Abstract:

Objective To study the mechanism of pyrifazimine (TBI-166) accumulating reactive oxygen species (ROS) to kill Mycobacterium tuberculosis(MTB) through quinone oxidoReductase (QOR). Methods MtbQOR protein was expressed and purified. The decrease of NADPH (ΔA) at A_{340 nm} in blank control group and TBI-166 (0.03、0.3、3μg/ml) groups were detected by microplate reader; NADPH/NADP⁺ ratio of H37Rv after exposure to TBI-166 of different concentrations were detected by coenzyme Ⅱ NADP (H) detection kit; ROS fluorescence value of H37Rv strains and clinical collected strains were tested by bacterial ROS detection kit. Results The ΔA of control group (0.316±0.032) was lower than that of 0.03, 0.3, 3μg/ml TBI-166 groups (0.506±0.107, 0.531±0.054, 0.801±0.079) (t=-3.799,P<0.05;t=-7.634,P<0.01;t=-12.683,P<0.01). Within 48 hours, the NADPH/NADP⁺ratio of TBI-166 groups (0.847±0.229) was lower than that of the control group (1.728±0.384). ROS fluorescence value of H37Rv strains in TBI-166 groups (23072±2584) was higher than that of the control group (8282±448) (F=33.334, P<0.01). ROS fluorescence value of the strain number 12897 in TBI-166 groups (18369±3118) was higher than that of the control group (12268±2735) (F=5.026, P<0.05). ROS fluorescence value of the strain number 15171 in 3μg/ml TBI-166 group (17572±249) was higher than that of the control group (11109±343)(F=122.601, P<0.01). There was no significant difference detected in ROS fluorescence value between TBI-166 groups and blank control group in the strains numbered 11492 and 11195 (F=161.440, P>0.05; F=44.788, P>0.05). Conclusion TBI-166 could enhance redox reaction mediated by MtbQOR, decrease the ratio of NADPH/NADP⁺ and accumulate intra-bacterial ROS to kill MTB.

Key words: Mycobacterium tuberculosis, Molecular mechanism of pharmacological action, Quinone oxidoreductase, TBI-166