Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64 µg/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.

Micafungin Elicits an Immunomodulatory Effect in Galleria mellonella and Mice.

The echinocandin family of drugs is well characterized for antifungal function that inhibits ß-D-glucan synthesis. The aim of this work was to study whether micafungin, a member of the echinocandin family, elicits additional activities that prime the host's immune response. We found that in a Galleria mellonella model, prophylactic treatment with micafungin extended the life of Staphylococcus aureus-infected larvae (a pathogen to which the drug demonstrates no direct antimicrobial activity) compared to insects that did not receive micafungin (P < 0.05). The inhibition of pathogens in the G. mellonella infection model was characterized by a 2.43-fold increase in hemocyte density, compared to larvae inoculated with PBS. In a murine model where animals were provided micafungin prophylaxis 3 days prior to macrophage collection, macrophages were found associated with an average 0.9 more fungal cells per macrophage as compared to saline-treated animals. Interestingly, micafungin-stimulated macrophages killed 11.6 ± 6.2 % of fungal cells compared to 3.8 ± 2.4 % of macrophages from saline-treated animals. The prophylactic provision of micafungin prior to Candida albicans infection was characterized by an increase in the proinflammatory cytokines CXCL13 and SPP1 by 11- and 6.9-fold, respectively. In conclusion, micafungin demonstrated the ability to stimulate phagocytic cells and promote an immune response that can inhibit microbial infections.

Undetectable role of oxidative DNA damage in cell cycle, cytotoxic and clastogenic effects of Cr(VI) in human lung cells with restored ascorbate levels.

Cultured human cells are invaluable biological models for mechanistic studies of genotoxic chemicals and drugs. Continuing replacement of animals in toxicity testing will further increase the importance of in vitro cell systems, which should accurately reproduce key in vivo characteristics of toxicants such as their profiles of metabolites and DNA lesions. In this work, we examined how a common severe deficiency of cultured cells in ascorbate (Asc) impacts the formation of oxidative DNA damage by hexavalent chromium (chromate). Cr(VI) is reductively activated inside the cells by both Asc and small thiols but with different rates and spectra of intermediates and DNA adducts. We found that Cr(VI) exposure of H460 human lung epithelial cells in standard culture (<0.01 mM cellular Asc) induced biologically significant amounts of oxidative DNA damage. Inhibition of oxidative damage repair in these cells by stable XRCC1 knockdown strongly enhanced cytotoxic effects of Cr(VI) and led to depletion of cells from G(1) and accumulation in S and G(2) phases. However, restoration of physiological levels of Asc (≈ 1 mM) completely eliminated Cr(VI) hypersensitivity of XRCC1 knockdown. The induction of chromosomal breaks assayed by the micronucleus test in Asc-restored H460, primary human lung fibroblasts, and CHO cells was also unaffected by the XRCC1 status. Centromere-negative (clastogenic) micronuclei accounted for 80-90% of all Cr(VI)-induced micronuclei. Consistent with the micronuclei results, Asc-restored cells also showed no increase in the levels of poly(ADP-ribose), which is a biochemical marker of single-stranded breaks. Asc had no effect on cytotoxicity of O(6)-methylguanine, a lesion produced by direct DNA alkylation. Overall, our results indicate that the presence of physiological levels of Asc strongly suppresses pro-oxidant pathways in Cr(VI) metabolism and that the use of standard cell cultures creates a distorted profile of its genotoxic properties.

XPA impacts formation but not proteasome-sensitive repair of DNA-protein cross-links induced by chromate.

DNA-protein cross-links (DPCs) are caused by a large number of human carcinogens and anti-cancer drugs. However, cellular processes involved in decreasing a burden of these genotoxic lesions remain poorly understood. Here, we examined the impact of nucleotide excision repair (NER), which is a principal repair pathway for bulky DNA adducts, and the main cellular reducers on removal of chromium(VI)-induced DPC. We found that standard and ascorbate-restored cultures of isogenic XPA-null (NER deficient) and XPA-complemented human fibroblasts had very similar repair of Cr-DPC (60-65% average DPC removal after 24 h). However, XPA absence caused depletion of G1 and accumulation of G2 cells at low Cr(VI) doses, suggesting that Cr-DPC were not a significant cause of cell cycle perturbations. Interestingly, although pro-oxidant metabolism of Cr(VI) in glutathione-depleted cells generated significantly fewer DPC, they were repair resistant irrespective of the NER status of cells. Inhibition of proteasome activity by MG132 abolished DPC repair in both XPA-null and XPA-complemented cells. XPA loss caused two to three times higher initial DPC formation, demonstrating the importance of NER in removal of the precursor lesions. Our results indicate that human NER is not involved in removal of Cr-DPC containing non-histone proteins but it acts as a defence mechanism against these large lesions by preventing their formation. Therefore, individual differences in NER activity are expected to alter sensitivity but not persistence of DPC as a biomarker of hexavalent Cr.

The chromate-inducible chrBACF operon from the transposable element TnOtChr confers resistance to chromium(VI) and superoxide.

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (TnOtChr) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long TnOtChr of the mixed Tn21/Tn3 transposon subfamily contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC, were essential for establishment of high resistance in chromium-sensitive O. tritici. The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA. Expression of chrB, chrC, or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. TnOtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to TnOtChr-containing cells in the environments with multiple redox-active contaminants.

Reduction with glutathione is a weakly mutagenic pathway in chromium(VI) metabolism.

Although reductive metabolism of Cr(VI) always results in the production of Cr(III) and extensive Cr-DNA binding, cellular studies have indicated that different reduction processes are not equivalent in the induction of mutagenic events. Here, we examined mutagenicity and formation of Cr-DNA damage by Cr(VI) activated in vitro by one of its important reducers, glutathione (GSH). Our main focus was on reactions containing 2 mM GSH, corresponding to its average concentration in CHO (1.8 mM) and V79 (2.6 mM) mutagenicity models. We found that Cr(VI) reduction by 2 mM GSH produced only weak mutagenic responses in pSP189 plasmids replicated in human fibroblasts. Reductive activation of Cr(VI) with 5 mM GSH resulted in approximately 4-times greater DNA adduct-normalized yield of mutations. Mutagenic DNA damage formed in GSH-chromate reactions was caused by nonoxidative mechanisms, as blocking of Cr-DNA adduction led to a complete loss of mutagenesis. All GSH-mediated reactions also lacked significant DNA single-strand breakage. We developed a sensitive HPLC procedure for the detection of GSH-Cr-DNA cross-links based on the dissociation of DNA-conjugated GSH by Cr(III) chelation and its derivatization with monobromobimane. Weak mutagenicity of 2 mM GSH reactions was associated with a low production of mutagenic GSH-Cr-DNA cross-links (5.0% of total Cr-DNA adducts). In agreement with their greater mutation-inducing ability, 5 mM GSH reactions generated 4-5 times higher levels of GSH-DNA cross-linking. Overall, our results indicate that chromate reduction by physiological concentrations of GSH is a weakly mutagenic process, which is consistent with low mutagenicity of Cr(VI) in ascorbate-deficient cells.

Propyl-5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC): a new bacteriostatic agent against methicillin-resistant Staphylococcus aureus.

The emergence of Staphylococcus aureus strains resistant to 'last resort' antibiotics compels the development of new antimicrobials against this important human pathogen. We found that propyl 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) shows bacteriostatic activity against S. aureus (MIC = 4 µg/ml) and rescues Caenorhabditis elegans from S. aureus infection. Whole-genome sequencing of S. aureus mutants resistant to the compound, along with screening of a S. aureus promoter-lux reporter array, were used to explore possible mechanisms of action. All mutants resistant to HMPC acquired missense mutations at distinct codon positions in the global transcriptional regulator mgrA, followed by secondary mutations in the phosphatidylglycerol lysyltransferase fmtC/mprF. The S. aureus promoter-lux array treated with HMPC displayed a luminescence profile that was unique but showed similarity to DNA-damaging agents and/or DNA replication inhibitors. Overall, HMPC is a new anti-staphylococcal compound that appears to act via an unknown mechanism linked to the global transcriptional regulator MgrA.

Raf-kinase inhibitor GW5074 shows antibacterial activity against methicillin-resistant Staphylococcus aureus and potentiates the activity of gentamicin.

AIM: Increasing antimicrobial resistance has compromised the effectiveness of many antibiotics, including those used to treat staphylococcal infections like methicillin-resistant Staphylococcus aureus. The development of combination therapies, where antimicrobial agents are used with compounds that inhibit resistance pathways is a promising strategy. Results/methodology: The Raf kinase inhibitor GW5074 exhibited selective in vitro activity against Gram-positive bacteria, including clinical isolates of S. aureus with a minimum inhibitory concentration (MIC) of 2-8 µg/ml. GW5074 was effective in vivo in the Galleria mellonella infection model. The compound showed synergy with gentamicin by lowering MIC by fourfold, compared with gentamicin MIC alone. CONCLUSION: This work demonstrates the antimicrobial properties of GW5074 and supports further investigation of the kinase inhibitors as antibiotic adjuvants.

A Defensin from the Model Beetle Tribolium castaneum Acts Synergistically with Telavancin and Daptomycin against Multidrug Resistant Staphylococcus aureus.

The red flour beetle Tribolium castaneum is a common insect pest and has been established as a model beetle to study insect development and immunity. This study demonstrates that defensin 1 from T. castaneum displays in vitro and in vivo antimicrobial activity against drug resistant Staphylococcus aureus strains. The minimum inhibitory concentration (MIC) of defensin 1 against 11 reference and clinical staphylococcal isolates was between 16-64 µg/ml. The putative mode of action of the defensin peptide is disruption of the bacterial cell membrane. The antibacterial activity of defensin 1 was attenuated by salt concentrations of 1.56 mM and 25 mM for NaCl and CaCl2 respectively. Treatment of defensin 1 with the reducing agent dithiothreitol (DTT) at concentrations 1.56 to 3.13 mM abolished the antimicrobial activity of the peptide. In the presence of subinhibitory concentrations of antibiotics that also target the bacterial cell envelope such as telavancin and daptomycin, the MIC of the peptide was as low as 1 µg/ml. Moreover, when tested against an S. aureus strain that was defective in D-alanylation of the cell wall, the MIC of the peptide was 0.5 µg/ml. Defensin 1 exhibited no toxicity against human erythrocytes even at 400 µg/ml. The in vivo activity of the peptide was validated in a Caenorhabditis elegans-MRSA liquid infection assay. These results suggest that defensin 1 behaves similarly to other cationic AMPs in its mode of action against S. aureus and that the activity of the peptide can be enhanced in combination with other antibiotics with similar modes of action or with compounds that have the ability to decrease D-alanylation of the bacterial cell wall.

XRCC1 deficiency sensitizes human lung epithelial cells to genotoxicity by crocidolite asbestos and Libby amphibole.

BACKGROUND: Asbestos induces DNA and chromosomal damage, but the DNA repair pathways protecting human cells against its genotoxicity are largely unknown. Polymorphisms in XRCC1 have been associated with altered susceptibility to asbestos-related diseases. However, it is unclear whether oxidative DNA damage repaired by XRCC1 contributes to asbestos-induced chromosomal damage. OBJECTIVES: We sought to examine the importance of XRCC1 in protection against genotoxic effects of crocidolite and Libby amphibole asbestos. METHODS: We developed a genetic model of XRCC1 deficiency in human lung epithelial H460 cells and evaluated genotoxic responses to carcinogenic fibers (crocidolite asbestos, Libby amphibole) and nongenotoxic materials (wollastonite, titanium dioxide). RESULTS: XRCC1 knockdown sensitized cells to the clastogenic and cytotoxic effects of oxidants [hydrogen peroxide (H2O2), bleomycin] but not to the nonoxidant paclitaxel. XRCC1 knockdown strongly enhanced genotoxicity of amphibole fibers as evidenced by elevated formation of clastogenic micronuclei. Crocidolite induced primarily clastogenic micronuclei, whereas Libby amphibole induced both clastogenic and aneugenic micronuclei. Crocidolite and bleomycin were potent inducers of nuclear buds, which were enhanced by XRCC1 deficiency. Libby amphibole and H2O2 did not induce nuclear buds, irrespective of XRCC1 status. Crocidolite and Libby amphibole similarly activated the p53 pathway. CONCLUSIONS: Oxidative DNA damage repaired by XRCC1 (oxidized bases, single-strand breaks) is a major cause of chromosomal breaks induced by crocidolite and Libby amphibole. Nuclear buds are a novel biomarker of genetic damage induced by exposure to crocidolite asbestos, which we suggest are associated with clustered DNA damage. These results provide mechanistic evidence for the epidemiological association between XRCC1 polymorphisms and susceptibility to asbestos-related disease.

Rapid DNA double-strand breaks resulting from processing of Cr-DNA cross-links by both MutS dimers.

Mismatch repair (MMR) strongly enhances cyto- and genotoxicity of several chemotherapeutic agents and environmental carcinogens. DNA double-strand breaks (DSB) formed after two replication cycles play a major role in MMR-dependent cell death by DNA alkylating drugs. Here, we examined DNA damage detection and the mechanisms of the unusually rapid induction of DSB by MMR proteins in response to carcinogenic chromium(VI). We found that MSH2-MSH6 (MutSalpha) dimer effectively bound DNA probes containing ascorbate-Cr-DNA and cysteine-Cr-DNA cross-links. Binary Cr-DNA adducts, the most abundant form of Cr-DNA damage, were poor substrates for MSH2-MSH6, and their toxicity in cells was weak and MMR independent. Although not involved in the initial recognition of Cr-DNA damage, MSH2-MSH3 (MutSbeta) complex was essential for the induction of DSB, micronuclei, and apoptosis in human cells by chromate. In situ fractionation of Cr-treated cells revealed MSH6 and MSH3 chromatin foci that originated in late S phase and did not require replication of damaged DNA. Formation of MSH3 foci was MSH6 and MLH1 dependent, whereas MSH6 foci were unaffected by MSH3 status. DSB production was associated with progression of cells from S into G(2) phase and was completely blocked by the DNA synthesis inhibitor aphidicolin. Interestingly, chromosome 3 transfer into MSH3-null HCT116 cells activated an alternative, MSH3-like activity that restored dinucleotide repeat stability and sensitivity to chromate. Thus, sequential recruitment and unprecedented cooperation of MutSalpha and MutSbeta branches of MMR in processing of Cr-DNA cross-links is the main cause of DSB and chromosomal breakage at low and moderate Cr(VI) doses.