A Cysteine Protease is a Target for the Enzyme Structure-Based Design of Antiparasitic Drugs

Abstract

Proteases have been shown to be factors in the pathogenicity of many parasitic diseases, either by inducing tissue damage and facilitating invasion or by enabling the parasites to salvage metabolites from host proteins. To study genes encoding cysteine proteases of parasites, a general method for cloning fragments of thiol protease genes was developed using the polymerase chain reaction (PCR) with degenerate oligonucleotide primers. Subsequently, a PCR-amplified gene fragment of the protozoan parasite, Trypanosoma cruzi, was used to isolate a full-length gene encoding a cysteine protease. At least six copies of the gene are organized in the genome as a tandem array. The high degree of sequence identity with the papain family of enzymes suggested the name “cruzain.” A copy of the gene was expressed in bacteria as an inactive, insoluble fusion polypeptide. Subsequently, the fusion protein was solubilized in urea and refolded to produce a polyprotein which processed autocatalytically to yield active, recombinant enzyme. This expression method generated recombinant protease of sufficient quality and quantity for crystallization. Diffraction quality crystals of recombinant cruzain, inactivated with a peptide inhibitor shown to block growth of the parasites in infected human cells, have been produced and characterized. The studies presented herein will provide insight into the mechanism of action and structure of cruzain and may enable the development of specific inhibitors for antiparasitic chemotherapy in the treatment of Chagas' disease.