1 Institut Pasteur de Bruxelles, 642, rue Engeland, B-1180, Brussels, Belgium; 2 University of Maryland Biotechnology Institute, College Park, MD 20742, USA; 3 Technische Universität Dresden, Institut für Organische Chemie, Bergstraße 66, D-01069, Dresden, Germany
Received 22 April 2004; returned 9 June 2004; revised 3 July 2004; accepted 19 July 2004
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Abstract |
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Methods: Bioluminescent strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis, expressing the luxA and luxB genes from Vibrio harveyi were used for the comparison of the antimycobacterial activity of the two synthetic macrodiolides pamamycin-607 and pamamycin-621A and a non-naturally occurring cyclic dimer of pamamycin-607, i.e. yukomycin.
Results: Pamamycin-607 was the most active of the three macrocycles and was more active against M. tuberculosis than against M. smegmatis. Twenty-five clinical isolates of M. tuberculosis were susceptible to pamamycin-607 in a narrow MIC range of 1.52.0 mg/L. The new assay was also validated by comparison with the BACTEC radiometric test.
Conclusion: Rapid screening of a new class of macrocyclic antimycobacterials using bioluminescent mycobacteria identified pamamycin-607 as a potential antituberculous agent. The latter was active against clinical isolates of M. tuberculosis within a narrow MIC range of 1.52.0 mg/L irrespective of their resistance to isoniazid or rifampicin. Our findings warrant further investigations.
Keywords: bioluminescence , luciferase , Mycobacterium tuberculosis , macrodiolides
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Introduction |
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Preliminary but encouraging results were obtained on the antimycobacterial activity of pamamycin-607 and a mixture of two pamamycin-649 homologues purified from S. alboniger,4 but since these substances were difficult to produce and to purify this work was temporarily interrupted. Recently, the enantioselective chemical synthesis of pamamycin-6076,7 and pamamycin-621A (P. Fischer and P. Metz, unpublished work) was accomplished, through which sufficient amounts of these two agents can be secured in high purity for early biological evaluations.
In this study, we screened the antimycobacterial activity of the two macrodiolidespamamycin-607 and pamamycin-621Aand a non-naturally occurring cyclic dimer of pamamycin-607 (termed yukomycin), against bioluminescent Mycobacterium bovis BCG. We also compared the activity of the most active compound (pamamycin-607) against bioluminescent Mycobacterium tuberculosis and Mycobacterium smegmatis, as well as several clinical isolates of M. tuberculosis.
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Materials and methods |
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The chemical structures of pamamycin-607,5 pamamycin-621A8 and yukomycin are illustrated in Figure 1(a). The two macrodiolides only differ in the substitution at C(7') of the macrocycle.
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Bacterial strains and growth conditions
M. smegmatis (ATCC 607), M. bovis BCG (GL2) and M. tuberculosis H37Rv were grown in Middlebrook 7H9 medium (Difco) supplemented with 10% Middlebrook ADC (Difco) and 0.05% Tween 80. Time course experiments were carried out in 50 mL in 75 cm2 tissue culture flasks at 37°C and were inoculated by diluting log phase cultures prepared from 80°C glycerol stocks.
Bioluminescence detection and assay
Cultures of M. smegmatis, M. bovis BCG and M. tuberculosis transformed with pSMT9 were found to have concentrations of 4 x 109, 2 x 103 and 2 x 105 relative light units (RLU)/mL, respectively. We observed an equivalence of 1 RLU for 10 cfu of mycobacteria. To evaluate the activity of the two macrodiolides and yukomycin against M. bovis BCG, luciferase microdilution plate susceptibility assays were performed in triplicate by incubating 5 x 105 bacilli with different dilutions of the antimicrobial agents. Each well contained a total volume of 0.2 mL of Middlebrook 7H9 broth supplemented with OADC (Middlebrook OADC Enrichment; Becton Dickinson Microbiology Systems). To detect bacterial luciferase activity, 0.2 mL of mycobacterial suspension culture was added to 0.7 mL of phosphate-buffered saline and 0.1 mL of 1% n-decyl aldehyde (Sigma) in ethanol. Luminescence was measured in a Luminometer TD 20/20 Turner Designs (Promega) at day 2 or 3. Raw data were collected over 15 s. Results are expressed in RLU.
To determine the potential bacteriostatic or bactericidal activity of pamamycin-607 against M. smegmatis, M. bovis BCG and M. tuberculosis, assays were performed by incubating 104 bacilli with the antimicrobial agent dilutions. The bacterial luciferase activity was measured over 4 days. In each experiment, the baseline luciferase activity was determined immediately after inoculation (time zero). Bactericidal activity is defined as a reduction of minimum 2 log of the initial inoculum within a time span of 2 days.
MIC determination for clinical isolates
Since it would have been difficult to transform the clinical isolates of M. tuberculosis with the luciferase plasmid, we used the BACTEC method10 to evaluate the susceptibility of 25 clinical isolates of M. tuberculosis to pamamycin-607 in a concentration range of 0.110.0 mg/L (0.10.21.01.52.05.010.0 mg/L). Among these isolates, 14 were susceptible to both isoniazid and rifampicin, seven were isoniazid resistant and four were multidrug-resistant. For comparison, susceptibility of the bioluminescent M. tuberculosis H37Rv and M. bovis BCG strains was also determined by the radiometric method, according to the specifications proposed by Becton Dickinson (inoculum: a positive BACTEC culture with a growth index 500).
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Results |
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The two pamamycins and yukomycin were assayed for their effect on the growth of luminescent M. bovis BCG by measuring the luciferase activity by luminometry, as described above. We determined the antimycobacterial activity of the three agents by comparing their inhibitory effect on the luminescence of an exponentially growing culture. Figure 1(b) represents the doseresponse curves of the three compounds after 2 days of incubation. The MIC, which is represented by the intercept of the curve with the background activity at time zero or cut-off level (990, 3900 and 2700 mRLU for pamamycin-607, pamamycin-621A and yukomycin, respectively), was significantly lower for pamamycin-607 than for pamamycin-621A and yukomycin. MICs determined by this luciferase assay were as follows: pamamycin-607, 0.9 mg/L; pamamycin-621A, 3.7 mg/L; and yukomycin, 3.9 mg/L.
Activity of pamamycin-607 against bioluminescent mycobacteria
Pamamycin-607, being the most active compound, was further used to investigate the susceptibility of three species of mycobacteria (M. bovis BCG, M. tuberculosis and M. smegmatis). Pamamycin-607 showed equivalent inhibitory activity against M. bovis BCG and M. tuberculosis (H37Rv), whereas it was 510 times less active against M. smegmatis (Figure 2a). MICs were revealed to be: 4.7 mg/L for M. smegmatis, 0.9 mg/L for M. bovis BCG and 0.55 mg/L for M. tuberculosis (H37Rv).
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Clinical isolates
Using the BACTEC radiometric susceptibility method, we determined the pamamycin-607 MIC for 25 clinical isolates of M. tuberculosis. The MIC of pamamycin-607 was 1.5 mg/L for 14 isolates and 2.0 mg/L for the 11 remaining isolates. It is noteworthy that all the clinical isolates shared a very narrow response range, clearly independent of their resistance to first-line antimycobacterials (isoniazid and rifampicin) (data not shown).
To understand the difference between this range and the sensitivity observed with the bioluminescence method we also tested each bioluminescent strain in the radiometric assay. With the latter, the MICs for M. tuberculosis H37Rv and M. bovis BCG were 1.0 and 1.01.5 mg/L, respectively, as compared with 0.55 and 0.9 mg/L in the bioluminescence assay.
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Discussion |
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On the other hand, we observed that all the 25 clinical isolates of M. tuberculosis were less susceptible to pamamycin-607 with the radiometric assay than with the bioluminescence test. However, our comparative results indicate that this difference can be explained by both technical factors and differences in susceptibility between laboratory strains and clinical isolates.
Pamamycins may be very active antimycobacterials belonging to a totally new class of drugs. Therefore, their primary mode of action is worth studying. Indeed, in a preliminary screening we did not observe any cross-resistance with the two major first-line antimycobacterials, isoniazid and rifampicin. This topic certainly warrants further investigation, especially on multi-resistant strains. Furthermore, the lead molecule pamamycin-607 might be adapted or refined in order to improve its pharmacokinetics and reduce its potential toxicity. Eventually, the assays described here might emerge as useful tools to screen and investigate novel more potent pamamycin derivatives or other unrelated compounds.
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Acknowledgements |
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Footnotes |
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* Present address. INRA Tours, F-37380, Nouzilly, France
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References |
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2 . Natsume, M. (1999). Pamamycins, unique macrodiolides with aerial mycelium-differentiation-inducing activity in Streptomyces alboniger. Recent Research Developments in Agricultural & Biological Chemistry 3, 1122.
3 . Chou, W. G. & Pogell, B. M. (1981). Mode of action of pamamycin in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 20, 44354.[ISI][Medline]
4 . Braibant, M., Fauville, M., Pogell, B. et al. (1996). Two pamamycin derivatives inhibit phosphate uptake in M. bovis BCG and growth of M. tuberculosis. In Programs and Abstracts of the Third International Conference on the Pathogenesis of Mycobacterial Infections, Stockholm, Sweden, 1996. p. 52. Swedish Institute for Infectious Disease Control, Stockholm, Sweden.
5 . Natsume, M., Kondo, S. & Marumo, S. (1989). The absolute stereochemistry of pamamycin-607, an aerial mycelium-inducing substance of Streptomyces alboniger. Journal of the Chemical Society, Chemical Communications 24, 19113.[CrossRef]
6 . Wang, Y., Bernsmann, H., Gruner, M. et al. (2001). Total synthesis of pamamycin-607. Tetrahedron Letters 42, 78014.[CrossRef][ISI]
7 . Metz, P. (2004). Synthetic studies on the pamamycin macrodiolides. Topics in Current Chemistry 244, in press.
8 . Gräfe, U., Schlegel, R., Dornberger, K. et al. (1993). Isolation and structural elucidation of pamamycin-621, a new macrodiolide antibiotic from Streptomyces aurantiacus. Natural Products Letters 3, 26571.
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.
Snewin, V. A., Gares, M. P., Gaora, P. O. et al. (1999). Assessment of immunity to mycobacterial infection with luciferase reporter constructs. Infection and Immunity 67, 458693.
10 . Siddiqi, S. H., Libonati, J. P. & Middlebrook, G. (1981). Evaluation of rapid radiometric method for drug susceptibility testing of Mycobacterium tuberculosis. Journal of Clinical Microbiology 13, 90812.[ISI][Medline]
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