to the dependence of the mechanism of activity on glycerol metabolism [13, 14], a carbon source only available to the organism in
the form of glycerol-based host lipids. In all these studies, compounds were screened under the in vivo relevant conditions, hits
validated by dose titration with subsequent determination of minimum inhibitory concentrations (MIC) of those hits that inhibited
growth of the bacillus.
Determination of MIC values is critical in developing
structure-activity relationships (SAR) of scaffolds discovered in
such screens, to guide further biological evaluation of the compounds and to understand mechanism of action. Interpretation of
whole cell SAR is driven not only by target-based SAR but also
differences in uptake, efflux, metabolism, and protein binding.
Biological evaluations of growth inhibitors to understand mechanism of action are in turn, always interpreted as a function of MIC
values. In this respect, the MIC value is dependent on the growth
condition, for example, the concentration that inhibits full growth
of the bacillus can depend on nutrient sources as well as the physical
state of the medium (liquid versus agar). The determination of
MIC values on solid media is often overlooked in drug discovery
studies. However, this is an important consideration since agar
MIC values, important information for generation of resistant
mutants on solid media, do not always correspond to MIC values
observed during growth in liquid media [15].
This chapter describes methods for the determination of MIC
values during growth on different in vivo relevant carbon sources as
well as restriction of growth imposed by acidic pH combined with
nitrosative stress. In vivo relevant carbon sources include glucose,
cholesterol, and fatty acids. The nitrosative stress used in our methods is derived from reactive nitrogen intermediates formed by
acidified nitrite [16]. The methods in this chapter describe a limited
set of growth readouts but these assays can readily be adapted to
other established measurements of mycobacterial growth [17]. In
addition, methods to explore the role of detergent effects are
described since the MIC values of several compounds including
rifampicin and QcrB-targeting imidazopyridines are significantly
affected by the presence and type of detergent in the medium.
The relevance of detergent effects in tuberculosis drug discovery
are outside the scope of this discussion. In this chapter, we provide
methods for understanding the effects of the non-hydrolyzable
detergent, Tyloxapol, as opposed to Tween 80 which is hydrolyzed
by the mycobacterium to generate the carbon source, oleic acid.
Growth of M. tuberculosis to yield suspensions amenable for growth
inhibition assays necessarily requires use of detergent to generate
homogenous cell suspension. However, it should be noted that a
detergent-free MIC assay has been described [18]. Our experience
is also that the presence of bovine serum albumin (BSA) in the
medium may dramatically affect MIC values. Although the effect of
596
Qinglan Wang and Helena I. M. Boshoff
the form of glycerol-based host lipids. In all these studies, compounds were screened under the in vivo relevant conditions, hits
validated by dose titration with subsequent determination of minimum inhibitory concentrations (MIC) of those hits that inhibited
growth of the bacillus.
Determination of MIC values is critical in developing
structure-activity relationships (SAR) of scaffolds discovered in
such screens, to guide further biological evaluation of the compounds and to understand mechanism of action. Interpretation of
whole cell SAR is driven not only by target-based SAR but also
differences in uptake, efflux, metabolism, and protein binding.
Biological evaluations of growth inhibitors to understand mechanism of action are in turn, always interpreted as a function of MIC
values. In this respect, the MIC value is dependent on the growth
condition, for example, the concentration that inhibits full growth
of the bacillus can depend on nutrient sources as well as the physical
state of the medium (liquid versus agar). The determination of
MIC values on solid media is often overlooked in drug discovery
studies. However, this is an important consideration since agar
MIC values, important information for generation of resistant
mutants on solid media, do not always correspond to MIC values
observed during growth in liquid media [15].
This chapter describes methods for the determination of MIC
values during growth on different in vivo relevant carbon sources as
well as restriction of growth imposed by acidic pH combined with
nitrosative stress. In vivo relevant carbon sources include glucose,
cholesterol, and fatty acids. The nitrosative stress used in our methods is derived from reactive nitrogen intermediates formed by
acidified nitrite [16]. The methods in this chapter describe a limited
set of growth readouts but these assays can readily be adapted to
other established measurements of mycobacterial growth [17]. In
addition, methods to explore the role of detergent effects are
described since the MIC values of several compounds including
rifampicin and QcrB-targeting imidazopyridines are significantly
affected by the presence and type of detergent in the medium.
The relevance of detergent effects in tuberculosis drug discovery
are outside the scope of this discussion. In this chapter, we provide
methods for understanding the effects of the non-hydrolyzable
detergent, Tyloxapol, as opposed to Tween 80 which is hydrolyzed
by the mycobacterium to generate the carbon source, oleic acid.
Growth of M. tuberculosis to yield suspensions amenable for growth
inhibition assays necessarily requires use of detergent to generate
homogenous cell suspension. However, it should be noted that a
detergent-free MIC assay has been described [18]. Our experience
is also that the presence of bovine serum albumin (BSA) in the
medium may dramatically affect MIC values. Although the effect of
596
Qinglan Wang and Helena I. M. Boshoff