defects, while the more slowly dividing bradyzoite form can remain latent within the tissues for many years, representing a threat to immunocompromised patients. We have developed a genetic screen to identify regulatory genes that control parasite differentiation and have isolated mutants that fail to convert to bradyzoites. One of these mutants has an insertion disrupting a locus that encodes a developmentally regulated non-coding RNA transcript, named Tg-ncRNA-1. Microarray hybridizations suggest that Tg-ncRNA-1 is involved in the early steps of bradyzoite differentiation. Since Tg-ncRNA-1 does not contain an open reading frame, we used the algorithm Coding Potential Calculator (CPC) that evaluates the protein-coding potential of a transcript, to classify Tg-ncRNA-1. The CPC results strongly indicate that Tg-ncRNA-1 is a non-coding RNA (ncRNA). Interestingly, a previously generated mutant also contains an insertion in Tg-ncRNA-1. We show that both mutants have a decreased ability to form bradyzoites, and complementation of both mutants with wild-type Tg-ncRNA-1 restores the ability of the parasites to differentiate. It has been shown that an important part of bradyzoite differentiation is transcriptionally controlled, but this is the first time that a non-coding RNA is implicated in this process.

Initiation and termination of NF-kappaB signaling by the intracellular protozoan parasite Toxoplasma gondii.

Signaling via the NF-kappaB cascade is critical for innate recognition of microbial products and immunity to infection. As a consequence, this pathway represents a strong selective pressure on infectious agents and many parasitic, bacterial and viral pathogens have evolved ways to subvert NF-kappaB signaling to promote their survival. Although the mechanisms utilized by microorganisms to modulate NF-kappaB signaling are diverse, a common theme is targeting of the steps that lead to IkappaB degradation, a major regulatory checkpoint of this pathway. The data presented here demonstrate that infection of mammalian cells with Toxoplasma gondii results in the activation of IKK and degradation of IkappaB. However, despite initiation of these hallmarks of NF-kappaB signaling, neither nuclear accumulation of NF-kappaB nor NF-kappaB-driven gene expression is observed in infected cells. However, this defect was not due to a parasite-mediated block in nuclear import, as general nuclear import and constitutive nuclear-cytoplasmic shuttling of NF-kappaB remain intact in infected cells. Rather, in T. gondii-infected cells, the termination of NF-kappaB signaling is associated with reduced phosphorylation of p65/RelA, an event involved in the ability of NF-kappaB to translocate to the nucleus and bind DNA. Thus, these studies demonstrate for the first time that the phosphorylation of p65/RelA represents an event downstream of IkappaB degradation that may be targeted by pathogens to subvert NF-kappaB signaling.

Toxoplasma gondii Hsp90 is a potential drug target whose expression and subcellular localization are developmentally regulated.

Two replicative forms characterize the asexual cycle of the protozoan parasite Toxoplasma gondii: rapidly growing tachyzoites and slowly dividing encysted bradyzoites. The mechanisms that regulate the transition between these two stages are not clearly understood. However, stress inducers that also activate heat shock protein expression can trigger formation of bradyzoites in vitro. Here, we studied the association of the T.gondii Hsp90 with modulation of parasite differentiation and response to stress stimuli using RH DeltaUPRT parasites and the cystogenic strain ME49 and a clone derivative of that strain, PK. Our results show that Hsp90 transcript and protein levels increase under stress or bradyzoite differentiation conditions. Moreover, fluorescence microscopy studies revealed that Hsp90 is present in the cytosol of tachyzoites and both in the nucleus and cytosol of mature bradyzoites, suggesting a correlation between its subcellular organization and these two developmental stages. To further characterize the role for Hsp90 in bradyzoite differentiation, T.gondii tachyzoite mutants that are defective in differentiation showed the same staining pattern as tachyzoites under differentiation conditions. In addition, geldanamycin, a benzoquinone ansamycin antibiotic capable of binding and disrupting the function of Hsp90, blocked conversion both from the tachyzoite to bradyzoite and the bradyzoite to tachyzoite stage, suggesting an essential role for this protein in the regulation of stage interconversion. These results thus suggest Hsp90 may play a role in stage switch.

Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii.

Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6-thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.