Schistosoma Mansonii aspartic protease expression and refolding trials

Abstract

Schistosomiasis is a parasitic disease that causes considerable socio-economic losses in affected areas due to loss of productive capacity of affected individuals and high rates of morbidity and mortality. Therapeutic controls for this parasitic disease have shown some drawbacks with resistance emergence to praziquantel, the drug of choice for treatment, being reported in recent years. Thus, new chemotherapeutic targets are been investigated, aiming to develop drugs that are more effective, with lower cost and fewer adverse reactions. Among these new targets, aspartic proteases are among the most promising, since their involvement in other diseases such as Alzheimer's and diabetes have been proven and a chemotherapeutic arsenal for AIDS treatment has been developed based on the structure and function of the HIV1 aspartic protease. The rational design of new drugs requires knowledge of the structures of target proteins. In this study we have examined the activation of pro-enzyme, recombinant forms of two putative cathepsin D-like aspartic proteases from the helminth Schistosoma mansonii (SmCDs). Extensive folding trials were undertaken in attempts to determine the potential for activation of the proteolytic activities of the enzymes. A recombinant protein disulfide isomerase from the same organism was also prepared for use in protease folding trials. Preliminary evidence suggests that an activated form of one of the two proteases (SmCD2) may be obtained by introducing a solubilizing maltose-binding tag on the N-terminal end resulting in soluble expression of the enzyme. This result has raised the prospect of developing an in vitro screening tool to identify potential lead compounds for new drug development. Although PDI samples were purified and active in rearrange disulfide bridges of insulin, no evidence of assistance in refolding SmCDs were observed. Since PDI itself may be considered as a drug target, crystallization attempts in order to obtain its structures were done. Both crystallization of PDI and its influence in SmCDs refolding must be further be investigated in different conditions.