Cloning and Expression of Methione Aminopeptadse-1 from Trypanosoma Cruzi

Trypanosoma cruzi is the causative agent of Chagas disease and affects millions of people. The high toxicity of existing treatments and the lack of vaccines clearly demonstrate the demand for new drugs to treat this parasitosis. In this study we intend to validate Methionine Aminopeptidase-1 (TcMetAP1) as chemotherapeutic target. This metalloprotease removes the N-terminal methionine during protein synthesis. We hypothesize that specific inhibitors of the TcMetAP1 will clear the infection produced by this parasite. A proliferation assay was performed using Mycobacterium tuberculosis MetAP1 inhibitors. In a 96 well plate, infected U2OS cells (human osteocytes) with T. cruzi Y strain trypomastigotes were incubated for 48 hr. After they were fixed and stained with Draq5. The percentage of infected cells was determined using high content imaging the proliferation of amastigotes was inhibited 85 – 68 % at 780 nM in compounds OT004, OT005, OT006, OT007 and OT008. The inhibitor OT004 showed to be the most effective. The TcMetAP1 gene was cloned into the yeast expression vector PichiaPink (pPink). The expression of the protein was performed using four different yeast strains ade2, ade2 pep4, ade2,prb1 and ade2 prb1 pep. At this point we successfully expressed the recombinant TcMetAP. To determine the induction profile over time, samples of the culture were taken at specific time points over a four day period. Once the recombinant TcMetAP1 is purified we will develop the enzymatic assay, determine its kinetics characteristics and assay for specificity of our lead inhibitor as well as to be used for HST.