Screening Inhibitors Of Babesia Microti Pyruvate Kinase

Babesia microti is an intraerythrocytic apicomplexan protozoa,which infects human or domestic animals mainly through ixodes,blood transfusion or blood products,and in rare cases,it is transmitted through placenta from mother to child.Healthy Human infected usually have no obvious clinical symptoms,but immunodeficiency population caused by B-cell lymphoma,organ transplantation,HIV/AIDS or the use of immunosuppressive drugs will increase the risk of babesiosis,and even lead to multiple organ failure or even death in serious cases.At present,with the rapid development of anti-babesial drugs,supportive measures are taken to use atovaquone,particularly in combination with azithromycin,but due to the high resistance of B.microti,it is easy to lead to treatment failure and high recurrence rate.Glycolysis is the main way of energy metabolism of dipteroides.In the life cycle of red blood cells,B.microti consumes glucose to obtain energy and mainly relies on anaerobic glucose metabolism to produce ATP and lactate,so the acquisition of pyruvate is very important,because pyruvate kinase?Py K?is the critical glycolytic enzyme for energy acquisition of B.microti,which is expected to be a drug target,it can block the production of pyruvate to limit the energy supply of B.microti and to inhibit the growth of B.microti.Based on Py K of B.microti,this study explored the catalytic activity of Py K and screened its specific inhibitors.Details as follows:Using the c DNA and g DNA of B.microti as templates,the fragments of Py K?and Py K?were amplified,1470 bp and 1977 bp,respectively.The prokaryotic expression vectors of p GEX-6p-1-Py K?and p GEX-6p-1-Py K?containing GST tags were constructed and transformed into E.coli BL21 for recombinant expression.The purified proteins of Py K?and Py K?were obtained from the supernatant.Western blot analysis showed that Py K?and Py K?had good immunogenicity,and the natural protein size of the parasites was about 80 k Da and 100 k Da,respectively.Due to difficulty to purify the recombinant protein of Py K?,only the GST tag of the recombinant protein Py K?was removed to explore its catalytic activity.By changing p H and the concentrations of Mg²⁺,K⁺and substrate,the enzyme kinetic parameters of Py K?were obtained,such as Michaelis constant K_m,K_cat and V_max,and the optimum conditions of enzyme catalytic reaction were determined,3 m M PEP,1 m M ADP,p H=7.0.In inhibitor screening tests,13 compounds were screened by LDH enzyme coupling method,the data displayed that 6 compounds inhibited the activity of recombinant protein Py K?,including Tannic acid,Shikonin,Apigenin,PKM2inhibitor,Rosiglitazone and Pioglitazone.Then we repeated the effects of 13 drugs on the activity of Py K?with pyruvate kinase activity test kit,and the results were basically the same as before.With the screening of Tannic acid,Shikonin,Apigenin and PKM2 inhibitor in vitro,the four compounds had obvious inhibitory effect on the growth of B.microti at the micromole level,and the cytotoxic test demonstrated Tannic acid had low poisonous.In addition,in the inhibition test of Tannic acid in vitro,the supplement of pyruvate products can partially recover the parasite infection rate of the Tannic acid group.In conclusion,in this study we amplified the genes of Py K?and Py K?and explored the catalytic activity of Py K?.In addition,we successfully screened out four compounds,Tannic acid,Shikonin,Apigenin and PKM2 inhibitor,through enzyme activity mechanics test and culture test in vitro to inhibit the catalytic activity of Py K?and the growth of B.microti in vitro.The results will provide a theoretical basis for the design and development of new anti-babesial drugs and provide a reference for the study of other pathway of glycometabolism.