Identi fi cation of Novel Inhibitors of M . tuberculosis Growth Using Whole Cell Based High-Throughput Screening

Despite the urgent need for new antitubercular drugs, few are on the horizon. To combat the problem of emerging drug resistance, structurally unique chemical entities that inhibit new targets will be required. Here we describe our investigations using whole cell screening of a diverse collection of small molecules as a methodology for identifying novel inhibitors that target new pathways for Mycobacterium tuberculosis drug discovery. We find that conducting primary screens using model mycobacterial species may limit the potential for identifying new inhibitors with efficacy against M. tuberculosis. In addition, we confirm the importance of developing in vitro assay conditions that are reflective of in vivo biology for maximizing the proportion of hits from whole cell screening that are likely to have activity in vivo. Finally, we describe the identification and characterization of two novel inhibitors that target steps in M. tuberculosis cell wall biosynthesis. The first is a novel benzimidazole that targets mycobacterial membrane protein large 3 (MmpL3), a proposed transporter for cell wall mycolic acids. The second is a nitrotriazole that inhibits decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1), an epimerase required for cell wall biosynthesis. These proteins are both among the small number of new targets that have been identified by forward chemical genetics using resistance generation coupled with genome sequencing. This suggests that methodologies currently employed for screening and target identification may lead to a bias in target discovery and that alternative methods should be explored. T alarming rise in resistance to drugs currently used to treat tuberculosis has spurred a significant increase in efforts geared toward identifying new drugs effective against Mycobacterium tuberculosis. Currently there are only six new compounds in the clinical development pipeline that were specifically developed for treating tuberculosis. Of these potential drugs, only two function by inhibiting a completely novel target. Two of the compounds are inhibitors of ribosomal function, and two have complex mechanisms with multiple targets. In order to meet future therapeutic needs, the discovery of new chemotypes and the chemical and biological validation of new targets are required. Although several promising inhibitors have been identified that encompass both novel chemical scaffolds and novel targets, more work is needed to ensure that a sufficient number of new drugs will be available to combat the continual emergence of drug resistance. There is as yet no clear consensus on the best methods for identifying novel inhibitors of M. tuberculosis that will be effective in vivo. Approaches range from target-based discovery to whole cell screening efforts against M. tuberculosis or related mycobacteria. Target-based approaches rely on knowledge of protein essentiality and function, either through direct studies in mycobacterial species or through homology to proteins in other species. One challenge with target-based approaches is that knowledge of protein essentiality is not sufficient to guarantee “druggability”. In addition, compounds identified in target-based screens often lack activity against whole cells. Whole cell screening approaches circumvent these issues but also have inherent challenges. Screening directly against M. tuberculosis is extremely challenging as a BSL3 level containment facility equipped with high-throughput screening equipment is required, and procedures required for handling this organism are laborious. Many groups have therefore relied upon the use of model mycobacterial species such as Mycobacterium smegmatis. However, it is unclear to what degree these organisms can be relied upon as adequate surrogates for Received: April 1, 2012 Accepted: May 11, 2012 Articles pubs.acs. org/acschemicalbiology © XXXX American Chemical Society A dx.doi. org/10.1021/cb300151m | ACS Chem. Biol. XXXX, XXX, XXX−XXX M. tuberculosis. In addition, despite the fact that it is becoming clear that the conditions used for culturing M. tuberculosis during whole cell screening are critical to identifying inhibitors that will be effective in vivo, there is no clear consensus on the conditions that best reflect in vivo biology. Finally, the elucidation of the target of a promising small molecule identified in a whole cell screen can be extremely challenging. Herein we report our experience in whole cell screening using a library of ∼20,000 diverse small molecules from the Broad Institute collection against M. tuberculosis and the related mycobacterial species M. smegmatis and Mycobacterium bovis BCG. We found a surprisingly small overlap in the small molecules that are effective against these three species, highlighting the importance for directly screening against M. tuberculosis. In addition, we describe the characterization of 91 hits derived from this screen. We also describe testing of 1113 possible leads obtained from a 341,808 compound screen conducted by the NIAID and Southern Research Institute. From these combined screening efforts we identified novel inhibitors that target decaprenylphosphoryl-β-D-ribose 2′epimerase (DprE1), an enzyme required for cell wall biosynthesis, and mycobacterial membrane protein large 3 (MmpL3), a proposed exporter of newly synthesized cell wall mycolic acids. Interestingly, these two proteins now appear to be the targets of several chemically unrelated small molecules. Notably, these targets were all identified using a similar method of resistance generation and whole genome sequencing of mutants. This repeated identification of the same targets by the same methodology, albeit using multiple structurally unrelated inhibitors, may imply either that these targets are highly “druggable” and/or the prioritization of molecules whose targets are identified by resistance generation leads to a bias in identified targets. ■ RESULTS AND DISCUSSION Screening To Identify M. tuberculosis Growth Inhibitors. To identify compounds with activity against M. tuberculosis strain H37Rv, we employed a high-throughput screen using constitutive GFP fluorescence as a reporter of bacterial number to screen a total of 20,000 compounds selected from the larger screening collection of the Broad Institute for maximal chemical diversity and consisting of commercially available compounds, natural products, compounds generated by Diversity Oriented Synthesis, and other synthetic compounds. The complete data from the screen has been deposited in the publicly accessible database Chembank (http://chembank.broadinstitute.org/). Cells were seeded into 384-well plates at a low density and were incubated in the presence of ∼25 μM small molecules for a total of 3 days at which time GFP fluorescence was used as a readout of bacterial growth (Z′-factor = 0.8 for rifampicin and Z′-factor = 0.7 for clofazimine, coefficient of variation = 0.05). Dose responses of the known antitubercular drugs clofazimine and rifampicin were used as positive controls for selection of a z-score cutoff to define hits from the screen (see Methods). Although the absolute magnitude of the z-scores obtained from day to day using this assay varied, the relative performance of a given compound from day to day was highly reproducible. Independent screening of the same set of 4608 compounds on different days showed extremely highly correlated z-scores with R = 0.95 (Figure 1a). Using a z-score cutoff of −4, we identified 91 hits that were cherry-picked for further analysis. The 91 hits obtained from the primary screen were cherry picked and retested in a dose−response assay. Seventy-nine of the 91 compounds (87%) retested at concentrations at or below the screening concentration (Supplementary Tables S1 and S2). Forty-five compounds retested with an IC90 of 10 μM or less, and 6 compounds retested with an IC90 of less than 1 μM. The active hits were subjected to a number of secondary screens to assess mammalian cell toxicity and activity against M. tuberculosis in infected macrophages (Supplementary Tables S1 and S2). The majority of the hit compounds are unique structures that have not been reported to have antimicrobial activity. We thus sought to further characterize these small Figure 1. Screening validation and cross-species comparison of hits identified in primary screens against M. tuberculosis, M. smegmatis, and M. bovis BCG. (a) Reproducibility of a GFP reporter based screen for identifying inhibitors of M. tuberculosis replication. To determine the reproducibility of the GFP-based assay, the same set of 4308 unique compounds was screened against M. tuberculosis on two different days. A high degree of correlation is observed for z-scores obtained using the same assay against M. tuberculosis on different screening days. (b) Correlation of hits identified in primary screens against M. tuberculosis and M. smegmatis. 11,147 unique compounds were screened against M. tuberculosis using the GFP assay and M. smegmatis using an OD600 based assay. Plotted are composite z-scores calculated for each compound in both the M. tuberculosis (x-axis) and M. smegmatis (yaxis) screens. (c) Correlation of hits identified in primary screens against M. tuberculosis and M. bovis BCG. 13,715 unique compounds were screened against M. tuberculosis and M. bovis BCG using GFPbased assays. Plotted are composite z-scores calculated for each compound in both the M. tuberculosis (x-axis) and M. bovis BCG (yaxis) screens. All screens were conducted using duplicate wells for each compound, and composite z-scores for the duplicates are plotted. R values were calculated using the two-tailed Pearson product moment correlation test. ACS Chemical Biology Articles dx.doi. org/10.1021/cb300151m | ACS Chem. Biol. XXXX, XXX, XXX−XXX B molecules to assess their potential for development as tool compounds for the identification and validation of novel targets for drug development. Cross Speci