Nonsteroidal Anti-Inflammatory Drug Sensitizes Mycobacterium
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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.