2-Alkoxycarbonylaminopyridines: inhibitors of Mycobacterium tuberculosis FtsZ

E. Lucile White*, William J. Suling, Larry J. Ross, Lainne E. Seitz and Robert C. Reynolds

Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA

Received 1 October 2001; returned 3 January 2002; revised 1 March 2002; accepted 2 April 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Compounds originally designed as putative tubulin inhibitors were tested as antitubercular agents for inhibition of the Mycobacterium tuberculosis analogue of tubulin, FtsZ. Initial screening of 200 2-alkoxycarbonylpyridines found several that inhibited M. tuberculosis growth. Two compounds, SRI-3072 and SRI-7614, inhibited FtsZ polymerization and were equipotent against susceptible and single-drug-resistant strains of M. tuberculosis. In addition, SRI-3072 reduced the growth of M. tuberculosis in mouse bone marrow macrophages. Our results suggest that these types of compound might be developed into antitubercular drugs effective against the current multidrug-resistant strains of M. tuberculosis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
FtsZ is the first non-regulatory element to appear at the septum site of bacteria, and the function of the septum has been shown to depend on correct FtsZ function.1 FtsZ is a cytosolic protein that polymerizes in a GTP-dependent manner and has been shown to be the bacterial tubulin homologue.2 The sequence similarity between FtsZ and tubulin, however, is generally low (<20% identity). FtsZ is a very promising target for new antimicrobial drugs because of its central role in bacterial cell division.

Over the course of a 30 year programme to design synthetic inhibitors of tubulin polymerization, thousands of analogues have been prepared at the Southern Research Institute (SRI) and screened as polymerization inhibitors.3 These compounds were shown to compete with colchicine for its binding site.4 Our initial hypotheses were that this class of compounds, the 2-alkoxycarbonylaminopyridines, should inhibit M. tuberculosis FtsZ and that the low degree of sequence homology between FtsZ and tubulin was consistent with finding compounds specific for M. tuberculosis. Over 200 compounds from the SRI repository were submitted to the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) for screening, and several were found to have antimicrobial activity. We selected two of these, SRI-7614 and SRI-3072, for further study.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antimicrobial assay

Most antimicrobial assays were conducted by the TAACF screening facility against M. tuberculosis H37Rv (ATCC 27294; American Type Culture Collection, Manassas, VA, USA) using a Bactec 460 radiometric system to determine the MIC99.5 Rifampicin was the positive control. The MIC99 was also determined of SRI-7614 and SRI-3072 for strains of M. tuberculosis resistant to isoniazid, rifampicin, ethambutol, kanamycin, pyrazinamide, thiacetazone or cycloserine. Concurrent with the determination of MIC99, compounds were tested for cytotoxicity (IC50) in Vero cells after 72 h exposure. The selectivity index (SI) of a compound is defined as the IC50:MIC99 ratio.

MIC/MBC of SRI-3072 for M. tuberculosis H37Ra

The MIC of SRI-3072 was determined by us for M. tuberculosis H37Ra (ATCC 25177) as described elsewhere,6 using a colorimetric (Alamar Blue) microdilution broth assay. SRI-3072 was assayed in quadruplicate. Ethambutol was the positive control and had an MIC of 2 mg/L. Bactericidal activity of SRI-3072 was determined with the same microtitre plate used for MIC. The plate was examined visually just before the addition of the redox dye after 6 days of incubation. Each well with no visible growth was mixed by carefully drawing liquid in and out of a microlitre pipette and plated (10 µL) on to 7H11 agar. The plates were incubated for 21 days in polyethylene bags at 36–37°C and the colonies then counted with the aid of a dissecting microscope. The counts were compared with those of the initial inoculum to calculate the log10 reduction in survivors. The MBC was defined as the lowest drug concentration that reduced the cfu by 2 log10 ± S.D. (n = 4).

Activity of SRI-3072 in M. tuberculosis-infected macrophages

SRI-3072 was assayed at 0.25, 1, 4 and 16 x MIC (0.2 mg/L) through the TAACF for activity against M. tuberculosis Erdman (ATCC 35801) in monolayers of mouse bone marrow macrophages, as described previously.7 Activity was reported as the lowest drug concentration yielding a 90% (EC90) and 99% (EC99) reduction in cfus after 7 days relative to drug-free controls. Toxicity was determined by visual inspection.

Purification of FtsZ and assays

The M. tuberculosis FtsZ was expressed and purified as described previously8 with the following modification. A polymerization/depolymerization step was added before preparative gel filtration. This improved purification gave a protein preparation that was >99% pure with a 1:1 molar ratio of GDP to FtsZ.

The polymerization and depolymerization of purified FtsZ at 0.5 mg/mL (13 µM) was followed by the previously described light scattering method.8 Compounds were initially evaluated at 100 µM. If inhibition was observed, the compounds were retested at several concentrations. The percentage control activity was calculated by comparison with an assay without compound. Three independent curves were run. The GTPase activity of 25 µL of sample containing FtsZ was followed by monitoring the radioactive release of inorganic phosphate using the method described by Mukherjee et al.9 Compounds were tested at 100 µM only.

The GTP-initiated polymerization of purified bovine tubulin (1 mg/mL, 20 µM; Sigma) was followed by a light scattering assay. Compounds were initially evaluated at 100 µM. If inhibition was observed, the compounds were retested at several concentrations.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antimicrobial activity

Two SRI compounds, as shown in Table 1 together with two other representatives of the types of structure screened, were selected for study. Ethyl [6-amino-2,3-dihydro-4-phenyl-1H-pyrido(4,3-b)(1,4)diazepin-8-yl]carbamate (SRI-7614) had an MIC99 of 6.25 mg/L (19 µM) in the Bactec assay (TAACF) and an SI > 32. Ethyl {8-[[4-(diethylamino)-1-methylbutyl]-amino]-2,3-diphenylpyrido(2,3-b)pyrazin-6-yl}carbamate (SRI-3072), with an MIC99 of 0.15 mg/L (0.28 µM), was the more active compound. The antitubercular activity of both compounds was confirmed in our laboratories.


View this table:
[in this window]
[in a new window]
 
Table 1.  TAACF test results for inhibition of M. tuberculosis H37Rv growth
 
The MICs of SRI-7614 and SRI-3072 were determined for drug-resistant M. tuberculosis through the TAACF. Strains of M. tuberculosis that were resistant to either isoniazid, rifampicin, ethambutol, kanamycin, pyrazinamide, thiacetazone or cycloserine were uniformly susceptible to SRI-7614 and SRI-3072, with MICs similar to those of the wild-type strain. Thus, no cross-resistance was evident among these various types of drug and either SRI compound. This result was as expected, as FtsZ is a new target not inhibited by the clinically active antituberculosis drugs.

The MIC and MBC of SRI-3072 for M. tuberculosis H37Ra were 0.25 mg/L (0.47 µM) and 1–2 mg/L (1.9–3.8 µM), respectively. The log reduction in cfu/mL in the presence of 0.95, 1.9 and 3.8 µM drug was 0.98 ± 0.11, 1.7 ± 0.06 and 2 ± 0.12, respectively. Growth in the viability control without drug was estimated to have increased >=1 log10 cfu/mL after 6 days of incubation. Our results indicated that the drug was bactericidal. A compound is generally considered to be bactericidal if the ratio of MIC to MBC is <=4. For SRI-3072, this ratio was 4 using a value for the MBC of 1.9 µM.

SRI-3072 inhibited the growth of M. tuberculosis Erdman in infected mouse bone marrow macrophages. The concentration effecting 90% and 99% reduction in mycobacterial growth after 7 days was 0.23 µM (0.12 mg/L, EC90) and 2.7 µM (1.42 mg/L, EC99). The EC90 was similar to the MICs for H37Rv, H37Ra, Erdman and the drug-resistant strains, indicating adequate permeability into macrophages.

Inhibition of FtsZ polymerization and GTP hydrolysis

SRI-3072, SRI-7614 and colchicine inhibited M. tuberculosis FtsZ polymerization in a dose-dependent manner, with ID50 values of 52 ± 12, 60 ± 0 and 104 ± 2 µM (Table 2). GTP hydrolysis by FtsZ is required for polymerization to proceed.8 One hundred micromolar SRI-3072, SRI-7614 and colchicine inhibited GTP hydrolysis by 20, 25 and 30%, respectively.


View this table:
[in this window]
[in a new window]
 
Table 2.  Inhibitors of FtsZ and tubulin polymerization and GTP hydrolysis
 
SRI-3072, SRI-7614, colchicine and two other 1-deaza-pteridines (SRI-5713 and SRI-20158) were evaluated as inhibitors of tubulin polymerization (Table 2). As expected, colchicine was more effective as an inhibitor of tubulin (ID50 6.5 µM) than FtsZ (ID50 100 µM). Of our two lead compounds, SRI-3072 was specific for FtsZ; no inhibition of tubulin was seen at 100 µM. Like colchicine, SRI-7614 inhibited polymerization of both proteins (ID50 60 µM FtsZ, ID50 4 µM tubulin). 1-Deaza-7,8-dihydropteridines were the subclass of compound that was pursued at SRI as potential antimitotic agents. Two of these compounds (SRI-5713 and SRI-20158) had no antitubercular activity (Table 1) and also failed to inhibit FtsZ polymerization (Table 2). However, these two compounds inhibited tubulin polymerization.

Development of new antitubercular agents is of critical importance worldwide. Our programme has identified a new class of small-molecule inhibitors of M. tuberculosis that we believe inhibit a protein target, FtsZ, which differs from the mechanism of action used by current clinical drugs. Since M. tuberculosis is an intracellular parasite, it is important that a drug is able to penetrate into macrophages and inhibit the growth of the infecting bacteria. SRI-3072 reduced the growth of M. tuberculosis Erdman in macrophages, and our data indicated that this class of compounds would enter macrophages readily. Our two lead compounds, SRI-7614 and SRI-3072, inhibited the in vitro polymerization and GTP hydrolysis of M. tuberculosis FtsZ, whereas compounds inactive in the tuberculosis screen did not. One of these compounds, SRI-3072, was specific for FtsZ and did not inhibit tubulin polymerization. These data support our hypothesis that the biological target of the 2-alkoxycarbonylaminopyridine analogues with antitubercular activity is FtsZ. More definitive studies to define better the mechanism of action of these compounds are underway.


    Acknowledgements
 
This work was supported by an internal research and development grant awarded by the SRI. The 2-alkoxycarbonylaminopyridines were from the SRI repository. The TAACF screening facilities located at the National Hansen’s Disease Program in Baton Rouge, LA, USA, provided antimycobacterial data through a research and development contract with the US National Institute of Allergy and Infectious Diseases.


    Footnotes
 
* Corresponding author. Tel: +1-205-581-2344; Fax: +1-205-581-2877; E-mail: white{at}sri.org Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . de Boer, P. A., Cook, W. R. & Rothfield, L. I. (1990). Bacterial cell division. Annual Review of Genetics 24, 249–74.[ISI][Medline]

2 . Erickson, H. P. (1995). FtsZ, a prokaryotic homolog of tubulin? Cell 80, 367–70.[ISI][Medline]

3 . Temple, C. G. (1990). Synthesis and evaluation of 1-deaza-7,8-dihydropteridines and ring analogs. In Chemistry and Biology of Pteridines (Curtis, H.-C., Ghisla, S. & Blau, N., Eds), pp. 1009–14. Walter de Gruyter, Berlin, Germany.

4 . Bowdon, B. J., Waud, W. R., Wheeler, G. P., Hain, R., Dansby, L. & Temple, C., Jr (1987). Comparison of 1,2-dihydropyrido[3,4-b]pyrazines (1-deaza-7,8-dihydropteridines) with several other inhibitors of mitosis. Cancer Research 47, 1621–6.[Abstract]

5 . Collins, L. A. & Franzblau, S. G. (1997). Microplate Alamar Blue assay versus BACTEC 460 system, for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrobial Agents and Chemotherapy 41, 1004–9.[Abstract]

6 . 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, 2784–93.[Abstract/Free Full Text]

7 . Skinner, P. S., Furney, S. K., Jacobs, M. R., Klopman, G., Ellner, J. J. & Orme, I. M. (1994). A bone marrow-derived murine macrophage model for evaluating efficacy of antimicrobial drugs under relevant physiological conditions. Antimicrobial Agents and Chemotherapy 38, 2557–63.[Abstract]

8 . 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, 4028–34.[Abstract/Free Full Text]

9 . Mukherjee, A., Dai, K. & Lutkenhaus, J. (1993). Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein. Proceedings of the National Academy of Sciences, USA 90, 1053–7.[Abstract]