Systems biology-guided identification of synthetic lethal gene pairs and its potential use to discover antibiotic combinations.

Mathematical models of metabolism from bacterial systems biology have proven their utility across multiple fields, for example metabolic engineering, growth phenotype simulation, and biological discovery. The usefulness of the models stems from their ability to compute a link between genotype and phenotype, but their ability to accurately simulate gene-gene interactions has not been investigated extensively. Here we assess how accurately a metabolic model for Escherichia coli computes one particular type of gene-gene interaction, synthetic lethality, and find that the accuracy rate is between 25% and 43%. The most common failure modes were incorrect computation of single gene essentiality and biological information that was missing from the model. Moreover, we performed virtual and biological screening against several synthetic lethal pairs to explore whether two-compound formulations could be found that inhibit the growth of Gram-negative bacteria. One set of molecules was identified that, depending on the concentrations, inhibits E. coli and S. enterica serovar Typhimurium in an additive or antagonistic manner. These findings pinpoint specific ways in which to improve the predictive ability of metabolic models, and highlight one potential application of systems biology to drug discovery and translational medicine.

Model-driven discovery of synergistic inhibitors against E. coli and S. enterica serovar Typhimurium targeting a novel synthetic lethal pair, aldA and prpC.

Mathematical models of biochemical networks form a cornerstone of bacterial systems biology. Inconsistencies between simulation output and experimental data point to gaps in knowledge about the fundamental biology of the organism. One such inconsistency centers on the gene aldA in Escherichia coli: it is essential in a computational model of E. coli metabolism, but experimentally it is not. Here, we reconcile this disparity by providing evidence that aldA and prpC form a synthetic lethal pair, as the double knockout could only be created through complementation with a plasmid-borne copy of aldA. Moreover, virtual and biological screening against the two proteins led to a set of compounds that inhibited the growth of E. coli and Salmonella enterica serovar Typhimurium synergistically at 100-200 µM individual concentrations. These results highlight the power of metabolic models to drive basic biological discovery and their potential use to discover new combination antibiotics.

3-(2-oxoethylidene)indolin-2-one derivatives activate Nrf2 and inhibit NF-κB: potential candidates for chemoprevention.

Induction of cytoprotective phase 2 enzymes through inhibition of Keap1, a repressor of transcription factor Nrf2, is a cancer-prevention strategy. Compounds that elicit antiinflammatory and cytoprotective effects are promising candidates for chemoprevention. Novel analogues of 1-methyl-3-(2-oxopropylidene)indolin-2-one ('supercinnamaldehyde'; SCA) were synthesized, and their abilities to induce cytoprotective responses through Nrf2 induction and to suppress inflammatory responses were examined. 1-Methyl-3-(2-oxo-2-phenylethylidene)indolin-2-one (6) was identified as the lead compound. The compounds showed induction of Nrf2-dependent phase 2 enzymes in Keap1+/+ mouse embryonic fibroblasts (MEFs), which was abrogated in Keap1-/- MEFs. The compounds also displayed a suppressive effect on NF-κB signaling that was at least partly responsible for inhibition of lipopolysaccharideinduced inflammatory responses. These SCA analogues exhibited cytoprotective and anti-inflammatory activities and may be developed further as chemopreventive agents.

Induction of tumor cell death through targeting tubulin and evoking dysregulation of cell cycle regulatory proteins by multifunctional cinnamaldehydes.

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G(2)/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G(2) phase. G(2) arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G(2) to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G(2) phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.

Shogaols at proapoptotic concentrations induce G(2)/M arrest and aberrant mitotic cell death associated with tubulin aggregation.

Shogaols have been previously reported to induce cancer cell death via multiple mechanisms, among which one analog 6-shogaol has been reported to cause microtubule damage through specific reaction with sulfhydryl groups in tubulin. In this study, a series of shogaols with different side chain lengths (4-, 6-, 8- and 10-shogaol) was synthesized and evaluated for antiproliferative activity in HCT 116 colon carcinoma and SH-SY5Y neuroblastoma cells. 4- and 6-shogaol were identified as lead compounds possessing the strongest antiproliferative activity. In the soft agar assay, the lead shogaols displayed dose-dependent inhibition on cancer cell colony formation under anchorage-independent conditions. Using HCT 116 as the selected cancer cell line, the molecular events linking shogaols-induced G(2)/M cell cycle arrest to apoptosis characterized by caspase 3 and PARP cleavage were investigated. At sublethal concentrations, the halt at G(2)/M phase was alleviated along time and cells survived. Conversely, proapoptotic concentrations of 4- and 6-shogaol induced irreversible G(2)/M arrest that was at least in part associated with down-regulation of cell cycle checkpoint proteins cdk1, cyclin B and cdc25C, as well as spindle assembly checkpoint proteins mad2, cdc20 and survivin. A dose- and time-dependent accumulation of insoluble tubulin in the insoluble fractions of cell lysates provided evidence that G(2) checkpoint failure led to disruption of microtubule turnover. In summary, our results conclude that shogaols cause apoptosis by inducing aberrant mitosis at least through the attenuation of cell cycle and spindle assembly checkpoint proteins.

Efficacy study of vesicular gel containing methotrexate and menthol combination on parakeratotic rat skin model.

Methotrexate (MTX) is indicated in the symptomatic control of severe, recalcitrant, and disabling psoriasis. The oral or parenteral route of administration causes systemic toxicity. The topical route of delivery, though, reduces systemic toxicity and has limited applicability due to restricted permeability. Liposomal and niosomal MTX topical formulations have also been investigated with limited success to achieve drug localization in the skin. Menthol has been suggested in conditions of psoriasis, in addition to its skin-penetration-enhancing effect on drugs. The present work aimed at investigating the potential benefits of combining menthol with MTX in a vesicular gel base for not only improving the penetration and dermal availability of MTX, but also to render such a formulation more effective with greater patient acceptability. MTX liposomes were prepared by thin-film hydration, and the vesicles were characterized for drug-entrapment efficiency, size, and morphology. These liposomal vesicles were incorporated in a gel base, and this vesicular gel was evaluated for transdermal drug permeation and extent of drug accumulation in the skin, using a rat skin ex vivo model. Skin histology studies were carried out to investigate any structural changes caused by the permeation enhancers. Antipsoriatic efficacy of the formulations was tested in vivo, using the rat tail model. The results indicated that the vesicular gel containing menthol could cause maximum drug retention in the skin. The skin treated with menthol had a disrupted epidermis and microcavities. The in vivo studies also ascertained the effectiveness of the formulation in inducing a normal pattern of differentiation in the rat tail skin that initially showed parakeratosis, which is also characteristic of psoriatic epidermis. These results show the potential of vesicular gel containing MTX and menthol to improve penetration into the skin and cause drug retention in skin appendages.

Cinnamaldehydes inhibit thioredoxin reductase and induce Nrf2: potential candidates for cancer therapy and chemoprevention.

Trans-cinnamaldehyde (CA) and its analogs 2-hydroxycinnamaldehyde and 2-benzoyloxycinnamaldehyde have been reported to possess antitumor activity. CA is also a known Nrf2 activator. In this study, a series of ortho-substituted cinnamaldehyde analogs was synthesized and screened for antiproliferative and thioredoxin reductase (TrxR)-inhibitory activities. Whereas CA was weakly cytotoxic and TrxR inhibiting, hydroxy and benzoyloxy substitutions resulted in analogs with enhanced antiproliferative activity paralleling increased potency in TrxR inactivation. A novel analog, 5-fluoro-2-hydroxycinnamaldehyde, was identified as exhibiting the strongest antitumor effect (GI(50) 1.6 microM in HCT 116 cells) and TrxR inhibition (IC(50) 7 microM, 1 h incubation with recombinant TrxR). CA and its 2-hydroxy- and 2-benzoyloxy-substituted analogs possessed dual TrxR-inhibitory and Nrf2-inducing effects, both attributed to an active Michael acceptor pharmacophore. At lethal concentrations, TrxR-inhibitory potencies correlated with the compounds' antiproliferative activities. The penultimate C-terminal selenocysteine residue was shown to be a possible target. Conversely, at sublethal concentrations, these agents induced an adaptive antioxidant response through Nrf2-mediated upregulation of phase II enzymes, including TrxR induction. We conclude from the results obtained that TrxR inactivation contributes at least partly to cinnamaldehyde cytotoxicity. These Michael acceptor molecules can potentially be exploited for use in different concentrations in chemotherapeutic and chemopreventive strategies.