Bacterial enzymes DNA gyrase and topoisomerase IV are validated targets for the discovery of new antibacterial agents. Due to their structural similarities, it is possible to design compounds that can simultaneously inhibit both enzymes. Such compounds could be more successful in the fight against bacterial resistance which is rapidly spreading worldwide and poses a great threat to public health. We can target catalytic or ATP-binding sites of these enzymes. Catalytic inhibitors include clinically successful fluoroquinolones while ATP-competitive inhibitors currently do not have a representative on the market. Another group of DNA gyrase and topoisomerase IV inhibitors are the so-called new bacterial topoisomerase inhibitors.
In the framework of the doctoral dissertation, we focused on the ATP-competitive inhibitors and developed several series of new benzothiazole-based dual inhibitors of DNA-gyrase (GyrB) and topoisomerase IV (ParE). In addition to the classic series of molecules, we have also developed two smaller series of conjugates of our inhibitors with the catalytic inhibitor ciprofloxacin or with siderophore mimics, as hybrid compounds can also contribute to the slower emergence of resistant strains. The compounds were designed by structure-based design and chemically optimized, achieving stronger inhibition of both enzymes than previously published compounds, improved antibacterial activity and improved physicochemical properties. We have also patented all new inhibitors (PCT/EP2019/073412). We prepared compounds with low nanomolar inhibition of GyrB and ParE and very good antibacterial activity against Gram-positive (MIC values of the best compounds: 0.0078-0.25 µg/mL) and Gram-negative bacteria (MIC values of the best compounds: 0.5-16 µg/mL).
For compounds from different series, with the strongest antibacterial activities in vitro, we investigated their in vitro safety profile (cytotoxicity, genotoxicity, mitotoxicity, hERG and NaV1.5 inhibition, hemolysis, reactive metabolites) and selectivity against related enzymes such as human topoisomerase IIα and protein kinases. The compounds showed good selectivity and no significant toxicity. For three inhibitors, we obtained their crystal structures in the binding site of GyrB from Staphylococcus aureus or Pseudomonas aeruginosa, which confirm their predicted binding mode in the ATP-binding site. The three best compounds were tested in vivo in mouse models of dermal and systemic infections and displayed good efficacy. An in vivo effective inhibitor with morpholine attached to the benzothiazole bicycle was highlighted as the most promising compound, which, in addition to potent enzyme inhibitory activity and antibacterial activity, also exhibits relatively good physicochemical properties, pharmacokinetic profile, safety and selectivity. The newly discovered benzothiazole inhibitors thus represent important progress in the field of discovering new agents for the treatment of infections with resistant bacterial strains.
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