A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy

Abstract

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

Figure 1. In vivo activity of compound 3 in the mouse model.

Balb/C mice were infected intranasally with ∼10³ M. tuberculosis H37Rv. Drug treatment was initiated 1 week after infection and was administered orally for 28 days daily at 25 mg kg⁻¹ (red circles) and 100 mg kg⁻¹ (blue diamonds). Bacterial loads were determined after 14 and 28 days of drug treatment and compared with the control group (HPβCD formulation alone, black squares). Isoniazid (green triangles, 25 mg kg⁻¹) was used as a positive control.

Figure 2. Glycerol dissimilation is required for the anti-tubercular activity of the PI compounds.

M. tuberculosis H37Rv (red squares) and spontaneous mutants resistant to 2 (clones 1–9, other symbols) were incubated in a medium containing a complex mixture of carbon sources (7H9 medium, a), or in a medium containing glycerol as the sole carbon source (Sauton medium, b). Growth was monitored by following the OD₆₀₀ over time. The MIC₅₀ of 1 (red circles) and 2 (blue squares) was tested against the parental H37Rv strain (c, f), H37Rv ΔglpK strain (d) and H37Rv ΔglpK pMV306-glpK (e) in the presence (c–e) or absence (f) of glycerol. Streptomycin (black triangles) was used as a reference compound.

Figure 3. Glycerol metabolism is not required for the virulence of M. tuberculosis in vivo.

Balb/C mice were infected intranasally with ∼10³ bacilli of the parental H37Rv (black circles) or of the ΔglpK (red squares) M. tuberculosis strains. The bacterial burden was followed in the lungs by CFU enumeration. Four mice per time point of each group were used and the standard deviations are shown.

Figure 4. Accumulation of glycerol phosphate and rapid ATP depletion in PI-treated M. tuberculosis.

(a) MIC₅₀ values of glycerol phosphate (black squares), DHAP (blue triangles) and methylglyoxal (red diamonds) were determined for M. tuberculosis H37Rv. M. tuberculosis H37Rv was exposed to 1 and 2 in the presence of glycerol for 24 h. The relative abundances of glycerol phosphate (b) and of pyruvate (c) are shown. M. tuberculosis H37Rv was exposed to 1 and 2 in the presence of glycerol for 24 h. The intracellular ATP level was quantified (d). Streptomycin and moxifloxacin were used as reference compounds.

Figure 5. Glycerol metabolites and the putative methylglyoxal-detoxification pathway in M. tuberculosis.

The generation of methylglyoxal from DHAP is usually spontaneous (dotted line). Glo I: glyoxylase I, Glo II: glyoxylase II, lldD: lactate dehydrogenase (cytochrome), G3P: glyceraldehyde-3-phosphate, DHAP: dihydroxyacetone phosphate.