Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition

Abstract

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

Fig. 1.. Antimycobacterial activity of 8918 in vitro and in mice.

(A) Structures of proguanil, 8918 and lidamidine. (B) In vitro activity against Mtb H37Rv. Colony-forming units (CFU) remaining after treatment with 8918. Inoc = inoculum at day 0. Means ± SD of triplicates in 1 experiment representative of 3. (C) In vivo activity against Mtb H37Rv. Histogram represents log₁₀ CFU from the lungs of BALB/c mice. One day after intranasal inoculation of 10⁶ Mtb H37Rv Mtb, mice were treated via oral gavage, 4 days per week over 2 weeks and then euthanized. Results are means ± SD for 5 mice per group in 1 experiment representative of 2. Results for the 8918-treated groups were statistically significantly different than for the vehicle-treated group (*; P < 0.001; one-way ANOVA).

Fig. 2.. Impact of 8918 on Mtb metabolites and lipids.

(A) Impact on metabolites. Filter-grown Mtb was transferred to plates containing 7H9 medium with or without 8918 and lysates collected 24 hours later for LC-MS. The Y-axis indicates raw intensity values for the area under the curve for each metabolite peak. Boxes represent the range of peak areas of triplicate samples. The center bar represents median raw intensity value for each metabolite peak. (B) Impact on synthesis of TDMs and TMMs. M. bovis var. Bacille Calmette-Guérin was incubated with DMSO vehicle alone, isoniazid (INH, 0.6 μM), proguanil (prog., 6.25 or 20 μM), or 8918 (6.25 or 20 μM) for 12 hours, then exposed for 24 hours to ¹⁴C acetate. Extracted lipids were subjected to TLC as described (30) (24-hour Phosphorimager exposure). (C) Impact on synthesis of PDIMs. The experiments were done as in (B) but using ¹⁴C propionate (5-day Phosphorimager exposure). (D) Quantitation of results from (B) and (C) (24-hour exposures for both). Results for (A-D) are each from one experiment representative of two independent experiments.

Fig. 3.. Functional relevance of PptT as the target for the antimycobacterial activity of 8918.

(A) Resistance of pptT mutant Mtb to 8918. Inhibition of growth of wild-type Mtb by 8918 but not of the pptT mutant W170S or wild-type Mtb expressing the pptT W170S mutant. Means ± SD of triplicates in 1 experiment representative of 5. (B) Increased susceptibility of Mtb to growth inhibition by 8918 upon knockdown of PptT in 4 different strains. The degree of sensitization to 8918 correlates with the extent of the knockdown. Means ± SEM of at least 12 data points from 4 independent experiments. (C) Complementation with atc. Means ± SEM of at least 5 data points from 2 independent experiments. (D) Knockdown of PptT does not affect the potency of isoniazid (INH), BTZ043, chlorpromazine (CPZ), or bedaquiline (BDQ). Means ± SEM of at least 5 data points from 2 independent experiments.

Fig. 4.. Inhibition of PptT through binding of 8918 to the Ppt channel in the active site.

(A, B) Impact of 8918 on activity of purified recombinant PptT. (A) V_max for recombinant PptT in the absence of 8918 (gray) or with 8918 at 5, 10, 20, 40 or 80 μM (shown in decreasing intensities of red). Chart displays V_max and K_m ± the standard error as a function of 8918 concentration in μM. (B) Inhibition of PptT by 8918. IC₅₀ was 2.5 μM, with maximum inhibition of 82%. (C-F) Structure of PptT with 8918 and adenosine 3’,5’-bisphosphate (PAP) moiety of CoA. (C) Simulated annealing composite omit electron density map (contoured at 2σ) of the PptT hydrophobic pocket showing protein residues in the active site with 8918 and PAP. (D) Active site of PptT with residues forming the Ppt binding pocket depicted with beige carbons. 8918 forms van der Waals interactions with Tyr160, Phe173 and Trp170. Glu157 is oriented towards the hydrophobic pocket and hydrogen bonds with 8918. Mg ions are depicted as green spheres. (E) Slice through the molecular surface of the active site of PptT colored according to the electrostatic potential (+/− 10 kT/e). (F) 2D interaction map indicating 8918’s interactions with PptT. Green shading represents hydrophobic regions, blue shading represents hydrogen bond interactions and grey shading represents accessible surface area.

Fig. 5.. Resistance to 8918 from LOF mutations in Rv2795c; identification of Rv2795c as a PptH.

(A) Resistance to 8918 in the rv2795c mutant H246N and rv2795c knock out and restoration of sensitivity with complementation of the knockout with the wild-type allele to a level matching that of wild-type Mtb. Means ± SD of triplicates in 1 experiment representative of 3. (B) Schematic of the reaction carried out in vitro by AcpH’s and detected in (C) and (D) with Rv2795c (PptH), which is not homologous to AcpH’s. (C) Conversion of holo-AcpM to apo-AcpM on incubation with C-terminally truncated Rv2795c for up to 4 hours. The experiment shown is representative of 5. (D) Detection of Ppt during incubation of AcpM alone, Rv2795c alone or the combination over the number of hours indicated. The experiment shown is representative of 3. The asterisk indicates statistically significant differences at all time points from each of the controls by unpaired Student’s t test (p < 0.002 for the 0.1 hour time point). Decreasing recovery of Ppt with time reflects instability of the compound. AUC, area under the curve (ion counts).