High-performance liquid chromatography evaluation of lipophilicity and QSRR modeling of a series of dual DNA gyrase and topoisomerase IV inhibitors

Bacterial DNA gyrase and topoisomerase IV control the topological state of DNA during replication and represent important antibacterial drug targets. To be successful as drug candidates, newly synthesized compounds must possess optimal lipophilicity, which enables efficient delivery to the site of action. In this study, retention behavior of twenty-three previously synthesized dual DNA gyrase and topoisomerase IV inhibitors was tested in RP-HPLC system, consisting of C8 column and acetonitrile/phosphate buffer (pH 5.5 and pH 7.4) mobile phase. logD was calculated at both pH values and the best correlation with logD was obtained for retention parameter φ0, indicating that this RP-HPLC system could be used as an alternative to the shake-flask determination of lipophilicity. Subsequent QSRR analysis revealed that intrinsic lipophilicity (logP) and molecular weight (bcutm13) have a positive, while solubility (bcutp3) has a negative influence on this retention parameter

Exploring the 5-Substituted 2-Aminobenzothiazole-Based DNA Gyrase B Inhibitors Active against ESKAPE Pathogens

We present a new series of 2-aminobenzothiazole-basedDNA gyraseB inhibitors with promising activity against ESKAPE bacterial pathogens.Based on the binding information extracted from the cocrystal structureof DNA gyrase B inhibitor A, in complex with Escherichia coli GyrB24, we expanded the chemicalspace of the benzothiazole-based series to the C5 position of thebenzothiazole ring. In particular, compound E showedlow nanomolar inhibition of DNA gyrase (IC50 < 10 nM)and broad-spectrum antibacterial activity against pathogens belongingto the ESKAPE group, with the minimum inhibitory concentration <0.03 & mu;g/mL for most Gram-positive strains and 4-16 & mu;g/mLagainst Gram-negative E. coli, Acinetobacter baumannii, Pseudomonasaeruginosa, and Klebsiella pneumoniae. To understand the binding mode of the synthesized inhibitors, acombination of docking calculations, molecular dynamics (MD) simulations,and MD-derived structure-based pharmacophore modeling was performed.The computational analysis has revealed that the substitution at positionC5 can be used to modify the physicochemical properties and antibacterialspectrum and enhance the inhibitory potency of the compounds. Additionally,a discussion of challenges associated with the synthesis of 5-substituted2-aminobenzothiazoles is presented

New N-phenylpyrrolamide DNA gyrase B inhibitors: Optimization of efficacy and antibacterial activity

The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several small-molecule inhibitor classes have been discovered but none has so far reached the market. We present here the discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC50 values against DNA gyrase, and submicromolar IC50 values against topoisomerase IV from Escherichia coil and Staphylococcus aureus. The most potent compound in the series has an IC50 value of 13 nM against E. coil gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive bacteria are in the low micromolar range. The oxadiazolone derivative with an IC50 value of 85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC values of 1.56 mu M against Enterococcus faecalis, and 3.13 mu M against wild type S. aureus, methicillinresistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PA beta N) is 4.6 mu M. (C) 2018 Elsevier Masson SAS. All rights reserved.Peer reviewe

An optimised series of substituted N-phenylpyrrolamides as DNA gyrase B inhibitors

ATP competitive inhibitors of DNA gyrase and topoisomerase IV have great therapeutic potential, but none of the described synthetic compounds has so far reached the market. To optimise the activities and physicochemical properties of our previously reported N-phenylpyrrolamide inhibitors, we have synthesized an improved, chemically variegated selection of compounds and evaluated them against DNA gyrase and topoisomerase IV enzymes, and against selected Gram-positive and Gram-negative bacteria. The most potent compound displayed IC50 values of 6.9 nM against Escherichia coli DNA gyrase and 960 nM against Staphylococcus aureus topoisomerase IV. Several compounds displayed minimum inhibitory concentrations (MICs) against Gram-positive strains in the 1-50 mu M range, one of which inhibited the growth of Enterococcus faecalis, Enterococcus faecium, S. aureus and Streptococcus pyogenes with MIC values of 1.56 mu M, 1.56 mu M, 0.78 mu M and 0.72 mu M, respectively. This compound has been investigated further on methicillin-resistant S. aureus (MRSA) and on ciprofloxacin non-susceptible and extremely drug resistant strain of S. aureus (MRSA VISA). It exhibited the MIC value of 2.5 mu M on both strains, and MIC value of 32 mu M against MRSA in the presence of inactivated human blood serum. Further studies are needed to confirm its mode of action. (C) 2019 Elsevier Masson SAS. All rights reserved.Peer reviewe

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. 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 mg/mL) and Gram-negative pathogens (MICs: range, 1-2 mg/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. (C) 2021 Elsevier Masson SAS. All rights reserved.Peer reviewe

Hybrid Inhibitors of DNA Gyrase A and B: Design, Synthesis and Evaluation

The discovery of multi-targeting ligands of bacterial enzymes is an important strategy to combat rapidly spreading antimicrobial resistance. Bacterial DNA gyrase and topoisomerase IV are validated targets for the development of antibiotics. They can be inhibited at their catalytic sites or at their ATP binding sites. Here we present the design of new hybrids between the catalytic inhibitor ciprofloxacin and ATP-competitive inhibitors that show low nanomolar inhibition of DNA gyrase and antibacterial activity against Gram-negative pathogens. The most potent hybrid 3a has MICs of 0.5 &micro;g/mL against Klebsiella pneumoniae, 4 &micro;g/mL against Enterobacter cloacae, and 2 &micro;g/mL against Escherichia coli. In addition, inhibition of mutant E. coli strains shows that these hybrid inhibitors interact with both subunits of DNA gyrase (GyrA, GyrB), and that binding to both of these sites contributes to their antibacterial activity

New dual DNA gyrase and topoisomerase IV inhibitors active against resistant bacterial strains and with low potential for resistance development

Bakterijska encima DNA-giraza in topoizomeraza IV sta validirani tarči za odkrivanje novih protibakterijskih učinkovin. Zaradi svoje strukturne podobnosti omogočata načrtovanje spojin, ki lahko sočasno zavirajo oba encima, take spojine pa so lahko uspešnejše v boju proti bakterijski odpornosti, ki se hitro širi po celem svetu in predstavlja veliko grožnjo javnemu zdravju. Med drugimi poznamo katalitične zaviralce teh encimov, med katere uvrščamo klinično uspešne spojine fluorokinolone, t.i. nove zaviralce bakterijskih topoizomeraz ter ATP-kompetitivne zaviralce, ki trenutno še nimajo svojega predstavnika na trgu. V okviru doktorske disertacije smo se posvetili ATP-kompetitivnim zaviralcem in razvili več serij novih dvojnih zaviralcev DNA-giraze (GyrB) in topoizomeraze IV (ParE) benzotiazolnega tipa. Razvili smo tudi dve manjši seriji konjugatov naših zaviralcev s katalitičnim zaviralcem ciprofloksacinom ali s siderofornimi fragmenti, saj lahko tudi hibridne spojine pripomorejo k počasnejšemu pojavu odpornih sevov. Spojine smo načrtovali s strukturno podprtim načrtovanjem in jih kemijsko optimizirali, s čimer smo dosegli boljšo jakost zaviranja obeh encimov kot predhodno objavljene spojine, izboljšano protibakterijsko delovanje in izboljšane fizikalno-kemijske lastnosti. Vse nove zaviralce smo tudi patentno zaščitili (PCT/EP2019/073412). Pripravili smo spojine z nizko nanomolarno jakostjo zaviranja GyrB in ParE ter zelo dobrim protibakterijskim delovanjem na po Gramu pozitivne (MIK vrednosti najboljših spojin: 0,0078-0,25 µg/mL) in po Gramu negativne bakterije (MIK vrednosti najboljših spojin: 0,5-16 µg/mL). Pri spojinah iz različnih serij, z najmočnejšim protibakterijskim učinkom in vitro, smo raziskovali njihov varnostni profil in vitro in selektivnost napram sorodnim encimom ter pokazali, da izkazujejo dobro selektivnost napram humani topoizomerazi II? ter protein kinazam, prav tako niso problematične z vidika varnosti (citotoksičnost, genotoksičnost, mitotoksičnost, zaviranje hERG in NaV1.5, hemoliza, nastanek reaktivnih metabolitov). Za tri zaviralce smo razrešili kristalne strukture spojin v vezavnem mestu GyrB iz bakterij Staphylococcus aureus ali Pseudomonas aeruginosa, ki prikazujejo in potrjujejo predvideni način vezave naših spojin v ATP-vezavno mesto. Tri najboljše spojine smo testrali in vivo na mišjih modelih dermalnih in sistemskih okužb in dokazali njihovo učinkovitost. Kot najobetavnejšo spojino lahko izpostavimo in vivo učinkoviti zaviralec, ki ima na benzotiazol pripet morfolin, in ki poleg dobre encimske zaviralne aktivnosti in protibakterijskega delovanja izkazuje tudi ustrezne fizikalno-kemijske lastnosti, farmakokinetični profil, varnost in selektivnost. Novo odkriti benzotiazolni zaviralci tako predstavljajo pomemben napredek na področju odkrivanja novih učinkovin za zdravljenje okužb z odpornimi bakterijskimi sevi.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

Estrogenic potency of endocrine disrupting chemicals and their mixtures detected in environmental waters and wastewaters

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 EC$_{50}$ 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