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Človeška DNA-topoizomeraza II je dobro znana in pomembna tarča protirakavih učinkovin. Zaradi omejitev trenutno klinično uporabljenih zaviralcev te tarče poteka intenzivno raziskovanje z namenom iskanja zaviralcev z alternativnimi mehanizmi delovanja, med katerimi velik potencial kažejo ATP-kompetitivni zaviralci. ATP-vezavno mesto človeške DNA-topoizomeraze II vsebuje edinstvene strukturne elemente, ki so značilni za naddružino ATPaz GHKL. Proteini znotraj te naddružine, med katere spadajo tudi bakterijske topoizomeraze tipa II, si delijo visoko trodimenzionalno podobnost ATP-vezavnih mest. S pomočjo sistematičnega rešetanja hišne knjižnice ATP-kompetitivnih zaviralcev bakterijske DNA-giraze in topoizomeraze IV smo odkrili nov kemotip ATP-kompetitivnih zaviralcev človeške DNA-topoizomeraze II. Prvotno smo identificirali 20 spojin zadetkov različnih strukturnih tipov, kot najbolj obetajoče smo za nadaljnjo optimizacijo izbrali 1,2,4-substituirane N-fenilpirolamide. S pomočjo preliminarnega odnosa med strukturo in delovanjem (SAR), ki je temeljil na primarnih N-fenilpirolamidnih zadetkih in strukturno podprtem načrtovanju, smo pripravili nove serije zaviralcev DNA-topoizomeraze II. Nove zaviralce lahko razdelimo na šest strukturnih tipov, pri čemer smo pri vseh ohranili N-fenilpirolamidni skelet. Izpostavimo lahko spojine tipa IV in VI zaradi njihove zelo dobre zaviralne aktivnosti na DNA-topoizomerazi II (vrednosti IC50 v submikromolarnem in nizkem mikromolarnem območju), pa tudi zaradi odlične citotoksične aktivnosti na rakavih celičnih linijah MCF-7 in HepG2, pri čemer izstopa spojina 130g s submikromolarno vrednostjo IC50 na celični liniji HepG2. Spojina 46d prav tako kaže odlično citotoksično aktivnost na rakavih celičnih linijah MCF-7 in HepG2 kljub slabši zaviralni aktivnosti na DNA-topoizomerazi II. Visoka citotoksičnost te spojine je najverjetneje posledica dualnega zaviralnega delovanja na DNA-topoizomerazo II in Hsp90, ki je tudi znana tarča v razvoju novih protirakavih učinkovin. Nadaljnji razvoj dualnih zaviralcev DNA-topoizomeraze II in Hsp90 bi bil zanimiv pristop razvoja novih protirakavih spojin, ki je zaenkrat glede na literaturo še precej neraziskan. Spojine 46d, 112a in 112b (slednji sta primera spojin strukturnega tipa VI) so bile ovrednotene tudi na panelu rakavih celičnih linij NCI-60, kjer so pokazale zelo dobre citotoksične aktivnosti, zlasti proti celičnim linijam raka debelega črevesa in centralnega živčnega sistema ter melanomu. Spojina 46d ter spojine strukturnih tipov IV in VI (še posebej 130g) tako kažejo potencial za nadaljnji razvoj visoko citotoksičnih zaviralcev DNA-topoizomeraze II kot potencialnih protirakavih učinkovin. Human DNA topoisomerase II is a well-known and important target of anticancer drugs. Due to limitations of the currently clinically used inhibitors of this target, there is intense research ongoing to find compounds with alternative mode of inhibition, among which ATP-competitive inhibitors show the highest potential. The ATP-binding site of human DNA topoisomerase II contains unique structural elements specific to the GHKL superfamily of ATPases. Members of the GHKL superfamily, which include bacterial topoisomerases type II, share a high three-dimensional similarity of ATP-binding sites. Through systematic screening of the in house library of ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV, a new chemotype of ATP-competitive inhibitors of human DNA topoisomerase II was discovered. Initially, we identified 20 hit compounds of different structural types. As the most promising, 1,2,4-substituted N- phenylpyrrolamides were selected for further optimization. New series of topoisomerase II inhibitors were prepared with the help of the preliminary structure-activity relationship (SAR) that was based on the primary N-phenylpyrrolamide hits and the structure-based design. New inhibitors can be divided into six structural types, all of which possess a N-phenylpyrrolammide scaffold. Compounds of types IV and VI were selected because of their very good inhibitoy activity on topoisomerase II (IC50 values in the submicromolar and low micromolar range) as well as ther excellent cytotoxic activity on MCF-7 and HepG2 cancer cell lines. The outstanding compound is 130g with submicromolar IC50 value on HepG2 cancer cell line. Compound 46d, despite weaker topo IIα inhibitory activity, also shows excellent cytotoxic activity on cancer cell lines MCF-7 and HepG2, which is probably due to its dual inhibitory activity on topoisomerase II and Hsp90, which is also a known target in new anticancer drug development. Further development of such dual topoisomerase II and Hsp90 inhibitors would be an interesting approach of development of new highly cytotoxic compounds, which has not yet been well investigated according to our literature search. Compounds 46d, 112a and 112b (the latter two represent compounds of structural type VI) were also evaluated on NCI-60 panel of cancer cell lines where they showed very good cytotoxic activities, especially against colon and central nervous system cancer and melanoma cell lines. Compound 46d and compounds of structural types IV and VI (especially 130g) therefore show potential to be further developed into potent topoisomerase II inhibitors as potential anticancer drugs.

DNA gyrase is an important target for the development of novel antibiotics. Although ATP-competitive DNA gyrase (GyrB) inhibitors are a well-studied class of antibacterial agents, there is currently no representative used in therapy, largely due to unwanted off-target activities. Selectivity of GyrB inhibitors against closely related human ATP-binding enzymes should be evaluated early in development to avoid off-target binding to homologous binding domains. To address this challenge, we developed selective 3D-pharmacophore models for GyrB, human topoisomerase IIα (TopoII), and the Hsp90 N-terminal domain (NTD) to be used in in silico activity profiling paradigms to identify molecules selective for GyrB over TopoII and Hsp90, as starting points for hit expansion and lead optimization. The models were used to profile highly active GyrB, TopoII, and Hsp90 inhibitors. Selected compounds were tested in in vitro assays. GyrB inhibitors 1 and 2 were inactive against TopoII and Hsp90, while 3 and 4, potent Hsp90 inhibitors, displayed no inhibition of GyrB and TopoII, and TopoII inhibitors 5 and 6 were inactive at GyrB and Hsp90. The results provide a proof of concept for the use of target activity profiling methods to identify selective starting points for hit and lead identification.

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 µg/mL against Klebsiella pneumoniae, 4 µg/mL against Enterobacter cloacae, and 2 µg/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.

PLoS biology 18(10), e3000819 (1-31) - (2020). doi:10.1371/journal.pbio.3000819, Antibiotics that inhibit multiple bacterial targets offer a promising therapeutic strategy against resistance evolution, but developing such antibiotics is challenging. Here we demonstrate that a rational design of balanced multitargeting antibiotics is feasible by using a medicinal chemistry workflow. The resultant lead compounds, ULD1 and ULD2, belonging to a novel chemical class, almost equipotently inhibit bacterial DNA gyrase and topoisomerase IV complexes and interact with multiple evolutionary conserved amino acids in the ATP-binding pockets of their target proteins. ULD1 and ULD2 are excellently potent against a broad range of gram-positive bacteria. Notably, the efficacy of these compounds was tested against a broad panel of multidrug-resistant Staphylococcus aureus clinical strains. Antibiotics with clinical relevance against staphylococcal infections fail to inhibit a significant fraction of these isolates, whereas both ULD1 and ULD2 inhibit all of them (minimum inhibitory concentration [MIC] ≤1 μg/mL). Resistance mutations against these compounds are rare, have limited impact on compound susceptibility, and substantially reduce bacterial growth. Based on their efficacy and lack of toxicity demonstrated in murine infection models, these compounds could translate into new therapies against multidrug-resistant bacterial infections., Published by PLoS, Lawrence, KS