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Compounds with novel modes of action are urgently needed to develop effective combination therapies for the treatment of tuberculosis. In this study, a series of compounds was evaluated for activity against replicating Mycobacterium tuberculosis and Vero cell line toxicity. Fourteen of the compounds with in vitro activities in the low micrometer range and a favorable selectivity index were classified using reporter strains of M. tuberculosis which showed that six interfered with cell wall metabolism and one disrupted DNA metabolism. Counter-screening against strains carrying mutations in promiscuous drug targets argued against DprE1 and MmpL3 as hits of any of the cell wall actives and eliminated the cytochrome bc1 complex as a target of any of the compounds. Instead, whole-genome sequencing of spontaneous resistant mutants and/or counter-screening against common isoniazid-resistant mutants of M. tuberculosis revealed that four of the six cell wall-active compounds, all pyridine carboxamide analogues, were metabolized by KatG to form InhA inhibitors. Resistance to two of these compounds was associated with mutations in katG that did not confer cross-resistance to isoniazid. Of the remaining seven compounds, low-level resistance to one was associated with an inactivating mutation in Rv0678, the regulator of the MmpS5-MmpL5 system, which has been implicated in non-specific efflux of multiple chemotypes. Another mapped to the mycothiol-dependent reductase, Rv2466c, suggesting a prodrug mechanism of action in that case. The inability to isolate spontaneous resistant mutants to the seven remaining compounds suggests that they act via mechanisms which have yet to be elucidated.

Compounds with novel modes of action are urgently needed to develop effective combination therapies for the treatment of tuberculosis. In this study, a series of compounds was evaluated for activity against replicating Mycobacterium tuberculosis and Vero cell line toxicity. Fourteen of the compounds with in vitro activities in the low micrometer range and a favorable selectivity index were classified using reporter strains of M. tuberculosis which showed that six interfered with cell wall metabolism and one disrupted DNA metabolism. Counter-screening against strains carrying mutations in promiscuous drug targets argued against DprE1 and MmpL3 as hits of any of the cell wall actives and eliminated the cytochrome bc 1 complex as a target of any of the compounds. Instead, whole-genome sequencing of spontaneous resistant mutants and/or counter-screening against common isoniazid-resistant mutants of M. tuberculosis revealed that four of the six cell wall-active compounds, all pyridine carboxamide analogues, were metabolized by KatG to form InhA inhibitors. Resistance to two of these compounds was associated with mutations in katG that did not confer cross-resistance to isoniazid. Of the remaining seven compounds, low-level resistance to one was associated with an inactivating mutation in Rv0678, the regulator of the MmpS5-MmpL5 system, which has been implicated in non-specific efflux of multiple chemotypes. Another mapped to the mycothiol-dependent reductase, Rv2466c, suggesting a prodrug mechanism of action in that case. The inability to isolate spontaneous resistant mutants to the seven remaining compounds suggests that they act via mechanisms which have yet to be elucidated. [ABSTRACT FROM AUTHOR]

Two flavonoid glycosides, kaempferol 3-O-(6″-acetyl)-β-glucopyranoside (KAG) and quercetin 3-O-(6″-acetyl)-β-glucopyranoside (QAG), were found to be inhibitors of eukaryotic DNA polymerases from a Japanese vegetable, Petasites japonicus. These compounds inhibited the activities of mammalian replicative DNA polymerases (i.e., pol α, δ, and ϵ), but not other pol β, η, κ, and λ activities. KAG was a stronger inhibitor and more selective to pol α than QAG. The IC50 values of KAG for pol α, δ, and ϵ were 41, 164, and 127 μM, respectively. The pol α inhibition by KAG was non-competitive with respect to both the DNA template-primer and the dNTP substrate. KAG and QAG did not influence the activities of prokaryotic DNA polymerases or other mammalian DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, human telomerase, human DNA topoisomerase I and II, T7 RNA polymerase, and bovine deoxyribonuclease I. Therefore, we concluded that these flavonoid glycosides are moderate replicative DNA polymerase inhibitors leaning more relatively to pol α, and could be used as chromatographic carriers to purify the DNA polymerases rather than cytotoxic agents. We then made a KAG-conjugated column such as the epoxy-activated Sepharose 6B. In the column, pol α was selectively adsorbed and eluted. [Copyright &y& Elsevier]

Lyophilised polysaccharide solutions and gels incorporating the broad spectrum antimicrobial compound, chlorhexidine digluconate (CHD), have potential application as self-adhering, topical delivery systems for the prophylaxis and treatment of wound infections. It is desirable to sterilise these dosage forms but they do not possess suitable thermal properties to be sterilised by autoclaving or dry heat procedures. Ionising radiation may offer a solution hence 'antimicrobial wafers' fabricated from guar (GG), xanthan (XG), karaya (KAG), sodium alginate (SA) and a 50:50 blend of SA:KAG, were gamma-irradiated (25 and 40 kGy). Wafer sterility was qualitatively determined and validated for bacterial and fungal species. The rheological properties of gels reconstituted from irradiated discs ('wafers') were measured and compared with control samples (non-irradiated) to assess changes to the flow properties. Diffusion of CHD from the resultant gels and viscous solutions, and efficacy against Pseudomonas aeruginosa, was determined using a disc diffusion test and release studies conducted in a diffusion cell apparatus. Although there was no change to cast shape, the rheological properties were drastically depleted for all irradiated samples except XG which showed no effective change in consistency, yield stress or efficacy, from the non-irradiated control. SA and SA-KAG samples displayed 'anomalous' release according to Korsmeyer-Peppas., (Copyright © 2013 Elsevier B.V. All rights reserved.)