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