We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.

Endocrine disrupting chemicals such as natural and synthetic steroid hormones and bisphenols are among the most important pollutants in the aquatic environment. We performed an environmental chemical analysis of five Slovenian water samples, two rivers, one groundwater, and the influent and effluent of wastewater treatment plants, with a highly sensitive analysis of twenty-five endocrine-disrupting compounds belonging to the groups of natural hormones, synthetic hormones, and bisphenols. Since these compounds are simultaneously present in the environment, it is important to study their individual effects as well as the effects of mixtures. We investigated in vitro the estrogenic potency of selected natural and synthetic steroid hormones and bisphenols detected in surface, ground and waste water in Slovenia using the OECD-validated transactivation assay on the cell line Hela9903. We predicted their mixture effects using the concentration addition model and compared them with experimentally determined values. Two mixing designs were used: a balanced design in which chemicals were combined in proportion to their individual EC₅₀ values, and an unbalanced design with compounds in proportion to their measured concentrations in the environmental samples. The estrogenic effects of the experimental mixtures followed the concentration addition model. Real water samples exhibited weaker estrogenic effects, showing the great heterogeneity of the real water samples.

A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125-0.25 μg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1-4 μg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated.

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.

Heat shock protein 90 (Hsp90) is a chaperone responsible for the maturation of many cancer-related proteins, and is therefore an important target for the design of new anticancer agents. Several Hsp90 N-terminal domain inhibitors have been evaluated in clinical trials, but none have been approved as cancer therapies. This is partly due to induction of the heat shock response, which can be avoided using Hsp90 C-terminal-domain (CTD) inhibition. Several structural features have been shown to be useful in the design of Hsp90 CTD inhibitors, including an aromatic ring, a cationic center and the benzothiazole moiety. This study established a previously unknown link between these structural motifs. Using ligand-based design methodologies and structure-based pharmacophore models, a library of 29 benzothiazole-based Hsp90 CTD inhibitors was prepared, and their antiproliferative activities were evaluated in MCF-7 breast cancer cells. Several showed low-micromolar IC₅₀, with the most potent being compounds 5g and 9i (IC₅₀, 2.8 ± 0.1, 3.9 ± 0.1 μM, respectively). Based on these results, a ligand-based structure–activity relationship model was built, and molecular dynamics simulation was performed to elaborate the binding mode of compound 9i. Moreover, compound 9i showed degradation of Hsp90 client proteins and no induction of the heat shock response.