Nonsteroidal Anti-Inflammatory Drug Sensitizes Mycobacterium

Nonsteroidal anti-inﬂammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials

Ben Golda, Maneesh Pinglea, Steven J. Bricknerb, Nilesh Shahc, Julia Robertsa, Mark Rundella, W. Clay Brackend, Thulasi Warriera, Selin Somersane, Aditya Venugopala, Crystal Darbya, Xiuju Jianga, J. David Warrend,f, Joseph Fernandezg, Ouathek Ouerfellic, Eric L. Nuermbergerh, Amy Cunningham-Bussela, Poonam Ratha, Tamutenda Chidawanyikaa, Haiteng Dengg, Ronald Realubiti, J. Fraser Glickmani, and Carl F. Nathana,1

Departments of aMicrobiology and Immunology, dBiochemistry, and eMedicine, and fMilstein Chemistry Core Facility, Weill Cornell Medical College, New York, NY 10065; bS. J. Brickner Consulting, LLC, Ledyard, CT 06339; cOrganic Synthesis Core, Memorial Sloan Kettering Cancer Center, New York, NY 10065; hDepartment of Medicine, Johns Hopkins Hospital, Baltimore, MD 21287; and gProteomics Resource Center and iHigh-Throughput Screening Resource Center, The Rockefeller University, New York, NY 10065

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2011.

Contributed by Carl F. Nathan, August 17, 2012 (sent for review May 29, 2012) Existing drugs are slow to eradicate Mycobacterium tuberculosis replicating (R) Mycobacterium tuberculosis (Mtb) (4). Mtb can (Mtb) in patients and have failed to control tuberculosis globally. occupy diverse microenvironments in the host. Evidence from One reason may be that host conditions impair Mtb’s replication, auxotrophs, analyses of gene expression, and direct and indirect reducing its sensitivity to most antiinfectives. We devised a high- biochemical measurements in vivo or ex vivo in experimental throughput screen for compounds that kill Mtb when its replica- animals and people suggest that such environments expose Mtb tion has been halted by reactive nitrogen intermediates (RNIs), to acid, hypoxia, reactive nitrogen intermediates (RNIs), oxida- acid, hypoxia, and a fatty acid carbon source. At concentrations tive stress, carbohydrate deficiency, and metal starvation or in- routinely achieved in human blood, oxyphenbutazone (OPB), an toxication, and require Mtb to metabolize fatty acids or cholesterol inexpensive anti-inflammatory drug, was selectively mycobacterici- (5–17). In vitro, many of the same conditions can make Mtb dal to nonreplicating (NR) Mtb. Its cidal activity depended on mild relatively refractory to killing by the standard agents, except for acid and was augmented by RNIs and fatty acid. Acid and RNIs pyrazinamide, which is only effective at a low pH. fostered OPB’s 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1- Thus, there is a need for a high-throughput screen (HTS) for (4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed compounds that kill Mtb when the Mtb has been rendered NR both replicating and NR Mtb, including Mtb resistant to standard by a combination of physiologically relevant host-imposed con- drugs. 4-OH-OPB depleted flavins and formed covalent adducts with ditions. We were encouraged to devise such a screen by recent N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically discoveries of a class of compounds that kill Mtb only when it is with oxidants and several antituberculosis drugs. Thus, conditions NR (18), an antibiotic in clinical use for other infections that kills that block Mtb’s replication modify OPB and enhance its cidal action. NR Mtb better than R Mtb (19), and a compound that kills NR Modified OPB kills both replicating and NR Mtb and sensitizes both and R Mtb equally well (20). Unfortunately, only one of those to host-derived and medicinal antimycobacterial agents. compounds is an approved drug, and even if it were of proven utility in TB, its price would preclude its use by most of those ome bacterial infections can be cured with a single dose of an who need it. We decided to screen other existing drugs that are Santibiotic, and most others can be cured with administration not regarded as antiinfectives for those that kill NR Mtb. Here, of one drug over several days or weeks. In contrast, routine we report finding such a drug in an HTS that combined four host- treatment of drug-sensitive tuberculosis (TB) takes 2 mo of therapy imposed conditions, some of which converted the drug into a form with four drugs and an additional 4 mo with two drugs to reduce active on both R and NR Mtb. the 2-y relapse rate below 5%. The difficulty of completing pro- longed treatment is a major reason for emergence of drug re- Results sistance. When the infecting strain is resistant to isoniazid and Screen for Compounds That Kill NR Mtb, R Mtb, or Both. We set out rifampin, the two drugs recommended for all 6 mo of treatment, to identify drugs that can kill Mtb in the face of replication- fi cure often requires 2 y of daily administration of toxic, expensive, inhibiting conditions. HTSs depend on robots that are dif cult second-line agents that are often unavailable at the point of care. to accommodate in the biological safety level (BSL) 3 conditions When the causative strain is additionally resistant to a quinolone required for work with pathogenic strains of Mtb. We took ad- 2 Δ Δ and an aminoglycoside, the resultant “extensively drug-resistant” vantage of mc 6220, a panCD lysA double-auxotrophic strain of TB was fatal to 80% of patients in a leading center (1), although Mtb H37Rv (a kind gift of W. Jacobs, Jr., Albert Einstein College of Medicine, New York), which has been found to cause no dis- complex multidrug regimens have achieved higher cure rates in fi populations not previously exposed to the additional drugs (2). ease when injected into immunocompetent or immunode cient In addition to sharing air with someone with TB, leading risk factors for contracting the disease are malnutrition, HIV infection, fi Author contributions: B.G. and C.F.N. designed research; B.G., M.P., J.R., M.R., W.C.B., diabetes, and exposure to smoke from cigarettes or cooking res T.W., S.S., A.V., C.D., X.J., J.D.W., J.F., E.L.N., A.C.-B., P.R., T.C., H.D., and R.R. performed (3). These epidemiological challenges exacerbate problems of in- research; N.S. and O.O. contributed new reagents/analytic tools; B.G., M.P., S.J.B., W.C.B., adequate diagnostic technology and limited access to drug sus- J.F., H.D., J.F.G., and C.F.N. analyzed data; and B.G. and C.F.N. wrote the paper. ceptibility testing and to drugs. Control of the pandemic is not in The authors declare no conflict of interest. sight (3). Freely available online through the PNAS open access option. It is widely hypothesized that treatment of TB is protracted 1To whom correspondence should be addressed. E-mail: [email protected]. because nonreplicating (NR) subpopulations of bacilli are phe- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. notypically tolerant to drugs that were selected for activity against 1073/pnas.1214188109/-/DCSupplemental.

16004–16011 | PNAS | October 2, 2012 | vol. 109 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1214188109 Downloaded by guest on October 2, 2021 mice, guinea pigs, or monkeys (21, 22). This strain has been not at up to 200 μM when tested against R Mtb in liquid cultures

deemed to be safe for use in BSL2 laboratories by the Institutional (Fig. 1A). The closely related pyrazolidinediones phenylbutazone INAUGURAL ARTICLE Biosafety Committees of the Albert Einstein College of Medicine (PB), suxibuzone, and sulfinpyrazone (Fig. 1B) did not kill either and Weill Cornell Medical College, as approved by the US Na- NR or R Mtb (Fig. 1A). Results using OD as a read-out were tional Institutes of Health. Provision of pantothenate and lysine confirmed by cfu assays with pure, resupplied OPB (Fig. 1 C and D). allows the auxotroph to grow like WT in vitro. The HTS under R Killing by OPB was concentration-dependent, time-dependent, conditions identified 24 actives that have known anti-Mtb activity; and extensive against both the auxotrophic strain (Fig. 1 C and minimal inhibitory concentrations (MICs) were determined for D)andWTMtb(Fig. S1B). In contrast, neither OPB nor 11 and were similar when tested against WT Mtb and the PB was bactericidal to Escherichia coli, Salmonella enterica var. auxotroph (Table S1), validating use of the auxotroph. Typhimurium, Pseudomonas aeruginosa, Staphylococcus au- It is a challenge to detect compounds that kill NR Mtb when reus,orCandida albicans up to 100 μM, nor was OPB cytotoxic the criterion for death is inability to replicate. We solved this to monkey kidney epithelial (Vero) cells or murine bone problem by conducting the assay in two stages. In the first stage, marrow-derived macrophages at 200 μM. we halted Mtb’s replication by incubation in 96- or 384-well Next, we determined the individual contributions of the con- microplates in modified Sauton’s minimal medium at pH 5.5 in ditions used to prevent Mtb from replication. At pH 7, OPB was 1% O2 and 5% CO2 with 50 μM butyrate or isobutyrate as the inactive in the presence or absence of butyrate or nitrite with either sole carbon source in the presence of 0.5 mM nitrite. These 1% or 21% O2. OPB’s mycobactericidal potency was markedly conditions prevented growth yet led to little or no decline in cfu increased as the pH was lowered from 5.5 to 4.5 (Fig. 2A), the over 6 d (Fig. S1A). We exposed Mtb to test compounds at 12.5 μM level in the phagosome of activated macrophages (8). OPB’s for 6 d and then diluted the contents of each well 21-fold into mycobactericidal activity was modestly enhanced by nitrite, but Middlebrook 7H9 medium containing dextrose and glycerol as even though decreasing the pH enhances the rate of RNI pro- carbon sources at pH 6.6 in 21% O2 and 5% CO2 without nitrite, duction from nitrite, activity became largely nitrite-independent conditions that support Mtb’s exponential replication. After at pH 4.5 (Fig. 2B). Butyrate also increased activity. The activity 7–10 d, we recorded the OD in each well in comparison to at low pH was indistinguishable at O2 levels of 21% and 1%. negative and positive control cultures that contained 0.25% DMSO or 12.5 μM rifampin, respectively, in the first stage. This two-stage Reactions of OPB Under Conditions That Impose Nonreplication. Mild screen had an average Z′ value of 0.7. In a parallel screen, we acidity, RNIs, and fatty acid might alter either OPB or Mtb. As

tested the same compounds against R Mtb in a one-stage assay monitored by liquid chromatography (LC)-MS and NMR, OPB MEDICAL SCIENCES using the same medium as for the second stage of the two-stage was stable at pH 7.0 with or without butyrate, nitrite, or dieth- assay. The one-stage screen had an average Z′ value of 0.9. ylenetriamine NONOate (DETA-NO), which donates NO at pH “ ” fi 7.0. In contrast, at pH 5.0, OPB rapidly and quantitatively con- Based on the distribution of results (Fig. S2), hits were de ned + as compounds that produced 60% inhibition of growth compared verted to a species whose mass [MH = 341.27 absolute mass units (amu)] was consistent with the addition of an oxygen to OPB with the DMSO controls. We screened overlapping sets of past + and present pharmaceuticals in the Spectrum and Johns Hopkins (MH = 325.15 amu) (Fig. 3A). Nitrite increased the rate of con- University collections (23), numbering ∼3,600, along with ∼2,000 version (Fig. 3A). We identified the unknown species as 4-butyl-4- natural products of bacterial and plant origin, with a confirmed hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4- hit rate of 2.7%. Of the actives among these ∼5,600 compounds, OH-OPB) (Fig. 3 B and C). After a brief incubation in aqueous 35% were active only against R Mtb, 17% only against NR Mtb, solution at 37 °C, 4-OH-OPB underwent opening of the N-N bond and 48% against both (Fig. 1A). Most were antiinfectives already in the pyrazolidinedione ring to form a quinonimine (Fig. 3 B and known to kill Mtb; cytotoxic drugs, such as 5-fluorouracil and C), a Michael acceptor. NMR spectra for 4-OH-OPB and its ring- gliotoxin; or compounds likely to be toxic, such as the quinones opened quinonimine were consistent with those described by juglone and lawsone. One active drug stood out because it fit Dekkers et al. (24). The addition of a hydroxyl to the pyr- 13 none of those categories. azolidinedione ring was consistent with a large C shift of the C4 carbon from 42 ppm (seen in OPB) to 81 ppm (seen in Oxyphenbutazone Selectively Kills NR Mtb. Oxyphenbutazone (OPB) 4-OH-OPB) using 2D 13C-heteronuclear single-quantum co- (Fig. 1B), an extensively used nonsteroidal anti-inflammatory drug herence (HSQC) and 2D 13C-heteronuclear multiple-bond cor- (NSAID), inhibited growth of NR Mtb by 100% at 12.5 μM but relation (HMBC) spectra.

A B C D

R inactives PB day 7 & 14 DMSO phenylbutazone 30 µM (PB) OPB day 7 & 14 100 µM 200 µM R &NR NR limit of 300 µM detection oxyphen- DMSO butazone (OPB) suxibuzone

Fig. 1. 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.

Gold et al. PNAS | October 2, 2012 | vol. 109 | no. 40 | 16005 Downloaded by guest on October 2, 2021 A liquid culture demonstrated that Mtb exposed to PB contained no detectable intrabacterial PB after 24 h. In contrast, Mtb cul- tured with OPB contained both OPB and 4-OH-OPB. We used pH 5.5 the filter-based method to study the kinetics of uptake. OPB was detectable in Mtb by 15 min, the first time point tested. The concentration increased steeply over 60 min and gradually over the next 23 h (Fig. 4A). pH 5.0 In addition to OPB and 4-OH-OPB, several chemical species appeared in OPB-treated Mtb that were not observed in vehicle- pH 4.5 treated Mtb (Table S2). None matched metabolites in the Kyoto Encyclopedia of Genes and Genomes database. To test if these were covalent adducts of OPB or its derivatives with Mtb metabolites, we synthesized an analog of OPB, 4-(butyl-2,2,3,3,4,4,4-d7)- 1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (OPB-d7) (Fig. S4) containing seven deuterium atoms in place of hydro- gens on the butyl chain, conferring an increased mass of 7.0439 B amu (Fig. 4B). Numerous molecular species were undetectable in untreated Mtb, present in OPB-treated Mtb, and present but ± pH 5.5 increased in mass by 7.0439 amu ( 5 ppm error) in Mtb treated pH 5.5 + NaNO with OPB-d7, as illustrated in Fig. 4C. The characterization of 2 over 30 major species that fit these criteria is summarized in Table S2. These features identified them as adducts with OPB pH 5.0 or its derivatives. The most abundant species had masses cor- pH 5.0 + NaNO responding to predicted reaction products (Figs. S5–S7) of the 2 quinonimine forms of OPB and 4-OH-OPB with the NAc-cysteine pH 4.5 (NAC)–ligated disaccharide mycothiol (MSH) and/or its pre- pH 4.5 + NaNO cursor, NAC (Fig. 4D and Table S2), the former as confirmed by 2 DMSO in vitro reaction of 4-OH-OPB with authentic MSH (Fig. 4E). Where tested, these tentative assignments were strongly supported by the masses of fragments in MS/MS (Table S2). Thus, Mtb fi Fig. 2. NR conditions that support mycobactericidal activity of OPB. We detoxi es OPB and 4-OH-OPB, in part, by forming covalent adducts with the intracellular thiols NAC and MSH. testedall16setsofconditions(pH5.5or7.0,±NaNO2, ±butyrate, 1% or 21% O2). Those with greatest impact were pH 5.5, as illustrated by further lowering Among the other most conspicuous metabolomic changes, − − fl the pH in the presence of NO2 (A), and NO2 , the effect of which was chemical species with properties of avin nucleotides were pH-dependent (B), as shown in an outgrowth assay (A) or a cfu assay (B) with abundant in untreated Mtb but depleted in OPB-treated Mtb. an inoculum of 1.6 × 107 cfu/mL and a limit of detection of 100 cfu/mL. An enzyme-coupled fluorometric assay confirmed depletion of Results are means ± SD of triplicates from one experiment representative the flavin pool (Fig. 4F). of two (A)orthree(B) independent experiments. OPB Sensitizes Mtb to Oxidants and Exogenous Antimicrobials. MSH, Bioactivity of 4-OH-OPB. Because OPB rapidly formed 4-OH- an abundant thiol in Mtb, functions like glutathione (26). Genetic OPB under conditions in which OPB was mycobactericidal, we disruption of its biosynthesis sensitizes Mtb to oxidative stress synthesized 4-OH-OPB, which proved to be equipotent to OPB (27). Consistent with competition for MSH, sublethal 4-OH-OPB in killing NR Mtb (Fig. 3D). The potency of each was modestly treatment rendered R Mtb eightfold more sensitive than un- enhanced by omission of BSA (Fig. 3D). Although butyrate did treated Mtb to growth inhibition by H2O2 and fourfold more not contribute to conversion of OPB to 4-OH-OPB, it did en- sensitive to the oxidants plumbagin, methyl viologen, and copper hance the mycobactericidal activity of 4-OH-OPB on NR Mtb. (12). Deletion of genes of the MSH biosynthetic pathway sen- In contrast to OPB, 4-OH-OPB killed R Mtb in liquid culture sitizes Mtb to diverse antibiotics (27, 28). To see if OPB also with an MIC of 100 μM in the presence of 0.5% BSA and an sensitizes Mtb to other agents, we rescreened the original com- MIC of 25 μM at lower BSA concentrations (Fig. 3E). Assays pound library in the presence of a single sublethal concentration performed independently at SRI International confirmed that of OPB. Of the compounds that appeared to synergize with OPB, the MIC of 4-OH-OPB against drug-sensitive R Mtb H37Rv was we identified those that appeared to synergize with 4-OH-OPB 26 μM and that 4-OH-OPB had comparable MICs against R Mtb as well. We picked four of them, including three TB drugs, for strains that were resistant to streptomycin (13 μM), ethionamide checkerboard analysis. 4-OH-OPB exhibited robust synergy with (13 μM), isoniazid (26 μM), p-aminosalicylate (26 μM), kanamycin all four, p-aminosalicylate, fenamisal, nitrofurazone, and PA-824 (26 μM), or ethambutol (26 μM). (29), as defined by fractional inhibitory concentrations of <0.5 The bactericidal spectrum of 4-OH-OPB was remarkably nar- (Fig. 5 A–D). In contrast, there was no synergy with amikacin or row, with weak activity against Mycobacterium smegmatis (MIC of with isoniazid. 200 μM in 7H9 lacking BSA and >200 μM in 7H9 with oleic acid, albumin, dextrose, catalase (OADC) or albumin, dextrose, sodium OPB in Mice. Mice were given OPB by gavage at a dose (20 mg/kg chloride (ADN) supplement) and S. aureus (MIC of 50 μM). of body weight) far above standard practice in humans (4–8 mg/ 4-OH-OPB was inactive on E. coli, S. enterica var. Typhimurium, kg in a 70-kg person). The serum concentration peaked at only P. aeruginosa,andC. albicans up to 200 μM, even when the tests 2 μM with a half-life of 3 h. The i.p. injections of 300–600 mg/kg with C. albicans were conducted at pH 5.5 or 4.5 (Fig. S3). of OPB, about 100-fold the human dose, achieved peak serum levels of only 160–205 μM with a half-life <2 h. In contrast, Identification of Intramycobacterial Targets of OPB and 4-OH-OPB. standard oral dosing of humans with OPB was reported to Targeted metabolomics of R Mtb growing on filters on drug- result in a peak serum concentration of 77–308 μMwitha containing agar (25) and LC-MS analysis of lysates of NR Mtb in half-life of 50–75 h (30–34). Thus, exposure to OPB was vastly

16006 | www.pnas.org/cgi/doi/10.1073/pnas.1214188109 Gold et al. Downloaded by guest on October 2, 2021 A C INAUGURAL ARTICLE

H H O O O

O N HN OPB + 16 amu N N 2 N N H+ O NO - O O

% conversion of OPB to O O 2 O HO H+ H HO

B QI D E

4-OH-OPB OPB OPB 11 9

7 4-OH-OPB MEDICAL SCIENCES

5 Hours 3 1

7.1 7.0 6.9 6.8 f1 (ppm)

Fig. 3. 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.InD, error bars fall within the symbols.

lower in mice than in humans, making the mouse an unsuitable Mtb within 15 min of exposure. When Mtb was exposed to OPB model in which to test OPB. under NR conditions, 4-OH-OPB also accumulated rapidly in the bacteria. Provision of fatty acid enhanced OPB’smycobacter- Discussion icidal activity in the presence of BSA. Fatty acids may displace fi We identi ed OPB, an inexpensive, off-patent NSAID, as a narrow- OPB from BSA. Alternatively, OPB may sensitize Mtb to the ’ spectrum antimycobacterial agent. OPB s mycobactericidal activity toxicity of fatty acids (9, 39, 40). was conferred by conditions that impose nonreplication on Mtb. The inability of PB to kill Mtb can be attributed to two features. The most important of these conditions, low pH, is obtained in the ’ fl First, it failed to accumulate within Mtb. Second, PB s lack of a phagosome of activated macrophages (8) and at in ammatory phenolic hydroxyl precludes 4-OH-PB from forming a quinoni- sites. RNIs also contributed to the activity of OPB. RNIs are mine. Thus, 4-OH-PB is less chemically reactive than 4-OH-OPB. produced by human macrophages in tuberculous lesions (35). In OPB or its derivatives, most likely the 4-OH-OPB quinonimine, granulomas, hypoxia may limit RNI production by host enzymes, depleted flavins. Many enzymatic reactions could be compromised but O2-starved Mtb respires nitrate (which is abundant in body fluids) and produces its own nitrite (36, 37). Mild acid converted as a result. Moreover, OPB or its products formed adducts with OPB to a form that was active against R Mtb as well as NR Mtb, MSH and NAC, an intermediate in MSH synthesis. This may ’ and RNIs accelerated the conversion. contribute to OPB s sensitization of Mtb to acid, oxidant stress, The primary product of OPB exposed to mild acidity and RNIs fatty acids in combination with low pH and RNIs, and certain was 4-OH-OPB. OPB did not kill R Mtb in liquid cultures, al- antibiotics (27, 41). Numerous other adducts were formed that though we confirmed an observation made over 40 y ago (38) that we did not identify. The mechanism of killing is thus likely to OPB was active against R Mtb on agar plates (MIC of 10 μg/mL). be multifactorial. In contrast, 4-OH-OPB killed R Mtb both in liquid culture and With respect to its mycobactericidal activity, OPB can be con- on agar plates, and it killed NR Mtb as well. Although PB was sidered a prodrug. However, OPB does not require an enzyme for not detectable in Mtb over a 24-h period, OPB was detectable in its activation. This, plus its multiplicity of targets, may explain why

Gold et al. PNAS | October 2, 2012 | vol. 109 | no. 40 | 16007 Downloaded by guest on October 2, 2021 A D E 100

703.27

o l

4-OH-OPB aBP 341.15

1043.42 Oe relative 1383.56 681.29

abundance O

538.18 1075.35 -

849.21 1021.44 1189.35 4H- 0 4-OH-OPB-(MSH) 100 2 1313.45 4-OH-OPB-(MSH) B 1

S HO 827.30 oi

849.28 +B r

N N relative

1335.44 m

-HO- abundance O O 647.24

: 684.19

D 1 1097.84

D 4 D D 0 D D D 4-OH-OPB-(MSH) F OPB-d7 100 2

mass = OPB + 7.04394 amu 1313.45

to M+

C ra

r a

Adduct Mass OPB OPB-d7 mass l om

number (m/z) (m/z) (m/z) shift (da) relative 4-OH-OPB-(MSH) 1335.44 1 abundance 684.19 827.30 3: 829.3118 present absent 849.28 1

16 7.0436 4-OH-OPB 836.3554 absent present 0 1313.4447 present absent 31 7.0435 400 600 800 1000 1200 1400 1600 1320.4882 absent present Mass (m/z)

Fig. 4. 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)for3d 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).

our extensive efforts to select OPB-resistant mutants of Mtb tablets or antacids (44). Less frequent are rash, gastric ulcers, hy- − + failed, such that the frequency of resistance appears to be <10 9. persensitivity reactions, Na retention, hepatitis, renal disorders, OPB’s antimicrobial spectrum was narrow, and its cytotoxic leukopenia, agranulocytosis, and aplastic anemia. The most serious action against mammalian cells was minimal. In part, this may toxicities are more frequent in the elderly. The incidence of acute reflect that conversion to the more reactive 4-OH-OPB quinoni- bone marrow failure was estimated at less than 1 in 50,000 (45) and mine depends on conditions that are likely to be relatively re- at 1 in 66,000 (44). In the setting of drug-resistant TB, the major stricted to certain pathological microenvironments. However, OPB toxicities of OPB should be considered in the context of the dire did not kill C. albicans even at pH 4.5 or 5.5, where formation of prognosis and compared with those of second-line drugs, which 4-OH-OPB was rapid and extensive, and preformed 4-OH-OPB averaged 16% in a recent report (46). Of practical concern, most was inactive on C. albicans and other microbes. Investigators patients with TB who need second-line drugs fail to receive them studying 4-OH-OPB as an anti-inflammatory agent reported it to because of cost. OPB is exceedingly inexpensive. be nontoxic in mice at serum concentrations <20 mM (42) and to We expected that some patients experiencing pain and fever be safe in people (43). OPB’s narrow antimicrobial and cytotoxic associated with TB may have been given OPB for symptomatic spectrum may be defined chiefly by differential abilities of vari- relief. We found three such reports involving a total of 84 patients, ous cells to take up, export, or metabolize OPB and 4-OH-OPB. who were reported to experience clearance of drug-resistant PB was introduced into clinical medicine in 1949 (32). It was Mtb from sputum (47), faster gain of weight (48), or improved soon demonstrated that humans metabolize PB, in part, to OPB, tolerance to conventional therapy (49). These studies neither and OPB was introduced as a drug a few years later. PB and OPB establish nor exclude that OPB’s apparent impact in patients were both widely used until less toxic but more costly NSAIDs with TB reflected an anti-inflammatory action, a microbiological replaced them in many countries. However, PB and OPB are still action, augmentation of the microbiological action of other drugs, used in regions where cost governs access. Gastrointestinal dis- or some combination of these effects. Nonetheless, in 84 subjects tress with OPB is common (∼10%) but avoidable with coated receiving 300–600 mg of OPB per day for 2–6 mo, addition of

16008 | www.pnas.org/cgi/doi/10.1073/pnas.1214188109 Gold et al. Downloaded by guest on October 2, 2021 A B Materials and Methods 61.0=CIF 61.0=CIF Strains and Growth Conditions. WT Mtb H37Rv was cultivated in Middlebrook INAUGURAL ARTICLE 7H9 medium with 0.2% glycerol, tyloxapol (0.02%), and 10% ADN supplement. Mtb strain mc26220 ΔpanCDΔlysA was passaged in Middlebrook 7H9 supplemented with glycerol (0.5%), OADC, tyloxapol (0.02%), casein hydro- lysate (CAS) amino acids (0.05%), L-lysine (240 μg/mL), and pantothenate (24

μg/mL). R conditions included incubation in 20% O2 and 5% CO2. NR conditions used minimal Sauton’s-based medium [per liter: 0.5 g of KH2P04, 0.5 g of MgS04, and 0.05 g of ferric ammonium citrate (this trace amount [0.005%] of citrate does not support growth)] supplemented with BSA (0.5%), NaCl (0.085%), tyloxapol (0.02%), L-lysine (240 μg/mL), pantothenate (24 μg/mL), and butyrate (50 μM), omitting glycerol, citrate, and asparagine from Sau- ’ C D ton s recipe, at pH 5.5 with 0.5 mM freshly prepared NaNO2. NR conditions FIC = 0.33 included incubation at 37 °C in chambers (BioSpherix) at 1% O2 and 5% CO2. The following microbes were grown in 150 μL in 96-well microtiter plates at 37 °C: uropathogenic E. coli TOP10 (Luria broth), S. enterica var. Typhimurium (Luria broth), S. aureus American Type Culture Collection (ATCC) 29213 (Mueller–Hinton broth), P. aeruginosa PAO1 (Luria broth), and C. albicans ATCC 90028 (YM broth) at pH 5.5. FIC = 0.30 HTS. HTS data were managed using Collaborative Drug Discovery software and JChem for Excel and MarvinView (ChemAxon). For HTS against NR Mtb,

100 μL of the NR medium with 0.5 mM NaNO2 was added to 96-well plates (Corning), followed by known drugs and bioactives in the collections from Prof. J. Liu (Johns Hopkins University, ∼2,000), Spectrum (∼2,000), and Analyticon 2 Fig. 5. Synergy between 4-OH-OPB and known TB drugs in killing Mtb. (∼2,000) as 0.5 μL of 5-mM stocks in DMSO. Mtb mc 6220 in log phase (∼0.5 A580) Synergistic killing of R Mtb by 4-OH-OPB (0 μM, black circles; 10 μM, red was washed twice with PBS containing tyloxapol (0.02%; PBS-Ty) and

circles) with the following drugs: p-aminosalicylate (PAS) (A), fenamisal (B), resuspended at an OD580 of 0.4 in NR medium containing 0.5 mM NaNO2 for nitrofurazone (C), and PA-824 (D). Drug concentrations are shown on 30–60 min. Mtb suspension (100 μL) was dispensed to each well, such that

a log10 scale. Results for Mtb treated with vehicle alone are indicated by the the ﬁnal volume was 200 μL, OD580 of 0.2, 0.25% DMSO, and ∼12.5 μMtest “ ” black circle corresponding to d (DMSO). The lowest fractional inhibitory agent. Wells were mixed, and plates were incubated under 1% O2 and 5% MEDICAL SCIENCES concentration (FIC), determined from large checkerboard assays in which CO2 in stacks between hydration plates containing only PBS. After 7 d, the each concentration of the known TB drugs was tested alone and together Mtb in each well was resuspended and diluted 21-fold (10 μL into 200 μL) with each of many concentrations of 4-OH-OPB, is shown. Cells at an OD580 of into fresh R medium. Test agents were thus carried over at ∼0.6 μM. After 0.01 were exposed to compounds for 7–14 d before reading the ﬁnal OD . 580 10–14 d of incubation at 37 °C in 20% O2 and 5% CO2, cells in outgrowth ± Results are means SD of triplicates from individual experiments represen- plates were resuspended and an aliquot (100 μL) was transferred to clear- tative of at least two independent experiments. bottomed, black, 96-well microtiter plates (Greiner) for OD580 determination by bottom-read. The HTS against R Mtb used R medium in a single-stage

assay with an initial OD of 0.1 and 7–8 d of incubation in 20% O2 and 5% OPB to standard TB treatment did not lead to additional toxicity CO2. During the course of this study, we converted to assays in black, clear- but, instead, was reported to confer clinical benefit. bottomed, 384-well plates (Greiner). To a final assay volume of 50 μL, we Although the mouse is the model of convention and conve- added either 0.25 μL or 0.50 μL of compounds at 5 mM in DMSO. Inhibition − − − nience for preclinical tests of candidate anti-TB drugs, differences of outgrowth was calculated as 100 (100[ODexp ODrifampin/(ODDMSO in metabolism of some drugs between mice and humans are too ODrifampin)]), where ODexp is the OD of the experimental well and ODDMSO great for the mouse to be of use in modeling these drugs’ exposure and ODrifampin are means for 32 negative and positive controls on each plate, respectively. in humans. This proved to be the case with OPB. In cases in which fi the mouse is pharmacokinetically unsuitable to test a particular The MIC was de ned as the lowest concentration at which there was no increase in OD beyond the starting value of 0.01. Synergy experiments were agent that otherwise holds promise and has already been used performed in R medium containing 0.005% BSA. Fractional inhibitory con- fi safely in patients with TB, it is our belief that the bene t-to-cost centrations for wells containing various concentrations of drug A and drug B

ratio of clinical trials in patients with TB will exceed that of were determined as the fractional inhibitory concentration = ([A]/MICA + [B]/ experiments in large animal models. MICB) when inhibition is ≥90% in the simultaneous presence of compound A In conclusion, we devised an HTS that finds compounds that and compound B, each used below its own MIC. MICs determined at SRI kill Mtb whose replication is inhibited by a combination of four International were based on resazurin reduction after 6 d of exposure of 4 pathophysiologically relevant conditions, extending the initial ob- ∼10 Mtb per well to eight different concentrations of 4-OH-OPB in 7H9 servation that antiinfectives need not be uniquely or preferentially medium with 10% OADC. 4 μ active on R bacteria (18). We demonstrated that use of an Mtb Vero monkey kidney cells (ATCC CRL-1587) seeded at 10 cells per 200 L per well in 96-well microtiter plates were incubated at 37 °C in DMEM with auxotroph allows robotic screening under BSL2 conditions. This 4.5 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL peni- may encourage others who lack access to robotics in BSL3 con- cillin, 100 μg/mL streptomycin, 0.5 mg/mL gentamicin, and 10% FBS. Cells ditions to conduct screens against Mtb. Testing known drugs, we were washed once with PBS, and medium was added containing 2% FBS identified OPB, a widely used, inexpensive, relatively safe NSAID and compound (0–100 μM). After 48 h, viability was measured by tetrazo- with narrow-spectrum mycobactericidal activity and a history of lium reduction (MTS assay; Promega). Bone marrow-derived macrophages clinical tolerability and potential benefit in patients with TB. We were obtained from femurs of C57BL/6 mice differentiated for 7 d in DMEM demonstrated that OPB’s mycobactericidal mechanism involves with 10% L-cell conditioned medium and 10% FBS. condition-dependent conversion to reactive species that deplete ’ fl Special Reagents. OPB was resupplied (MP Biomedicals), and its purity was Mtb s thiols and avins. OPB sensitizes Mtb to killing by two im- fi portant sets of factors: the same host chemistries that make Mtb con rmed by LC-MS, IR spectroscopy, and NMR spectroscopy. PB was from Sigma. MSH was a kind gift of R. Fahey and G. Newton (University of phenotypically resistant to many conventional anti-TB agents and California, San Diego, CA). some of those same drugs. Addition of OPB to currently avail- fi able treatments may bene t patients with drug-resistant TB Analysis of Metabolites. Mtb was incubated for 24 h under NR conditions with while we wait for the reemergent TB drug pipeline to deliver or without OPB or 4-OH-OPB, centrifuged, washed twice with PBS-tyloxapol, affordable new agents. resuspended in 500 μL of ice-cold methanol/acetonitrile/water (40:40:20

Gold et al. PNAS | October 2, 2012 | vol. 109 | no. 40 | 16009 Downloaded by guest on October 2, 2021 ratio), lysed by mechanical homogenization (3 × 30 s) with cooling on ice, concentrations were analyzed by LC/MS/MS. Serum concentrations of OPB filter-sterilized using 0.2-μ filters, and frozen at −80 °C until analysis. Vola- following i.p. injection were determined in-house. To each sample (100 μL) tiles were removed with a Speedvac (Labconco; 30 min, room temperature). was added 20 μL of citrate/phosphate buffer (0.1 M, pH 3) and 600 μLof Samples were then loaded on a Dionex 3000 nano-LC system coupled with chloroform. Mixed samples were centrifuged at 1,800 × g for 20 min at 4 °C. a Thermo Scientific LQ-Orbitrap XL Mass Spectrometer providing mass The recovered chloroform layer was evaporated, and the residue was dis- < measurement error 5 ppm. As standards, we reacted authentic MSH with solved in 20 μL of methanol and 2.5 μL of DMSO for HPLC analysis. 4-OH-OPB. The flavin adenine dinucleotide pool (oxidized plus reduced) was measured using a kit (BioVision). ACKNOWLEDGMENTS. We thank M. Larsen and W. R. Jacobs, Jr. (Albert Einstein College of Medicine) for the auxotrophic Mtb; T. Parker (Tuberculosis, NMR Spectroscopy. 1Hand13C NMR spectra were recorded at 11.7 T on Leprosy, and Other Mycobacterial Diseases Section, Respiratory Diseases a Bruker 500-MHz Avance III spectrometer equipped with a broad-band Branch, Division of Microbiology and Infectious Diseases, National Institute gradient probe. Spectra were externally referenced to tetramethylsilane of Allergy and Infectious Diseases, National Institutes of Health) for access (TMS) in appropriate solvent for 1D spectra. For 2D spectra, 1H and 13C to Resources for Researchers services at SRI International and Johns Hopkins University; Kathy N. Williams and Opokua Amoabeng (Johns Hopkins Uni- frequencies were referenced to the internal DMSO solvent signal calibrated versity) for assistance with the mouse studies; R. Fahey and G. Newton to TMS (50). Kinetic experiments were performed at 308 K. Spectra were (University of California, San Diego) for a MSH standard; D. J. Rouse (RTA acquired using 0.5-mM samples of OPB or 4OH-OPB in PBS prepared in D2O International) and The Global Alliance for Tuberculosis Drug Development at pH 5.0. Samples were warmed before initiation of kinetics. The time for PA-824; H. N. Sultan (The Rockefeller University HTS Resource Center) course was initiated on addition of an aliquot of 1 M NaNO2 in D2O to give for help with HTS; K. Rhee, L. P. S. de Carvalho, and S. Chakraborty for fi a final NaNO2 concentration 10-fold higher than the sample concentration. the metabolomic study on lter-grown cultures; G. Sukenick (NMR The 1D 1H spectra were collected at intervals for up to 18 h. Natural abun- Analytical Core) and G. Yang (Organic Synthesis Core, Memorial Sloan dance 13C chemical shifts were obtained at 298 K using 2D 13C-HSQC (51) and Kettering Cancer Center) for help with analysis and synthesis of OPB analogs; S. Ekins (Collaborative Drug Discovery) for cheminformatics work 2D 13C-HMBC (52) spectra. Natural abundance data were collected at sample not included here; Alfonso Mendoza-Losana (GlaxoSmithKline) for an concentrations from 25 to 100 mM using 25%, 58%, and 75% d6-DMSO experiment not included; J. Liu (Johns Hopkins University) for sharing solutions mixed with PBS prepared in D2O at pH 5.0. Spectra were processed, a collection of pharmaceutical agents; R. Elliott and K. Duncan (Bill and analyzed, and plotted using the NMR processing software MestReNova 5.3 Melinda Gates Foundation) and M. Reidenberg, S. Ehrt, E. Talley, G. Lin, (Mestrelab Research). Signal intensities were integrated for kinetic analysis R. Bryk, and L. P. S. de Carvalho (Weill Cornell Medical College) for of decay and growth rates. Curves were fitted and plotted to single expo- advice;H.A.Brown,K.Parikh,J.Pale,andS.Muffly (Weill Cornell nential models using Grace 5.1 (GNU general public license) software. Medical College) and L. Solla (Cornell University) for literature review; and F. Barany (Weill Cornell Medical College) for access to BSL2 facilities and robotics. This work was supported by the Tuberculosis Drug Acceler- Pharmacokinetics. Uptake of gavaged OPB was determined by Quest Phar- ator Program of the Bill and Melinda Gates Foundation and by the Abby maceutical Sciences under an institutional animal care and use committee- and Howard P. Milstein Program in Chemical Biology of Infectious Disease. approved protocol. OPB was dissolved in 5% DMSO/95% of 1% methylcellulose The Department of Microbiology and Immunology (Weill Cornell Medical in water and administered to adult female C57BL/6 mice. Plasma OPB College) is supported by the William Randolph Hearst Foundation.

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