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In this study, application of a dual absorbance/fluorescence assay to a chemical library screen identified several previously unknown inhibitors of mycobacteria. In addition, growth conditions had a significant effect on the activity profile of the library. Some inhibitors such as Se-methylselenocysteine were detected only when screening was performed under nutrient-limited culture conditions as opposed to nutrient-rich culture conditions. We propose that multiple culture condition library screening is required for complete inhibitory profiling and for maximal antimycobacterial compound detection.
Recent data from the World Health Organization show that there are more than 9 million new cases of tuberculosis (TB) each year, half a million of which are caused by drug-resistant Mycobacterium tuberculosis (26, 27, 28). The spread of antibiotic resistance has necessitated the identification of new anti-infective molecules for the treatment of TB (25). TB drug discovery research is often dependent on the robustness of the upstream biological assay used. In terms of whole-cell antimycobacterial assays, screens are commonly performed under optimal growth conditions such as in the presence of excess nutrients. Evidence suggests, however, that M. tuberculosis persists in a nutrient-deprived state in the host lung (4, 12-14, 21). Screening under nutrient-rich conditions may fail to detect compounds that are preferentially active against nutrient-limited M. tuberculosis during infection.
Traditionally, antimycobacterial assays use optical density (OD) as an indicator of growth, which can be distorted by the intrinsic absorbance of some compounds and the propensity of mycobacterial cells to form aggregates. Alternatives to OD measurement include the use of reporter molecules, such as the green fluorescent protein (GFP). Collins et al. (10) demonstrated that when expressed in M. tuberculosis, the levels of GFP paralleled the numbers of CFU during growth. The MICs of a range of antitubercular drugs determined using GFP were consistent with those obtained using Alamar Blue (7) and the BACTEC 460 system (9). In this work, we used both OD and GFP fluorescence to screen the library of pharmacologically active compounds (LOPAC) (LO1280; Sigma-Aldrich, St. Louis, MO) for antimycobacterial compounds.
For expression of GFP, a vector was constructed by PCR amplification of the gfpmut2 gene (11) from pOT11 (19) using primers GFP_RBS_F1 (5′-GGGGGTACCTTTAAGAAGATATACATATGAGTAAAGGAGAA-3′) and GFP_R1 (5′-GGGGGCATGCTTATTATTTGTATAGTTCATCCATGCC-3′). The product was cloned into the KpnI and SphI restriction sites of pTKmx (16), generating plasmid pTKmxGFP. The pAL5000 origin of replication of pTKmxGFP was excised by NheI restriction digestion and replaced with the replicon of the high-copy-number plasmid pHIGH100 (5), amplified using PCR primers OriM_F (5′-GGGGGCTAGCAACGAGGACAGTCGCACGAC-3′) and OriM_R (5′-GGGGGCTAGCATCGAGCCGAGAACGTTATC-3′), generating plasmid pSHIGH. The hsp60 gene promoter from Mycobacterium bovis BCG, amplified using PCR primers Hsp60_F (5′-GGGGGGTACCGGTACCGGTGACCACAACGACGCGCCCGCT-3′) and Hsp60_R (5′-GGGGGGTACCCGCAATTGTCTTGGCCATTGCGAA-3′), was cloned into the KpnI site of pSHIGH, generating plasmid pSHIGH+hsp60. (Underlining indicates position of restriction site for each primer.)
Mycobacterium smegmatis mc2155 harboring plasmid pSHIGH+hsp60 was inoculated into Luria-Bertani broth (LB) containing 50 μg/ml kanamycin and supplemented with 0.1% (vol/vol) Tween 80 and 100 μg/ml d-arabinose to reduce cell aggregation as previously described (2, 20). A selection of first- and second-line antitubercular drugs and tetracycline were used to test the validity of the antimycobacterial assay. The cultures were grown to the mid-logarithmic phase and diluted to an OD at 600 nm of 0.2 (10-mm path length). Two hundred microliters of sterile deionized water was added to each well on the perimeter of the plate to minimize evaporation of the growth medium during the assay. Fifty microliters of LB containing 50 μg/ml kanamycin, 0.1% Tween 80, and 100 μg/ml d-arabinose were added to the remaining wells. Starting at 50 μM, twofold serial dilutions were performed for the experimental compounds and control antibiotics. Fifty microliters of the cell culture, corresponding to approximately 5 × 106 CFU per well, was added to each one of the inner wells, except the medium control wells. The plates were sealed, wrapped in parafilm, and incubated at 37°C for 96 h with 200 rpm shaking. OD and GFP fluorescence measurements were performed at 0- and 96-h incubation using a Wallac Envision multilabel plate reader (Perkin-Elmer). Data were analyzed with SigmaPlot 11 (SYSTAT) using four-parameter logistic standard curve analysis. The MIC and 50% inhibitory concentration (IC50) values were determined with respect to the controls at 96 h. For each of the drugs tested, use of OD and GFP measurements produced identical MICs and good correlation for the IC50s (Table (Table11 and Fig. Fig.11).
The LOPAC library was screened for inhibitory activity toward M. smegmatis grown under nutrient-rich conditions. Two microliters of a 1 mM concentration of each compound from the 16 LOPAC stock plates was transferred to the wells of columns 2 to 11 using a Cybi-Well robotic liquid handling station (Cybio) to obtain a final chemical concentration of 20 μM. Two hundred microliters of sterile-distilled water were added to each well in column 1 to minimize evaporation and medium, solvent, and antibiotic controls were established in column 12 of each plate. Solvent controls, consisting of 2% dimethyl sulfoxide, did not produce any significant inhibition of M. smegmatis growth. Antibiotic controls, consisting of rifampin (rifampicin) and ciprofloxacin at 20 μM, yielded complete growth inhibition.
The library screens were performed three times, and the resulting data were normalized to control for plate-to-plate variation by linear scaling to match the most extreme plates in each data set and taking their natural log transforms. Control values that were more than 3 standard deviations from the mean were considered outliers and discarded. Z-factors were calculated for each assay control to give an indication of assay reliability, taking into account both dynamic range and assay variability (29). Under standard growth conditions, the Z-factors for capreomycin were Z = 0.87 (OD) and Z = 0.88 (GFP) and the Z-factors for rifampin were Z = 0.68 (OD) and Z = 0.87 (GFP), interpreted as an excellent assay. To enable comparison with previous work (7), the signal-to-noise ratios for capreomycin were 23.21 (OD) and 24.20 (GFP) and the ratios for rifampin were 9.44 (OD) and 22.78 (GFP). Following validation of potential hits using MIC and IC50 determination, 14 compounds that inhibited M. smegmatis growth under nutrient-rich conditions at concentrations of 12.5 μM or below were identified (Table (Table2).2). A number of these compounds, e.g., calcimycin, demeclocycline, doxycycline, lomefloxacin, minocycline, ofloxacin, and vancomycin, are known antibacterials, and therefore, activity against mycobacteria was not unexpected. In addition, antimycobacterial activity has previously been recorded for clotrimazole and niclosamide by the Southern Research Institute in Alabama (http://pubchem.ncbi.nlm.nih.gov) and other researchers (6, 22). From our review of the literature, antimycobacterial activity has not been previously reported for the remaining compounds, calmidazolium, diphenyleneiodonium, idarubicin, and methoctramine.
In the next stage of this work, we tested whether the inhibitory profile of the LOPAC library toward mycobacteria varied significantly under different culture conditions. M. smegmatis was grown in carbon- and nitrogen-limited Hartman-de Bonts (HdeB) medium (18, 21) in place of LB. Many hit compounds detected in rich media were also active under conditions of carbon and nitrogen limitation (Table (Table2).2). Clotrimazole was more active under nutrient-rich conditions. However, the screens identified additional inhibitors that were not detected using nutrient-rich culture conditions. These include Bay 11-7085, 4-chloromercuribenzoic, cisplatin, and Se-methylselenocysteine, which exhibit significantly lower MIC values under nutrient limitation (Table (Table2).2). These findings indicate that antimycobacterial activity contained within chemical libraries is significantly affected by the growth conditions used for screening. Screening protocols need to be able to detect drugs that are active under nutrient limitation and other conditions that are considered relevant to the host environment.
Of the compounds detected in the library screens against mycobacteria, a number of compounds may have activities relevant to TB drug development. Se-methylselenocysteine (MeSeCys) is used in the dietary chemoprevention of tumors (1). It is metabolized by selenocysteine lyase (β-lyase) producing methylselenol, which causes apoptosis in cancer cells by redox cycling and protein thiol modification (1, 15, 23). Selenocysteine lyases are widely distributed among bacteria (8). Enzymes annotated at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) as selenocysteine lyases are encoded in the genomes of a number of mycobacterial species, including M. smegmatis (e.g., genes MSMEG_1242 and MSMEG_4538) and M. tuberculosis (e.g., genes Rv1464 and Rv3025c). Hence, it is plausible that mycobacteria could convert MeSeCys into toxic selenium products, such as methylselenol. Mycobacteria may potentially use MeSeCys as a source of selenium or other nutrients, which may account for the more pronounced inhibitory activity seen for MeSeCys in nutrient-deprived conditions (Fig. (Fig.22).
We have been unable to find reports of inhibitory activity of MeSeCys or any organic forms of selenium toward mycobacteria or their use in the treatment of TB. Micronutrient supplementation with inorganic selenium can improve health outcomes for patients with TB by improving the nutritional status of the host (3, 24); however, an antibiotic role for selenium-based compounds has not been explored previously. Therefore, further investigations on MeSeCys as a potential antitubercular compound are being conducted.
In conclusion, the use of different growth media exerts a significant effect on the identification of active compounds in a chemical library screen. Incorporation of host-related physicochemical conditions into whole-cell screens could potentially augment the detection of alternative compounds that are active against M. tuberculosis infection.
We gratefully acknowledge the support of the Health Research Council of New Zealand (grant 07/379), the Wellington Medical Research Foundation (grant 2006/121), and the University Research Fund of the Victoria University of Wellington (grant 26211/1496). C.H.M. was supported by a postgraduate scholarship from the Victoria University of Wellington.
We are grateful for the valuable advice provided by Paul Teesdale-Spittle and David Bellows.
Published ahead of print on 28 September 2009.