Antimycobacterial action of B4128, a novel tetramethylpiperidyl-substituted phenazine

N. M. Matlola, H. C. Steel and R. Anderson*

Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, PO Box 2034, Pretoria 0001, South Africa


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The effects of the novel tetramethylpiperidyl (TMP)-substituted phenazine, B4128 (0.6–2.5 mg/L), on growth, phospholipase A2(PLA2) activity, cation (K+, Ca2+) fluxes and energy metabolism (ATP) of Mycobacterium aurum A+ and Mycobacterium tuberculosis (H37Ra) have been investigated in vitro. Growth, PLA2 and cation fluxes were measured radiometrically, while microbial ATP was assayed by means of a luciferin/luciferase chemiluminescence method. Exposure of the mycobacteria to B4128 resulted in immediate, dose-related enhancement of microbial PLA2 activity and inhibition of K+-influx, which preceded effects on microbial ATP, influx of Ca2+ and growth. These results demonstrate that B4128 possesses membrane-directed antimycobacterial properties that are similar to those of clofazimine.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In an effort to minimize the side-effects and to improve on the antimycobacterial activity of clofazimine, various 2,2,6,6-tetramethylpiperidyl (TMP)-substituted phenazine analogues of this prototype riminophenazine have been developed.1,2 The most important structural difference between these agents and clofazimine is the TMP group, as opposed to an isopropyl group, on the imino nitrogen at position 2 of the phenazine nucleus. The number and position of halogen atoms on the phenyl and anilino groups enable fine-tuning of the derivatives, depending on the target organism.1,2

Although the antimycobacterial action of clofazimine has been reported to be membrane directed and to involve phospholipase-mediated inactivation of microbial K+ uptake,3 the biochemical mechanism of the antimicrobial action of the TMP-substituted phenazines has not been established. The current study was undertaken to investigate the effects of B4128 on phospholipase A2 activity and K+ transport in mycobacteria, and to investigate any relationship between alterations in these and inhibition of bacterial growth.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Riminophenazines

B4128 [3-(2,4-dichloroanilino)-10-(2,4-dichlorophenyl)-2,10-dihydro-2-(2,2,6,6 tetramethylpiperid-4-ylimino) phenazine]2,4 (synthesized by Dr J. F. O'Sullivan, Department of Chemistry, University College, Dublin, Republic of Ireland) was dissolved in 100% ethanol to a stock concentration of 2 mg/L. Subsequent dilutions were made in absolute ethanol and B4128 was used at final concentrations of 0.6, 1.25 and 2.5 mg/L. Appropriate solvent controls were included in the various assays described.

Chemicals and reagents

Unless otherwise indicated, all chemicals used were obtained from the Sigma Chemical Co. (St Louis, MO, USA). Radiochemicals were purchased from Du Pont-NEN Research Products (Boston, MA, USA).

Mycobacteria

The rapidly proliferating, non-pathogenic, soil-dwelling mycobacterium Mycobacterium aurum A+, obtained from the Institut Pasteur (Paris, France), was used for the experiments described below, while Mycobacterium tuberculosis H37Ra was used in a limited number of confirmatory experiments.

Measurement of bacterial growth

The effect of B4128 (0.6 and 1.25 mg/L) on the growth of the test mycobacterium was investigated using the BACTEC TB system (Becton Dickinson Diagnostic Instrument Systems, Towson, MD, USA), as described previously.3

Uptake of rubidium-86 chloride

Rubidium-86 chloride (86Rb, 34 MBq) was used as tracer for measuring K+ uptake by mycobacteria. 86Rb has been described in several previous studies as being a useful tracer for the measurement of microbial transport of K+.3,5 The effects of B4128 on bacterial K+ transport were measured over a 5 min time course. The effects of pre-treatment of the bacteria with {alpha}-tocopherol (25 mg/L final), a lysophospholipid-neutralizing agent, on B4128-mediated inhibition of growth and K+ uptake were also investigated.

Ca2+ influx

Calcium-45 chloride (45Ca, 185 MBq) was used as a tracer for measuring Ca2+ uptake. The kinetics of Ca2+ transport in control mycobacteria and those treated with B4128 (0.6 and 2.5 mg/L) were also measured over a 5 min time course.4

Phospholipase A2 (PLA2) activity

Phosphatidylcholine 2-acyl-hydrolase (PLA2) activity in the mycobacteria was measured according to the release of [14C]arachidonate from the sn-2 position of added phosphatidylcholine [l-{alpha}-1-palmitoyl-2-arachidonyl (arachidonyl-1-14C), 1.48–2.22 GBq, 0.5 mCi/L]. The mycobacteria were treated with B4128 (0.6 and 2.5 mg/L) for 30 min at 37°C, after which [14C]arachidonate was extracted and measured using a high-performance thin layer chromatography (HPTLC) method.6

ATP levels

Microbial ATP levels were determined by means of a sensitive luciferin/luciferase chemiluminescence method.7 Bacterial cells (0.01 mg protein/10 mL) were co-incubated for 30 min at 37°C with or without B4128 (0.6 and 1.25 mg/L), after which the cells were concentrated by centrifugation, lysed with a nucleotide releasing agent (Lumac, Landgraaf, The Netherlands) and assayed for ATP, over a 10 s period with a chemiluminometer (Biocounter M2010 Multijet, Lumac Systems Inc., Titusville, FL, USA).

Expression of results

The results are expressed as the mean values ± S.E.M., as the percentage of the corresponding drug-free control system, for each series of experiments.


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 Abstract
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 Materials and methods
 Results
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 References
 
Effects of B4128 on mycobacterial cation transport, PLA2 activity, ATP and growth

The effects of B4128 at 0.6 and 2.5 mg/L on mycobacterial PLA2 activity and uptake of 86Rb and 45Ca are shown in Figures 1 and 2GoGo, respectively. B4128 caused immediate doserelated enhancement of PLA2 activity which coincided with inhibition of uptake of 86Rb, while influx of 45Ca was either unaffected (at 0.6 mg/L B4128) over the 5 min time course of the experiment, or increased after a lag period of 2–3 min following treatment of the bacteria with 2.5 mg/L B4128.



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Figure 1. The time-course of altered uptake of 86Rb ({blacksquare}) and 45Ca ({blacktriangleup}) by, and PLA2 ({blacktriangledown}) activity in, M. aurum A+ treated with B4128 at 0.6 mg/L. The results of three or four experiments are expressed as the mean percentages of the untreated control systems ± S.E.M.

 


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Figure 2. The time-course of altered uptake of 86Rb ({blacksquare}) and 45Ca ({blacktriangleup}) by, and PLA2 ({blacktriangledown}) activity in, M. aurum A+ treated with B4128 at 2.5 mg/L. The results of three or four experiments are expressed as the mean percentages of the untreated control systems ± S.E.M.

 
Microbial ATP levels were unaffected by B4128 at 0.6 mg/L, while at higher concentrations of this agent a reduction in mycobacterial ATP levels was observed. The absolute values for the control system and those for systems containing B4128 at 0.6 and 1.25 mg/L were 9.0 ± 0.9, 8.0 ± 1.0 and 3.5 ± 0.3 nmol ATP/mg protein, respectively.

The growth of M. aurum A+ was almost completely inhibited by B4128 at 0.6 and 1.25 mg/L (4.0 ± 0.2% and 2.9 ± 0.3% of control, respectively).

Pre-treatment of M. aurum A+ with {alpha}-tocopherol (25 mg/L) antagonized the inhibitory effects of B4128 (0.6 mg/L) on mycobacterial growth and uptake of 86Rb. In the absence of {alpha}-tocopherol, the mean percentages of growth and 86Rb uptake relative to the control were 25.1 ± 0.1 and 14.2 ± 2.5, respectively, while the corresponding values in the presence of {alpha}-tocopherol were 175.4 ± 3.5 and 79.0 ± 4.0 (P < 0.05 for antagonism of B4128-mediated inhibition of growth and uptake of 86Rb).

Brief exposure (5 min) of M. tuberculosis H37Ra to B4128 at 0.6 mg/L resulted in increased PLA2 activity (157 ± 34%) associated with decreased uptake of 86Rb (51 ± 8%) and inhibition of growth (56 ± 11%). Data from two experiments are presented for PLA2 activity whereas those from four experiments are presented in the case of 86Rb uptake and growth.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has recently been proposed that high-level accumulation in mononuclear phagocytes combined with anti-inflammatory properties, a low incidence of drug resistance and slow metabolic elimination make the riminophenazine group of compounds, of which clofazimine is the prototype, attractive candidates for the treatment of mycobacterial infections.2 Clofazimine has, however, proved to be disappointing in the treatment of both tuberculosis and Mycobacterium avium complex infections in humans. This has been attributed to several factors including dietary effects on absorption, low plasma concentrations and possibly the immunosuppressive properties of this agent, which may reduce efficacy in HIV-infected individuals.2,8 Moreover, side-effects, particularly gastrointestinal toxicity, have also restricted the clinical application of this agent in antimycobacterial chemotherapy.2

The TMP-substituted phenazines have been developed as possible alternatives to clofazimine.1,2 Although the side-effect profile of these novel compounds in humans has not been established,1,2 their activity against M. tuberculosis in vitro is superior to that of clofazimine, particularly in models of intracellular activity.4 This has been attributed to the higher level of accumulation of these agents in macrophages.2,4 Unlike clofazimine, which apparently inhibits the growth of mycobacteria by potentiating the activity of microbial PLA2, leading to lysophospholipid-mediated dysfunction of membrane cation transporters,3 the antimicrobial mechanism of action of the TMP-substituted phenazines has not been elucidated.

In the current study we have investigated the effects of B4128, a TMP-substituted phenazine which is approximately five- to 10-fold more active than clofazimine against intracellular M. tuberculosis in vitro, on PLA2 activity, cation (K+ and Ca2+) transport and growth of the non-pathogenic mycobacterium, M. aurum A+. Exposure to B4128, at concentrations that in the case of clofazimine are therapeutically relevant,9 resulted in immediate, dosage-related enhancement of PLA2 and inhibition of uptake of 86Rb, a marker of K+ transport.3,5 The relationship between potentiation of PLA2 activity and inhibition of K+ transport is well established in both prokaryotic4,9 and eukaryotic cells.8 Influx of Ca2+ into B4128-treated mycobacteria and depletion of microbial ATP were not observed at 0.6 mg/L B4128, indicating that altered PLA2 activity and K+ transport are probably primary and interrelated events. However, at 2.5 mg/L of this agent, influx of Ca2+ occurred after a lag period of 2–3 min, indicating that this was a secondary event, possibly due to depletion of microbial ATP. We have confirmed these findings in a limited series of experiments with the avirulent strain of M. tuberculosis H37R, and have also demonstrated that {alpha}-tocopherol, a membrane-stabilizing, lysophospholipid-neutralizing agent attenuates the effects of B4128 on mycobacterial cation transport and growth.

In conclusion, the TMP-substituted phenazines, which differ structurally from their predecessor clofazimine, appear to possess a similar mechanism of anti-mycobacterial action.


    Notes
 
* Corresponding author. Tel: +27-12-319-2425; Fax: +27-12-323-0732; E-mail: randerso{at}postillion.up.ac.za Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Franzblau, S. G. & O'Sullivan, J. F. (1988). Structure–activity relationship of selected phenazines against Mycobacterium leprae in vitro. Antimicrobial Agents and Chemotherapy 32, 1583–5.[ISI][Medline]

2 . Reddy, V. M., O'Sullivan, J. F. & Gangadharam, P. R. (1999). Antimycobacterial activities of riminophenazines. Journal of Antimicrobial Chemotherapy 43, 615–23.[Abstract/Free Full Text]

3 . Steel, H. C., Matlola, N. M. & Anderson, R. (1999). Inhibition of potassium transport and growth of mycobacteria exposed to clofazimine and B669 is associated with a calcium-independent increase in microbial phospholipase A2 activity. Journal of Antimicrobial Chemotherapy 44, 209–16.[Abstract/Free Full Text]

4 . van Rensburg, C. E., Jooné, G. K., Sirgel, F. A., Matlola, N. M. & O'Sullivan, J. F. (2000). In vitro investigation of the antimicrobial activities of novel tetramethylpiperidine-substituted phenazines against Mycobacterium tuberculosis. Chemotherapy 46, 43–8.[ISI][Medline]

5 . Bakker, E. P. & Harold, F. M. (1980). Energy coupling to potassium transport in Streptococcus faecalis. Interplay of ATP and the protonmotive force. Journal of Biological Chemistry 255, 433–40.[Abstract/Free Full Text]

6 . Bradova, V., Smid, F., Lednova, J. & Michalec, C. (1990). Improved one-dimensional thin-layer chromatography for the separation of phospholipids in biological material. Journal of Chromatography 533, 297–9.[Medline]

7 . Holmsen, H., Holmsen, I. & Bernhardsen, A. (1966). Microdetermination of adenosine diphosphate and adenosine triphosphate in plasma with firefly luciferase system. Analytical Biochemistry 17, 456–73.[ISI][Medline]

8 . Anderson, R. & Smit, M. J. (1993). Clofazimine and B669 inhibit the proliferative responses and Na+, K+-adenosine triphosphatase activity of human lymphocytes by a lysophospholipid-dependent mechanism. Biochemical Pharmacology 46, 2029–38.[ISI][Medline]

9 . Schaad-Lanyi, Z., Dieterle, W., Dubois, J. P., Theobald, W. & Vischer, W. (1987). Pharmacokinetics of clofazimine in healthy volunteers. International Journal of Leprosy and Other Mycobacterial Diseases 55, 9–15.[ISI][Medline]

10 . De Bruyn, E. E., Steel, H. C., van Rensburg, C. E. & Anderson, R. (1996). The riminophenazines, clofazimine and B669, inhibit potassium transport in Gram-positive bacteria by a lysophospholipid-dependent mechanism. Journal of Antimicrobial Chemotherapy 38, 349–62.[Abstract]

Received 28 February 2000; returned 21 June 2000; revised 11 September 2000; accepted 9 October 2000