Dequalinium, a new inhibitor of Mycobacterium tuberculosis mycothiol ligase identified by high-throughput screening.

Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol that is unique to actinomycetes and whose primary role is to maintain intracellular redox balance and remove toxins. MshC catalyzes the adenosine triphosphate (ATP)-dependent condensation of cysteine and glucosamine-inositol (GI) to produce cysteine-glucosamine-inositol (CGI). MshC is essential to Mycobacterium tuberculosis and therefore represents an attractive target for chemotherapeutic intervention. A screening protocol was developed to identify MshC inhibitors based on quantification of residual ATP using a coupled luminescent assay. The protocol was used to screen a library of 3100 compounds in a 384-well plate format (Z'>or=0.65). Fifteen hits (0.48%) were identified from the screen, and 2 hits were confirmed in a secondary assay that measures production of CGI. The structures of both hits contain N-substituted quinolinium moieties, and the more potent of the 2-namely, dequalinium chloride-inhibits MshC with an IC50 value of 24+/-1 microM. Further studies showed dequalinium to be an ATP-competitive inhibitor of MshC, to bind MshC with a KD of 0.22 microM, and to inhibit the growth of M. tuberculosis under aerobic and anaerobic conditions with minimum inhibitory and anaerobic bactericidal concentrations of 1.2 and 0.3 microg/mL, respectively. The screening protocol described is robust and has enabled the identification of new MshC inhibitors.

New method for the quantification of dequalinium cations in pharmaceutical samples by absorption and fluorescence diode array thin-layer chromatography.

A diode array HPTLC method for dequalinium chloride in pharmaceutical preparations is presented. For separation a Nano TLC silica gel plate (Merck) is used with the mobile phase methanol-7.8% aqueous NH(4)(+)CH(3)COO(-) (17:3, v/v) over a distance of 6 cm. Dequalinium chloride shows an R(F) value of 0.58. Pure dequalinium chloride is measured in the wavelength range from 200 to 500 nm and shows several by-products, contour plot visualized in absorption, fluorescence and using the Kubelka-Munk transformation. Scanning of a single track in absorption and fluorescence measuring 600 spectra in the range from 200 to 1100 nm takes 30s. As a sample pre-treatment of an ointment it is simply dissolved in methanol and can be quantified in absorption from 315 to 340 nm. The same separation can also be quantified using fluorescence spectrometry in the range from 355 to 370 nm. A new staining method for dequalinium chloride, using sodium tetraphenyl borate/HCl in water allows a fluorescence quantification in the range from 445 to 485 nm. The linearity range of absorption and fluorescence measurements is from 10 to 2000 ng. Sugar-containing preparations like liquids or lozenges with a reduced sample pre-treatment can be reliably quantified only in fluorescence. The total for the quantification of an ointment sample (measuring four standards and five samples), including all sample pre-treatment steps takes less than 45 min!

Flow injection spectrofluorimetric study of the supramolecular interaction between beta-cyclodextrin and dequalinium chloride and its analytical application.

The supramolecular interaction of dequalinium chloride (DQC) and beta-cyclodextrin has been studied by flow injection spectrofluorimetry. The results showed that beta-CD reacted with DQC to form a 1:1 host:guest complex with an apparent association constant of 4.99 x 10(2) L mol(-1). Based on the enhancement of the fluorescence intensity of DQC, a flow injection spectrofluorometric method for the determination of DQC in bulk aqueous solution in the presence of beta-CD was developed. The linear range was 0.0412-30.00 microg mL(-1). The detection limit was 12.3 ng mL(-1) with a sampling rate of 80 h(-1). There was no interference from the excipients normally used in tablets and serum compositions. The proposed method was successfully applied to the determination of DQC in tablets and serum.

Determination of antibacterial quaternary ammonium compounds in lozenges by capillary electrophoresis.

A method for the specific determination of three quaternary ammonium compounds, benzalkonium chloride, cetylpyridinium chloride and dequalinium chloride, used as antibacterial agents in candy-based lozenges, is described based on capillary zone electrophoresis. It is shown that, following optimisation of buffer composition with respect to organic modifier concentration. pH and buffer concentration together with the inclusion of sodium dodecylsulphate as an ion-pairing agent in the case of dequalinium chloride, these analytes migrate in less than 5 min. The resultant electrophoretic peaks are sharp and readily quantified. The individual alkyl components of benzalkonium chloride can be resolved as can related impurities in dequalinium chloride lozenges. The quantitative characteristics of the assay method, based on peak areas normalised with respect to migration times, are reported and the method is compared with a previously published method based on liquid chromatography.

Capillary electrophoresis and liquid chromatography in the analysis of some quaternary ammonium salts used in lozenges as antibacterial agents.

A comparison is made of the relative merits of high-performance liquid chromatography and capillary electrophoresis, both using direct UV detection, for the determination of three quaternary ammonium compounds used as the active antibacterial ingredient in lozenge formulations. While both techniques are capable of separating the compounds cetylpyridinium chloride, dequalinium chloride, and benzalkonium chlorides, the liquid chromatographic method involving ion pairing and using a 5-micron cyanopropyl stationary phase, was unable to resolve the benzalkonium chlorides from the lozenge excipients and quantitation was not possible. The capillary electrophoresis method using a 205-mm 50-micron-i.d. capillary with a running buffer of 50% vol/vol 50 mM phosphate buffer at pH 3 provided superior resolution of the three antibacterials in all lozenge formulations. This system was also capable of resolving impurities in the dequalinium chloride both in the standard and in lozenges containing this compound. On the basis of quantitative results previously published, both methods have adequate validation parameters since the relative insensitivity of capillary electrophoresis compared with liquid chromatography is not important at the concentration required to be determined following a single simple sample pretreatment.

Determination of the quaternary ammonium compounds dequalinium and cetylpyridinium chlorides in candy-based lozenges by high-performance liquid chromatography.

The retention behavior of the quaternary ammonium compounds benzalkonium chloride, cetylpyridinium chloride and dequalinium chloride on a 100 x 4.6 mm id cyanopropyl stationary phase column is reported as a function of organic modifier and ionic hydrophobic mobile phase additive concentrations. Optimum liquid chromatographic mobile phases using different mobile phase additives are reported which are suitable for the determination of cetylpyridinium chloride and dequalinium chloride in a variety of candy-based lozenge formulations. The quantitative aspects of assays based on the separation of active ingredients and formulation excipients were established. The generality of application of the assay methods was evaluated by determining the quaternary ammonium content of different lozenges and comparing the values obtained with the stated dose.