protein binding is complex and difficult to interpret in drug discovery with examples of highly protein-bound compounds being
in vivo efficacious compared to analogs with lower protein binding
[14], we simply provide a method to explore the effect of BSA
binding on MIC since the SAR can often be accentuated in the
absence of BSA in the medium. Synthesis of analogs that overcome
the protein binding to such an extent as to allow sufficient target
engagement remains empirical. Finally, we include methods for the
determination of MIC values of compounds on agar medium. The
reader is, however, encouraged to read previous descriptions of
MIC determination on solid media [17].
The MIC value in our methods is taken as the concentration of
compound that leads to complete growth inhibition. However, for
the assays that measure fluorescence or absorbance readouts, the
concentration that leads to 50, 90, and 99% growth inhibition can
be calculated as previously described [17]. The frontline antitubercular drugs show little variation in MIC as a function of
carbon source. However, rifampicin is affected by detergent composition of the medium showing a tenfold lower MIC value when
using Tween 80 as detergent compared to Tyloxapol (see Table 1).
Rifampicin also has a lower MIC value in the absence of BSA. The
stability of compounds in the medium over the duration of the
assay as well as to metabolism by the bacillus will affect interpretation and the understanding of the MIC values. A discussion of this
is outside the scope of this chapter but the measurement of parent
compound concentrations remaining in the assay will greatly facilitate the understanding of the SAR of analogs with inexplicable
absence of whole cell activity.
2 Materials
All aqueous solutions are made with distilled deionized water.
Media for cultivation of cells need to be filter-sterilized or autoclaved as described. Dimethyl sulfoxide (DMSO) does not need to
be filter-sterilized although DMSO source bottles are reserved for
cell culture purposes only and preferably dispensed in a biosafety
cabinet. All work with M. tuberculosis should be carried out in a
biosafety cabinet according to local guidelines.
2.1 Preparation of
Inoculum
1. Mycobacterium tuberculosis H37Rv (ATCC 27294),
M. tuberculosis pMSP12::GFP (Addgene), or other strain (see
Note 1).
2. 7H9/ADC/Tween 80 broth: Dissolve 4.7 g Middlebrook
7H9 broth base (Difco), 2 mL glycerol (add with a 3-mL sterile
syringe), and 0.5 mL Tween 80 (add with a 1-mL sterile
syringe) in 900 mL water (see Note 2). Aseptically add
100 mL albumin-dextrose complex (ADC) and filter-sterilize
MIC Determination for Mycobacterium Tuberculosis
597
in vivo efficacious compared to analogs with lower protein binding
[14], we simply provide a method to explore the effect of BSA
binding on MIC since the SAR can often be accentuated in the
absence of BSA in the medium. Synthesis of analogs that overcome
the protein binding to such an extent as to allow sufficient target
engagement remains empirical. Finally, we include methods for the
determination of MIC values of compounds on agar medium. The
reader is, however, encouraged to read previous descriptions of
MIC determination on solid media [17].
The MIC value in our methods is taken as the concentration of
compound that leads to complete growth inhibition. However, for
the assays that measure fluorescence or absorbance readouts, the
concentration that leads to 50, 90, and 99% growth inhibition can
be calculated as previously described [17]. The frontline antitubercular drugs show little variation in MIC as a function of
carbon source. However, rifampicin is affected by detergent composition of the medium showing a tenfold lower MIC value when
using Tween 80 as detergent compared to Tyloxapol (see Table 1).
Rifampicin also has a lower MIC value in the absence of BSA. The
stability of compounds in the medium over the duration of the
assay as well as to metabolism by the bacillus will affect interpretation and the understanding of the MIC values. A discussion of this
is outside the scope of this chapter but the measurement of parent
compound concentrations remaining in the assay will greatly facilitate the understanding of the SAR of analogs with inexplicable
absence of whole cell activity.
2 Materials
All aqueous solutions are made with distilled deionized water.
Media for cultivation of cells need to be filter-sterilized or autoclaved as described. Dimethyl sulfoxide (DMSO) does not need to
be filter-sterilized although DMSO source bottles are reserved for
cell culture purposes only and preferably dispensed in a biosafety
cabinet. All work with M. tuberculosis should be carried out in a
biosafety cabinet according to local guidelines.
2.1 Preparation of
Inoculum
1. Mycobacterium tuberculosis H37Rv (ATCC 27294),
M. tuberculosis pMSP12::GFP (Addgene), or other strain (see
Note 1).
2. 7H9/ADC/Tween 80 broth: Dissolve 4.7 g Middlebrook
7H9 broth base (Difco), 2 mL glycerol (add with a 3-mL sterile
syringe), and 0.5 mL Tween 80 (add with a 1-mL sterile
syringe) in 900 mL water (see Note 2). Aseptically add
100 mL albumin-dextrose complex (ADC) and filter-sterilize
MIC Determination for Mycobacterium Tuberculosis
597