PowerPoint Presentation

Developing DNA topoisomerases as targets for antibacterial chemotherapyCoombe Abbey, Sept 2016Tony MaxwellDept Biological ChemistryJohn Innes CentreNorwich, UK

Take-home messagesWhat we do is a very small slice of a very big cakeDNA topoisomerases (esp. DNA gyrase) are valuable targets for antibacterial chemotherapyFluoroquinolones are excellent antibiotics, but resistance is a serious problemThere is significant scope to further exploit topoisomerases as targets2

Inhibitors of DNA gyrase

InhibitorRelevanceCommentQuinolones (e.g. ciprofloxacin)Antibacterial agentsHighly successful billion dollar drugsAminocoumarins (e.g. coumermycin)Antibacterial agentsRelatively unsuccessful as drugsSimocyclinonePotential antibacterial agentNew amino coumarin compoundsMccB17Bacterial toxinPotential for design of new agents?CcdBBacterial toxinPotential for design of new agents?

Cyclothialidines, clerocidin, albicidin, naphthoquinones (e.g. diospyrin), etc.

3

OverviewDNA topology & DNA topoisomerasesDNA gyraseGyrase mechanismQuinolone antibioticsAminocoumarin antibioticsSimocyclinonesmm4tbENABLE4

DNA topology & DNA topoisomerases

Replication elongation

TranscriptionVarious biological processes lead to DNA topological problems, e.g. introduction of unwanted supercoiled and catenanes5

DNA topoisomerasesEssential in all cells to control DNA topology (e.g. in replication)All can relax DNA, but gyrase can catalyse supercoilingGyrase essential to bacteria but absent from most eukaryotes (plants and plasmodial parasites)Strand-passagemechanism; drugs canstabilise cleavage complex

6

DNA Topoisomerases

7There are currently no antibacterial agents targeted to type I topoisomerases

8

S. cerevisiae topo IIHuman topo IIHuman topo IIE. coli topo IVE. coli gyraseS. shibatae topo VIIIAIIBATPaseDNA binding/cleavage

N--CPar EPar CGyrBGyrATyrATPaseBATyr660116414286781171153169411831626630752804875530389550719Type II topoisomerases

Type II topoisomerase reactions

9

Type II topoisomerase mechanism

10

10

Mechanism of type II topos

11Joaquim Roca

Mechanism of DNA supercoiling by gyrasequinolonesaminocoumarinssimocyclinonesCostenaro, et al. (2007). Structure 15, 329-39.

12

13

Quinolone drugs

O

O

O

+ATP+DNAO

5'3'

3'

5'

3'

5'

5'3'

3'5'5'3'OH

OH

OH

O

Chemistry of gyrase-mediated DNA cleavage

3'

5'5'3'

Stabilisation of cleavage complex by quinolones14

Laponogov, et al., (2009) Nat. Struct. Mol. Biol., 16, 667-669.

Crystal structure of S. pneumoniae topo IV in complexes with DNA and quinolone drugs

15Fisher/Sanderson

A. baumannii ParE28-ParC58 complex with DNA and moxifloxacin; quinolone-Mg2+ complex is intercalated in the DNA at the cleavage site Interaction of moxifloxacin with Acinetobacter baumannii topoisomerase IV

Wohlkonig, et al. (2010) Nat Struct Mol Biol 17, 1152-3

16GSK

What are we looking for?New compounds with antibacterial activity that show inhibition of gyrase-catalysed supercoiling (and relaxation?)

Ideally compounds that also stabilise the gyrase-DNA cleavage complex, like quinolones, but that have a novel site of action

Involved in both MIC-based and target-based approaches

17

Aminocoumarin antibiotics

ClorobiocinNovobiocin

OCH3CH3CH3OOOHOOONHCOOHOHCONH2CH3

NH3CHOCH3CH3CH3OOOHOOONHCOOHOHCOCl

Coumermycin A1NHNH3CHOCH3CH3CH3OOOHOOONHOHCOCOH3CCONCH3HOH3COCH3OOHOOONHOHCOH3CCH3H3C

ATP18

Structure of DNA gyrase

Bush, et al. (2015). EcoSal. ASM Press

Wigley et al. Nature, 351, 624 (1991).Lewis, et al. EMBO J. 15, 1412-1420 (1996). ATP

ATP

Novobiocin

GyrB-NTD (43 kDa) with ADPNPGyrB-NTD-sub-domain (24 kDa) with novobiocin20

New aminocoumarins

21

Possible to generate large variety of novclobiocins using combinatorial biosynthesis, mutasynthesis and synthetic biology (Heide).Compounds with improved IC50s vs gyrase and topo IV.However, aminocoumarins tend to suffer from drawbacks:ToxicitySolubilityPoor Gm ve activity

Heide (2014) Int J Med Microbiol, 304, 31-36; Alt, et al. (2011) J Antimicrob Chemother, 66, 2061-2069

Simocyclinone D8

3-amino-4,7-dihydroxycoumarinD-olivoseTetraene dicarboxylic acidAngucyclic polyketideGenerally poor antibiotic activity, except vs certain Gram positivesPotent inhibitor of DNA gyrase, but not at ATPase site on GyrB but at DNA-binding site on GyrAFlatman et al. (2005) Antimicrob Agents Chemother 49, 1093-1100

OCH3CH3CH3OOOHOOONHCOOHOHCONH2CH3

NovobiocinIsolated from Streptomyces antibioticus (T 6040)22

OrganismMIC (g/mL)D4D8Bacillus subtilis DSM 10>100>100Bacillus brevis DSM 303010Staphylococcus aureus DSM 20231100100Streptomyces viridochromogenes T 5711Streptomyces coelicolor MLLER DSM 30301010Streptomyces antibiotics T 6040>100>100

Schimana et al. (2000) J. Antibiot. (Tokyo), 53, 779Antibacterial effects of simocyclinones23But more recent report suggests better activity of simocyclinone vs Gm ve clinical isolates (Richter, et al. (2010) Bioorg Med Chem Lett, 20, 1202-1204)

Edwards, et al. (2009) Science, 325, 1415-1418Crystal structure of the bifunctional antibiotic, simocyclinone D8, bound to DNA gyrase (GyrA59)

59 kDa35 kDa

H2NCOOHDNA breakage-reunion domainDNA wrappingE. coli GyrAMarcus Edwards24

24

25

Concerns about GyrA59 structureMutants dont all make senseNot clear that cross-linked tetramer could form in vivoMass spec data suggest that GyrA59 dimer can bind two SD8s

Mass spec of A59; SD8 titration. Shows that with increasing [SD8] a dimer species with 2 SD8s bound predominatesMarcus EdwardsAdam McKay26

Edwards, et al. (2011). Biochemistry 50, 3432.27

GyrA55-SD8 complexHearnshaw, S. J., et al. (2014). J. Mol. Biol., 426, 2023-2033Steve Hearnshaw

28

SimocyclinoneQuinolone29

Quinolone-aminocoumarin hybrids

Quinolone derivativesIC50 100 M IC50 75 M

Quinolone-aminocoumarin hybrids

IC50 50 MCC50 >100 MIC50 1 MCC50 10 MIC50 10 MCC50 10 M

Ciprofloxacin: IC50 1 MCC50 1 M30Austin, et al. (2016) Med. Chem. Commun., 7, 1387-1391

Austin/Searcey

OverviewDNA topology & DNA topoisomerasesDNA gyraseGyrase mechanismQuinolone antibioticsAminocoumarin antibioticsSimocyclinonesmm4tbENABLE31

WP1: hit generationScreen on TB model strainsH37Rv (multiplying)SS18b (dormant)(streptomycin-dependent)WP2: hit to targetTarget identificationResistant mutantsGenome sequencingTarget fishingWP3: target to drugTarget validationIsolationAssay designWP4: enabling chemistryHit to Lead chemistrySupporting chemistryEfficacy studyCandidate drugs32MM4TB pipelineTarget = gyrase?Mode of action?Kill TB

32EU consortium: more medicines for tuberculosis (25 labs)Fred CollinNatassja BushJenny PritchardSteve HearnshawSara HendersonClara Franch

mm4tb achievementsDevelopment of M.tub gyrase in terms of structure, function & drug targeting aspects (Nagaraja)>2000 compounds screened against M.tub gyrase>60 compounds found with IC50s