HTS of drugs and natural products for compounds that kill R or NR Mtb. (A) One of two independent screens of ∼5,600 compounds that were scored as active against R Mtb, NR Mtb, both, or neither. OPB (result circled) was uniquely active in NR conditions. (B) Chemical structures of OPB and three inactive nonhydroxylated congeners: PB, suxibuzone, and sulfinpyrazone. (C) Concentration and time dependence of killing of auxotrophic Mtb by OPB (red circles) but not PB (blue circles). Closed circles, day 7; open circles, day 14. (D) Rapid and extensive time-dependent killing of auxotrophic Mtb. Results in C and D are means ± SD of triplicates from one of two similar experiments.

NR conditions that support mycobactericidal activity of OPB. We tested all 16 sets of conditions (pH 5.5 or 7.0, ±NaNO₂, ±butyrate, 1% or 21% O₂). Those with greatest impact were pH 5.5, as illustrated by further lowering the pH in the presence of NO₂⁻ (A), and NO₂⁻, the effect of which was pH-dependent (B), as shown in an outgrowth assay (A) or a cfu assay (B) with an inoculum of 1.6 × 10⁷ cfu/mL and a limit of detection of 100 cfu/mL. Results are means ± SD of triplicates from one experiment representative of two (A) or three (B) independent experiments.

4-Hydroxylation of OPB under NR conditions. (A) Impact of acid and nitrite on 4-hydroxylation. OPB was incubated for 1 h (hatched bars) or 24 h (solid bars) in R conditions or at pH 5.0 ± NaNO₂ ± butyrate, all of which are NR conditions. Reaction mixtures were analyzed by LC-MS. Species with masses corresponding to that of OPB or that of OPB + 16 amu were quantified. (B) Conversion of OPB to 4-OH-OPB and conversion of 4-OH-OPB to a quinonimine were followed by 1D and 2D NMR. Reactions were carried out at 37 °C in 90% H₂O/10% D₂O in PBS at pH 5.0 with 1 mM OPB or 4-OH-OPB. (C) Mechanistic depiction of OPB oxidation to 4-OH-OPB and its quinonimine. (D) Equivalent potency of 4-OH-OPB (blue circles) and OPB (red circles) against NR Mtb and comparable enhancement of their potency by lowering BSA from 0.5% (solid circles) to zero (open circles). (E) Activity of 4-OH-OPB against R Mtb. Symbols are as in D. Results are means ± SD for triplicates from three experiments in D and E. In D, error bars fall within the symbols.

Uptake of OPB and impact on Mtb metabolites. (A) Rapid OPB uptake into Mtb from agar plates containing 100 μM OPB. Ion counts were normalized using residual peptides in each sample and a standard curve of OPB in DMSO. (B) Structure of OPB heptadeuterated on the butyl chain (OPB-d₇). (C) Illustration of strategy for identifying molecular species in Mtb that represent adducts with OPB or its derivatives. (D) In vitro confirmation of adduction between 4-OH-OPB and MSH. 4-OH-OPB was incubated in PBS for 15 min at 37 °C, followed by addition of MSH at a 1:1 or 1:3 molar ratio and immediate separation by LC-MS. The retention times, masses, and MS/MS fragments of the species observed in vitro matched those observed in Mtb. (E) In vivo confirmation of adduction between 4-OH-OPB and MSH. Mtb was exposed in NR conditions for 24 h to 0 or 50 μM OPB or OPB-d₇, washed, and lysed. Its metabolites were then analyzed by LC-MS. A molecular species was considered to represent an adduct with OPB or its derivatives if there were two m/z species differing by 7.0439 amu (<5 ppm error) between the two lysates, neither of which was observed in the untreated sample, the smaller of which was observed only in the OPB-treated sample and the larger of which was observed only in the OPB-d₇–treated sample. The examples shown correspond to adducts of 4-OH-OPB with one molecule of MSH (MH⁺ = 829.3118 and a reduced form with MH⁺ = 831.3272; the chemistry is proposed in Fig. S5) or two molecules of MSH (MH⁺ = 1313.4447). (F) Depletion of Mtb’s flavin pool. An Mtb suspension at an OD₅₈₀ of 0.7 was exposed to 0, 100, or 250 μM OPB in NR conditions (pH 4.5 + NaNO₂ + butyrate + 1% O₂) for 3 d before assay of the designated flavins was assessed collectively. Concentrations of OPB were higher than those used for viability assays because the Mtb suspension was 3.5-fold more concentrated. Error bars are means of triplicate samples ± SD in one experiment representative of four independent experiments. The impact of both concentrations of OPB was statistically significant, with P < 0.0001 (unpaired Student t test).

Synergy between 4-OH-OPB and known TB drugs in killing Mtb. Synergistic killing of R Mtb by 4-OH-OPB (0 μM, black circles; 10 μM, red circles) with the following drugs: p-aminosalicylate (PAS) (A), fenamisal (B), nitrofurazone (C), and PA-824 (D). Drug concentrations are shown on a log10 scale. Results for Mtb treated with vehicle alone are indicated by the black circle corresponding to “d” (DMSO). The lowest fractional inhibitory concentration (FIC), determined from large checkerboard assays in which each concentration of the known TB drugs was tested alone and together with each of many concentrations of 4-OH-OPB, is shown. Cells at an OD₅₈₀ of 0.01 were exposed to compounds for 7–14 d before reading the final OD₅₈₀. Results are means ± SD of triplicates from individual experiments representative of at least two independent experiments.