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4.3. Determination of growth inhibition (GI) percentage via
MABA
References and notes
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A primary screen was conducted at 6.25 lg/mL against M.
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the Microplate Alamar Blue Assay (MABA).27 Compounds exhibit-
ing fluorescence were tested in the BACTEC 460-radiometric sys-
tem. Compounds effecting <90% inhibition in the primary screen
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(MIC >6.25 lg/mL) were not further evaluated.
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4.4. Determination of minimum inhibitory concentration (MIC)
via MABA
10. Goldman, R. C.; Laughon, B. E.; Reynolds, R. C.; Secrist, J. A., III; Maddry, J. A.;
Guie, M. A.; Poffenberger, A. C.; Kwong, C. A.; Ananthan, S. Infect. Disord. Drug
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Compounds demonstrating at least 90% inhibition in the pri-
mary screen were re-tested against M. tuberculosis H37Rv at lower
concentrations in order to determine the actual minimum inhibi-
tory concentration (MIC) in the MABA. The MIC was defined as
the lowest concentration effecting a reduction in fluorescence of
90% relative to controls. Rifampicin (RIF) was used as the reference
compound (RIF MIC = 0.015–0.125 lg/mL).
14. Ortega, M. A.; Montoya, M. E.; Jaso, A.; Zarranz, B.; Tirapu, I.; Aldana, I.; Monge,
A. Pharmazie 2001, 56, 205.
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A. Arzneim.-Forsch. 2002, 52, 113.
4.5. Determination of 50% inhibitory concentrations (IC50) in
VERO cells
16. Zarranz, B.; Jaso, A.; Aldana, I.; Monge, A. Bioorg. Med. Chem. 2003, 11, 2149.
17. Jaso, A.; Zarranz, B.; Aldana, I.; Monge, A. Eur. J. Med. Chem. 2003, 38, 791.
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Lenaerts, A. J.; Franzblau, S. G.; Cho, S.-H.; Monge, A.; Goldman, R. C. J.
Antimicrob. Chemother. 2008, 62, 547.
Concurrent with the determination of MIC’s, compounds were
tested for cytotoxicity (IC50) in VERO cells at concentrations less
than or equal to 62.5 lg/mL or 10 times the MIC for M. tuberculosis
20. Vicente, E.; Villar, R.; Burguete, A.; Solano, B.; Perez-Silanes, S.; Aldana, I.;
Maddry, J. A.; Lenaerts, A. J.; Franzblau, S. G.; Cho, S.-H.; Monge, A.; Goldman, R.
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Heterocyclic Compounds: A Series of Monographs; Wiley: New York, 1979; vol.
.
H37Rv. After 72 h of exposure, viability was assessed on the basis of
cellular conversion of MTT into a formazan product using the Pro-
mega CellTiter 96 Non-radioactive Cell Proliferation Assay. The
Selectivity Index (SI = IC50/MIC) was also determined; it was con-
sidered significant when SI >10 (RIF IC50 >100 lg/mL, SI >800).
23. Ortega, M. A.; Morancho, M. J.; Martinez-Crespo, F. J.; Sainz, Y.; Montoya, M. E.;
de Cerain, A. L.; Monge, A. Eur. J. Med. Chem. 2000, 35, 21.
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Acknowledgments
Antimycobacterial data were provided by the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility (TAACF)
through a research and development contract with the U.S. Na-
tional Institute of Allergy and Infectious Diseases (NIAID). E. V. is
indebted to the Ministerio de Educación y Ciencia (Spain) for the
Grant AP2003-2175.
27. Collins, L. A.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.