Application of imb-ny compounds in the preparation of anti-tuberculosis mycobacterium drugs
A compound and lead compound technology, applied in the field of chemical drugs and drug screening, to achieve the effect of increasing the positive rate
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Embodiment 1
[0053] Example 1: Screening of potential lead compounds against the cell wall of Mycobacterium tuberculosis in vitro
[0054] By applying a high-throughput surrogate bacteria Corynebacterium glutamicum (CG) phenotypic screening model targeting the cell wall of Mycobacterium tuberculosis, to screen potential lead compounds against the cell wall of Mycobacterium tuberculosis in vitro, comprising the following steps:
[0055] 1. Preparation of strain seed solution: Streak inoculation of CG on the poured solid plate BHI medium (3.7% of BD company's brain heart infusion medium, add 1.5% agar, article number: 211065), recover and Cultivate fresh CG at 37°C, culture upside down for 12 hours, then pick a single colony and inoculate into 20mL liquid BHI medium (3.7% BD company's brain heart infusion medium, product number: 211065) at 37°C, 200rpm overnight nourish. After measuring the light absorption (OD) at visible wavelength at 600nm 600 ) is about 4.0 to 5.5. Dilute the bacteria...
Embodiment 2
[0092] Example 2: Expression, purification and application research of MTB IspD
[0093] By applying a high-throughput MTB IspD inhibitor screening model, new anti-tuberculosis drugs targeting MTB IspD were screened.
[0094] For the construction method of the cloning vector of IspD protein, please refer to the patent (patent name: IspD inhibitor screening model and new use of IspD inhibitor dumiphene, patent publication number CN 102277411A, invention date: 2010-06-10);
[0095] Apply the successfully constructed pET28a(+)-Rv3582c plasmid (the pET28a(+)-Rv3582c plasmid is constructed by conventional technical means) heat shock to transform E. MTB IspD protein pET28a(+)-Rv3582c::BL21(DE3)plysS strain with high expression of MTB IspD protein was obtained by culturing overnight at 37°C on an LB plate of Kan) to carry out protein expression, purification and application research. The specific method is:
[0096] 1) Streak inoculation of Escherichia coli pET28a(+)-Rv3582c::BL21(...
Embodiment 3
[0101] Example 3: Enzyme Activity Analysis
[0102] Reaction system: 10 μl ionic reagent (containing MgCl at a final concentration of 10 mM 2 , 20 mM NaF and 1 mM DTT); 1 μg of the purified IspD obtained in Example 2; CTP and MEP (the final concentrations thereof were 500 μM and 250 μM, respectively). The enzyme reaction buffer was 50 mM Tris-HCl (pH 8.0), and the total volume of the reaction system was 100 μl. Addition of heat-inactivated IspD was used as a control. At the same time, 100 μl PPi of 8 different concentrations (0 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 250 μM) were added, and the standard curve of absorption value and PPi concentration was drawn in the activity measurement, and the relevant straight line formula was fitted by Excel (see Figure 4 ); thereby calculating the reaction process of the enzyme according to the formula.
[0103] y=3.589x+16.418
[0104] R 2 =0.9997
[0105] Activity measurement: Incubate the above system at 37°C for 40 m...
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