Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA
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Abstract |
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Introduction |
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More recently, several of the deazapteridine derivatives were submitted to the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF; NIH, NIAID Contract AI45246) for screening against M. tuberculosis; some of them demonstrated activity against M. tuberculosis strain H37Rv. We later confirmed the antimicrobial activity of this class of compounds and report here on the activity of 1-deaza-7,8-dihydropteridines against both M. tuberculosis and M. avium in vitro. Also, we present data which suggest that these compounds do not inhibit mycobacterial dihydrofolate reductase. This class of compounds does not appear to inhibit the polymerization of mycobacterial FtsZ, the bacterial homologue of tubulin.7
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Materials and methods |
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The methods for the synthesis of 1-deaza-7,8-dihydropteridines have been reported elsewhere.3 A list of the compounds and their structures is presented in Tables I and II.
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The inhibitory activity of the test compounds against M. tuberculosis H37Ra (ATCC 25177; American Type Culture Collection, Rockville, MD, USA) and M. avium clinical isolates (serovar 1, 4 or 6; National Jewish Center for Immunology and Respiratory Diseases, Denver, CO, USA) was determined with a colorimetric microdilution broth assay described elsewhere.8 The compounds were dissolved in dimethylsulphoxide (DMSO), then diluted serially in assay medium with 10-fold dilutions. The greatest concentration of DMSO in the assay medium was 1.3%. This concentration did not affect the growth of the test organisms. The antimycobacterial drug ethambutol (Sigma, St Louis, MO, USA) was used as a positive control.
Dihydrofolate reductase inhibition
The inhibition of M. avium dihydrofolate reductase was determinined using purified recombinant enzyme as described elsewhere.8 Enzyme activity was measured by following the decrease in absorbance at 340 nm. Dihydrofolate (0.1 mM) was used to initiate the reaction after a 3 min preincubation of a mixture containing 10 mM 2- mercaptoethanol, 0.1 mM NADPH, 1 mM EDTA, 55 mM potassium phosphate (pH 7), enzyme and inhibitor as appropriate. The total assay volume was 1 mL and the temperature 30°C.
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Results |
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Overall, 17 of the 25 compounds tested were active (MIC 12.8 mg/L) against one or more of the bacterial strains. Growth inhibition by the positive control agent, ethambutol, for M. tuberculosis H37Ra was in the range >0.128 to
12.8 mg/L and that for the M. avium strains was in the range >1.28 to
12.8 mg/L (data not shown). Although most of the compounds tested contained an alkoxycarbonyl group (R1, Table I
) attached to the 2-amino nitrogen, this substitution was not required for activity. This is apparent by comparing compound 6 with compounds 3 and 4. Also, compounds with an acetoxycarbonyl substitution on the 2-N position and a phenyl substitution at position 6 (Table I
, compounds 1, 2, 3, 4, 5 and 11) were active against one or more of the strains only when a methyl or ethyl group was present on the 7 or 8 position (Table I
, R3 and R2, respectively). Interestingly, only the S-() enantiomer of the methyl substitution at position 6 was active (compare compound 4 with compound 5). In contrast to the observation that no substitutions on the 6-phenyl ring were required for activity as long as the heterocyclic ring contained an alkyl substitution at the 7- or 8-position, methyl-, methoxy-, chloro- and trifluoromethyl-6-phenyl substituted derivatives were active against one or more strains without an alkyl group at these positions (Table II
, compounds 12, 13, 18, 19, 20, 21 and 23). An amino- or fluoro-6-phenyl substitution, however, was inactive (compounds 22 and 24)
Inhibition of dihydrofolate reductase
We assayed compounds 2 and 6 for their ability to inhibit recombinant M. avium dihydrofolate reductase activity. No inhibition was seen with compound 2 or 6 at 32.5 and 40.4 µM, the highest concentrations assayed, respectively. Active pteridine derivatives, which have been assayed by us under the same conditions, inhibit enzyme activity by 50% at nanomolar concentrations.8
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Discussion |
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Certain 1-deaza-7,8-dihydropteridines cause mitotic arrest in cancer cells in vitro.5 Also, two of the compounds used for the present study, compounds 1 and 3, inhibit competitively the binding of colchicine to tubulin and the polymerization of tubulin.6 Recently FtsZ, a bacterial protein essential for cell division, has been proposed to be the tubulin homologue in eubacteria, and the solution of the crystal structure of recombinant FtsZ from Methanococcus jannaschii has revealed its strong similarity to that of tubulin.7 Since 1-deazadihydropteridines interact with tubulin, their mechanism of action in mycobacteria may involve a similar interaction with FtsZ. Recombinant FtsZ from M. tuberculosis was expressed recently in Escherichia coli and purified.10 Several 1-deaza-7,8-dihydropteridines that were active against M. tuberculosis H37Rv (TAACF, NIH, NIAID contract) did not inhibit M. tuberculosis FtsZ polymerization (E. Lucile White, personal communication). These results support the conclusion that these compounds did not interfere with the function of the FtsZ protein in mycobacteria.
In summary, our results demonstrate that 1-deaza-7,8-dihydropteridine derivatives inhibit the growth of M. tuberculosis and M. avium, and therefore could be useful as antimycobacterial drugs. Studies of the mechanism of action of these compounds indicate that they do not inhibit mycobacterial dihydrofolate reductase and do not interfere with the essential function of FtsZ in cell division. Further investigations are needed to determine how these compounds inhibit the growth of mycobacteria.
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Acknowledgments |
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Notes |
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References |
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2 . Snider, D. E. & La Montagne, J. R. (1994). The neglected global tuberculosis problem: a report of the 1992 World Congress on Tuberculosis. Journal of Infectious Diseases 169, 118996.[ISI][Medline]
3 . Temple, C., Wheeler, G. P., Elliott, R. D., Rose, J. D., Comber, R. N. & Montgomery, J. A. (1983). 1,2-Dihydropyrido[3,4-b]pyrazines: structureactivity relationships. Journal of Medicinal Chemistry 26, 915.[ISI][Medline]
4 . Wheeler, G. P., Bowdon, B. J., Werline, J. A. & Temple, C. (1981). 1-Deaza-7,8-dihydropteridines, a new class of mitotic inhibitors with anticancer activity. Biochemical Pharmacology 30, 23814.[ISI][Medline]
5 . Wheeler, G. P., Bowdon, B. J., Werline, J. A., Adamson, D. J. & Temple, C. G. (1982). Inhibition of mitosis and anticancer activity against experimental neoplasms by ethyl 5-amino-1,2-dihydro- 3-[(N-methylanilino)methyl]-pyrido[3,4-b]pyrazin-7-yl carbamate (NSC 181928). Cancer Research 42, 7918.[Abstract]
6 . Bowdon, B. J., Waud, W. R., Wheeler, G. P., Hain, R., Dansby, L. & Temple, C. (1987). Comparison of 1,2-dihydropyrido[3,4-b]pyrazines (1-deaza-7,8-dihydropteridines) with several other inhibitors of mitosis. Cancer Research 47, 16216.[Abstract]
7 . Lowe, J. & Amos, L. A. (1998). Crystal structure of the bacterial cell-division protein FtsZ. Nature 391, 2036.[ISI][Medline]
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Suling, W. J., Seitz, L. E., Pathak, V., Westbrook, L., Barrow, E. W., Zywno-Van Ginkel, S. et al. (2000). Antimycobacterial activities of 2.4-diamino-5-deazapteridine derivatives and effects on mycobacterial dihydrofolate reductase. Antimicrobial Agents and Chemotherapy 44, 278493.
9 . Williams, J. W., Duggleby, R. G., Cutler, R. & Morrison, J. F. (1980). The inhibition of dihydrofolate reductase by folate analogues: structural requirements for slow- and tight-binding inhibition. Biochemical Pharmacology 29, 58995.[ISI][Medline]
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White, E. L., Ross, L. J., Reynolds, R. C., Seitz, L. E., Moore,G. D. & Borhani, D. W. (2000). Slow polymerization of Mycobacterium tuberculosis FtsZ. Journal of Bacteriology 182, 402834.
Received 1 August 2000; returned 18 October 2000; revised 22 November 2000; accepted 1 December 2000