以结核分枝杆菌H_(37)Rv莽草酸脱氢酶为靶点的高通量筛选模型的建立及应用

目的建立以结核分枝杆菌莽草酸脱氢酶为靶点的新型抗结核药物高通量筛选模型;用此模型筛选莽草酸脱氢酶抑制剂;进一步评价化合物对莽草酸脱氢酶活性的影响。方法表达并纯化结核分枝杆菌H37Rv莽草酸脱氢酶;利用还原型辅酶II(NADPH)在溶液中的光吸收,测定酶的活性,构建了该酶抑制剂的高通量筛选模型;用Z′因子法评价该模型的可靠性,并对5万余个化合物进行筛选;测定了各抑制剂的IC50并对抑制剂6186050的酶抑制动力学进行了研究;用菌液稀释法评价了抑制剂对某些临床分离菌株包括耐药菌株的影响。结果得到了重组莽草酸脱氢酶;测得比活力为20987U/mg,所建的莽草酸脱氢酶高通量筛选模型Z′因子为0.76,符合高通量筛选的要求;对5万余个化合物进行筛选得到9个抑制率较高的化合物;抑制剂6186050为竞争性可逆抑制剂;抑制剂6230384和6186050对海分枝杆菌的最低抑菌浓度(MIC)都是32μg/ml。结论建立了稳定性好、灵敏度较高的结核分枝杆菌莽草酸脱氢酶抑制剂高通量药物筛选模型,应用该模型筛选得到的抑制剂可能具有抑菌活性。

Establishment and application of a high-throughput screening model targeting to shikimate dehydrogenase from Mycobacterium tuberculosis H37Rv

Objective To establish a high-throughput screening (HTS) model targeting Mycobacterium tuberculosis shikimate dehydrogenase (MtSD) for the discovery of novel antituberculosis drugs. The established model was used to screen inhibitors targeting MtSD and to explore the effects of compounds on the activity of SD. Methods After expression and purification, MtSD activity was measured by detecting the absorption of NADPH at 340 nm wavelength in solution. HTS was established based on the activity of SD and Z’ factors were used to evaluate the quality of the HTS model. 50 000 compounds were screened by using this model and IC 50 of inhibitors were determined, besides the enzyme kinetics of inhibitor 6186050 was investigated. Some bacterial strains segregated from clinical were evaluated for the effect of inhibitors by the method of diluting liquid. Results Recombinant MtSD was successfully obtained and has the optimal activity 20987 U/mg. The parameter Z’ factor was 0.76. It was suggested that the model was highly feasible and stable for HTS drug screening. 9 compounds were found to inhibit MtSD using the HTS model, and the inhibitory effect of inhibitor 6185020 on the activity of SD was reversible. Both of the MIC of inhibitors 6230384 and 6186050 were 32 μg/ml for Mycobacterium marinum. Conclusion A steady and sensitive HTS model for potential MtSD inhibitors was established. The hits of MtSD inhibitors possess bacteriostasis activity.