New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens.

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 µg/mL) and Gram-negative pathogens (MICs: range, 1-2 µg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.

Discovery of Novel Hsp90 C-Terminal Inhibitors Using 3D-Pharmacophores Derived from Molecular Dynamics Simulations.

Hsp90 C-terminal domain (CTD) inhibitors are promising novel agents for cancer treatment, as they do not induce the heat shock response associated with Hsp90 N-terminal inhibitors. One challenge associated with CTD inhibitors is the lack of a co-crystallized complex, requiring the use of predicted allosteric apo pocket, limiting structure-based (SB) design approaches. To address this, a unique approach that enables the derivation and analysis of interactions between ligands and proteins from molecular dynamics (MD) trajectories was used to derive pharmacophore models for virtual screening (VS) and identify suitable binding sites for SB design. Furthermore, ligand-based (LB) pharmacophores were developed using a set of CTD inhibitors to compare VS performance with the MD derived models. Virtual hits identified by VS with both SB and LB models were tested for antiproliferative activity. Compounds 9 and 11 displayed antiproliferative activities in MCF-7 and Hep G2 cancer cell lines. Compound 11 inhibited Hsp90-dependent refolding of denatured luciferase and induced the degradation of Hsp90 clients without the concomitant induction of Hsp70 levels. Furthermore, compound 11 offers a unique scaffold that is promising for the further synthetic optimization and development of molecules needed for the evaluation of the Hsp90 CTD as a target for the development of anticancer drugs.

In vitro estrogenic activity of binary and multicomponent mixtures with bisphenol A.

Bisphenol A and its analogs are environmental contaminants with well known estrogenic and anti-androgenic activities. In studies of human biomonitoring, simultaneous exposure to multiple bisphenols was shown in different biological samples, at picomolar to low nanomolar concentrations. Evaluation of their combined toxicities will therefore be a more realistic and reliable predictor for estimation of health risks than evaluation of only the single chemicals. In the present study, estrogenic activities of individual bisphenols were evaluated, along with their binary and multicomponent mixtures including three- and four-component mixtures, using the Organisation for Economic Co-operation and Development validated transactivation assay with the hERα-Hela9903 cell line. Concentration-dependent estrogenic activity was confirmed for all of the tested bisphenols, in the nanomolar to micromolar range. Estrogenic activities of binary and multicomponent mixtures followed a concentration addition model. Although exposure to individual bisphenols remains below their effective doses, we demonstrate that as a mixture, they can contribute additively to toxicity. This study thus emphasizes the importance of mixture toxicity evaluation for risk assessment of compounds that act like the bisphenols.

Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite.

Bisphenol AF (BPAF) is a fluorinated analog of bisphenol A (BPA), and it is a more potent estrogen receptor (ER) agonist. BPAF is mainly metabolized to BPAF-glucuronide (BPAF-G), which has been reported to lack ER agonist activity and is believed to be biologically inactive. The main goal of the current study was to examine the influence of the metabolism of BPAF via glucuronidation on its ER activity and adipogenesis. Also, as metabolites can have different biological activities, the effects of BPAF-G on other nuclear receptors were evaluated. First, in-vitro BPAF glucuronidation was investigated using recombinant human enzymes. Specific reporter-gene assays were used to determine BPAF and BPAF-G effects on estrogen, androgen, glucocorticoid, and thyroid receptor pathways, and on PXR, FXR, and PPARγ pathways. Their effects on lipid accumulation and differentiation were determined in murine 3T3L1 preadipocytes using Nile Red, with mRNA expression analysis of the adipogenic markers adiponectin, Fabp4, Cebpα, and PPARγ. BPAF showed strong agonistic activity for hERα and moderate antagonistic activities for androgen and thyroid receptors, and for PXR. BPAF-G was antagonistic for PXR and PPARγ. BPAF (0.1 µM) and BPAF-G (1.0 µM) induced lipid accumulation and increased expression of key adipogenic markers in murine preadipocytes. BPAF-G is therefore not an inactive metabolite of BPAF. Further toxicological and epidemiological investigations of BPAF effects on human health are warranted, to provide better understanding of the metabolic end-elimination of BPAF.

Comparison of in vitro hormone activities of novel flame retardants TBB, TBPH and their metabolites TBBA and TBMEPH using reporter gene assays.

The anti-androgenic and anti-thyroid hormonal activities of the two novel brominated flame retardants, TBB and TBPH and of their metabolites TBBA and TBMEPH have been compared using the luciferase reporter gene assays. Only the parent compounds TBB and TBPH exhibited anti-glucocorticoid activity with IC50 values of 1.9 µM and 0.3 µM. Furthermore, mode of action for these two compounds is by direct competing to the glucocorticoid receptor (GR) with IC50 values of 0.03 µM and 0.002 µM. All four tested compounds possess anti-androgenic and anti-thyroid hormonal activities, without agonist activities on the respective receptors. Anti-androgenic activities with IC50 values of 43.5 µM, 0.1 µM, 47.5 µM and 1.3 µM were found for TBB, TBPH, TBBA and TBMEPH. The anti-thyroid hormonal IC50 values of 37.5 µM, 0.1 µM, 22.8 µM and 32.3 µM for TBB, TBPH, TBBA and TBMEPH, together with the above quoted results, indicate that metabolism can modify anti-androgenic, anti-glucocorticoid and anti-thyroid hormonal effects of these novel brominated flame retardants. Furthermore, the parent flame retardants are shown to be able to disrupt the function of the GR as antagonists by direct competition to the receptor.

Influence of metabolism on endocrine activities of bisphenol S.

Bisphenol S (BPS; bis[4-hydroxyphenyl]sulfone) is commonly used as a replacement for bisphenol A in numerous consumer products. The main goal of this study was to examine the influence of different metabolic reactions that BPS undergoes on the endocrine activity. We demonstrate that hydroxylation of the aromatic ring of BPS, catalyzed mainly by the cytochrome P450 enzymes CYP3A4 and CYP2C9, is its major in-vitro phase I biotransformation. Nevertheless, coupled oxidative-conjugative reactions analyses revealed that glucuronidation and formation of BPS glucuronide is the predominant BPS metabolic pathway. BPS reactive metabolites that can be tracked as glutathione conjugates were not detected in the present study. Two in-vitro systems were used to evaluate the endocrine activity of BPS and its two main metabolites, BPS glucuronide and hydroxylated BPS 4-(4-hydroxy-benzenesulfonyl)-benzene-1,2-diol (BPSM1). In addition, we have tested two structural analogs of BPS, bis[4-(2-hydroxyetoxy)phenyl]sulfone (BHEPS) and 4,4-sulfonylbis(2-methylphenol) (dBPS). The test systems were yeast cells, for evaluating estrogenic and androgenic activities, and the GH3.TRE-Luc reporter cell line for measuring thyroid hormone activity. BPS and BPSM1 were weak agonists of the estrogen receptor, EC50 values of 8.4 × 10(-5) M and 6.7 × 10(-4) M, respectively. Additionally, BPSM1 exhibited weak antagonistic activity toward the thyroid hormone receptor, with an IC50 of 4.3 × 10(-5) M. In contrast to BPSM1, BPS glucuronide was inactive in these assays, inhibiting neither the estrogen nor the thyroid hormone receptors. Hence, glucuronidation appears to be the most important pathway for both BPS metabolism and detoxification.