Methanopyrus kandleri topoisomerase V contains three distinct AP ...
Introduction Mode of Inhibition Killing Kinetics Combination ......2019/04/15 · BWC0977, a novel...
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BWC0977, a novel dual target topoisomerase inhibitor: antimicrobial potency, spectrum and mechanism of action. S. Hameed, S. Sharma, R. Nandishaiah, N. Katagihallimath, N. Bharatham, A. Shanbhag, S. Rajagopal, P. Bhowmik, P. Amar, R. Narjari, V. Ramachandran, S. Datta & V. Balasubramanian Bugworks Research India Pvt. Ltd. Bengaluru 560065; email: [email protected] Killing Kinetics Combination Studies Introduction Mode of Inhibition Target Inhibition across Species Interaction Residues of Gyrase Acknowledgement References Effective against Ciprofloxacin Resistant Clinical Isolates Post Antibiotic Effect Resistant Mutant Generation SOS Response to DNA Damage • We have identified an antibacterial preclinical candidate BWC0977 that is equipotent against targets - Gyrase and Topoisomerase IV. It exhibits broad spectrum antibacterial activity against the ESKAPE pathogens. • BWC0977 inhibits gyrase by stabilizing single strand breaks in the DNA unlike Ciprofloxacin that stabilizes double strand breaks. • Computational studies predicted that the residues M120, D82 and R121 of GyrA are important for interacting with BWC0977. The upward shift in inhibition of supercoiling activity of the corresponding mutant enzymes confirms these interactions. • BWC0977 triggers an SOS response in bacterial cells similar to Ciprofloxacin through the induction of recA, recN, sulA and lexA promoters 1,2 . • BWC0977 is bactericidal, shows time dependent kill and a post antibiotic effect of 1.5 to 2 hrs. • Efforts to raise resistant mutants against BWC0977 in Escherichia coli and other Gram-negative organisms by single step methods yielded no mutants indicating very low resistance frequency. Though mutants obtained by long serial passage experiments showed a shift in MIC, sequencing revealed no target mutations. • Side chains of the M120 residues at the GyrA dimer interface forms strong hydrophobic interactions with the pyrazino-oxazinone ring of BWC0977. • -NH of pyrazino-oxazinone ring forms H-bond with D82. • R121 interacts with Oxazolidinone ring of BWC0977. R121 M120 D82 R121 M120 DG10 DG10 DC11 DC11 Major Interaction residues Ciprofloxacin : S83L BWC0977 : M120K & D82N E. coli gyrase SC, IC 50 in µM Compound WT S83L M120K D82N BWC0977 0.004 0.013 92 3.6 Ciprofloxacin 0.183 11.6 0.91 3.1 The shift in IC 50 of BWC0977 with M120K and D82N mutant gyrase enzymes confirms their interactions invitro. BWC0977 inhibits gyrase and topo IV with nanomolar potency across species. Ciprofloxacin shows higher micromolar inhibition on S. aureus gyrase activity. IC 50 (µM) Compound E. coli P. aeruginosa S. aureus Gyrase Topo IV Gyrase Topo IV Gyrase Topo IV BWC0977 0.004 0.01 0.009 0.071 0.01 0.095 Ciprofloxacin 0.189 16.3 0.244 22.9 25.5 8.5 Cleavage-complex assays were performed with E. coli gyrase using supercoiled plasmid in the absence of ATP to study the mode of inhibition of compounds. • E. coli SOS-inducible promoters fused to fast-folding green fluorescent protein (GFP) on a low-copy plasmid in E. coli MG1655 were used as reporter strains 2 . • The selected reporter strains were treated with varying concentrations of compounds during early growth phase (A 600 = 0.2, growing at 37°C ) and the GFP fluorescence was measured at the end of 6 hours of induction. • RFUs were normalized to the cell growth and fold induction was measured as a ratio of the treated vs. untreated cultures. C iprofloxacin B W C 0977 G epotidacin R ifam p ic in 0 5 10 15 20 recA Fold induction 2 X M IC 1 X M IC 1/2 X M IC 1/4 X M IC C iprofloxacin B W C 0977 G epotidacin R ifam p ic in 0 5 10 15 sulA Fold induction C iprofloxacin B W C 0977 G epotidacin R ifam p ic in 0 5 10 15 recN Fold induction C iprofloxacin B W C 0977 G epotidacin R ifam p ic in 0 5 10 lexA Fold induction • Single step selection methods to raise resistant mutants yielded no mutants. • A serial passage MIC protocol was done to obtain mutants in both wildtype and ∆tolC E. coli. Serial passage cycles (Days) MIC(μg/mL) 0 5 10 15 20 25 30 35 40 0.0001 0.001 0.01 0.1 1 10 E.coli WT E. coli DtolC After removal of drug pressure A 100-fold shift in MIC was observed in the wildtype E. coli at the end of 35 days while 10-fold shift was observed in ∆tolC. Individual colonies isolated from the wildtype culture were grown in the absence of drug pressure. The MIC of these stable mutants were 0.5µg/ml. The gyrA and parC genes of these mutants were sequenced. No mutations were identified in the target genes. BWC0977 was bactericidal in quinolone resistant clinical strains in a time dependent pattern. BWC0977 post antibiotic effect is similar to Ciprofloxacin. PAE in hours Concentration BWC0977 Ciprofloxaci n 1X MIC 0.28 0.82 2X MIC 1.35 1.1 4X MIC 1.6 1.6 8X MIC 2.5 >2.5 Antibiotic FIC (A) antibiotic FIC (B) BWC0977 FIC index (A+B) Interaction Amoxicillin 0.75 0.5 1.25 Indifference Ampicillin 0.5 0.5 1 Indifference Azithromycin 0.5 0.5 1 Indifference Aztreonam 0.5 0.5 1 Indifference Cefotaxime 0.25 0.5 0.75 Additive Cefepime 0.125 0.5 0.625 Additive Chloramphenicol 0.5 0.25 0.75 Additive Ciprofloxacin 0.125 0.5 0.625 Additive Clarithromycin 0.5 0.25 0.75 Additive Clindamycin 0.5 0.25 0.75 Additive Erythromycin 0.75 0.5 1.25 Indifference Kanamycin 0.25 0.5 0.75 Additive Levofloxacin 0.25 0.5 0.75 Additive Meropenem 0.5 0.5 1 Indifference Metronidazole 0.75 0.5 1.25 Indifference Moxifloxacin 0.5 0.5 1 Indifference Nitrofurantoin 0.75 0.5 1.25 Indifference Polymyxin 0.5 0.25 0.75 Additive Streptomycin 0.25 0.5 0.75 Additive Vancomycin 0.5 0.25 0.75 Additive BWC0977 exhibited additive and indifferent interactions. No antagonism was observed with any class of antibiotics. This study is supported by BIRAC, DBT, Govt. of India (BT/BIPP/0803/30/14, C- CAMP/BIG/98B) and CARB-X (6 IDSEP160030-01-02). We thank our partners Syngene International, St. John’s Research Institute and Narayana Health. 1. Zaslaver A. et al, 2006. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. 2. Scott D. mills et al, 2014. A Novel High-Throughput Cell-Based Assay Aimed at Identifying Inhibitors of DNA Metabolism in Bacteria. 3. PCT WO/2018/225097 No cross-resistance observed between fluroquinolones and BWC0977. E. coli SEC006 Time(hrs) Log 10 CFU 0 2 4 6 8 10 2 3 4 5 6 7 8 9 0.015 0.15 1.25 0.6 0.3 0.07 0.03 CC μg/mL E. coli SEC015 Time(hrs) Log 10 CFU 0 2 4 6 8 10 2 3 4 5 6 7 8 9 0.6 5 2.5 1.25 0.3 0.15 0.07 CC μg/mL A. baumannii SAB002 Time(hrs) Log 10 CFU 0 2 4 6 8 10 2 3 4 5 6 7 8 9 2.5 20 10 5 1.25 0.6 0.3 CC μg/mL A. baumannii SAB006 Time(hrs) Log 10 CFU 0 2 4 6 8 10 2 3 4 5 6 7 8 9 0.3 2.5 1.25 0.6 0.15 0.07 0.03 CC μg/mL K. pneumoniae SKB067 Time(hrs) Log 10 CFU 0 2 4 6 8 10 2 3 4 5 6 7 8 9 5 40 20 10 2.5 1.25 0.62 CC μg/mL K. pneumoniae SKB077 Time(hrs) Log 10 CFU 0 2 4 6 8 10 2 3 4 5 6 7 8 9 0.6 5 2.5 1.25 0.3 0.15 0.07 CC μg/mL P. aeruginosa SPA018 Time(hrs) Log 10 CFU 0 2 4 6 8 10 2 3 4 5 6 7 8 9 5 40 20 10 2.5 1.25 0.62 CC μg/mL P. aeruginosa SPA032 Time(hrs) Log 10 CFU 0 2 4 6 8 10 2 3 4 5 6 7 8 9 5 40 20 10 2.5 1.25 CC μg/mL Clinical isolate MIC (µg/mL) Emax (log10 CFU at 4X MIC) SEC006 0.015 2 SEC015 0.15 1.9 SKB067 1.25 2.8 SKB077 0.15 3.1 SAC002 1.25 2.3 SAC006 0.15 2.4 SPS018 1.25 3 SPS032 5 4.2 MIC values in µg/mL BWC0977 - 0.015 Ciprofloxacin - 0.01 R ecovery tim e (hrs) L o g 10 C FU 0 1 2 3 4 5 3 4 5 6 7 8 B W C 0977- 8X B W C 0977- 4X B W C 0977- 2X N o drug C iprofloxacin- 8X C iprofloxacin- 4X C iprofloxacin- 2X C iprofloxacin- 1X B W C 0977- 1X BWC0977 induces SOS response similar to Ciprofloxacin and Gepotidacin.
Transcript of Introduction Mode of Inhibition Killing Kinetics Combination ......2019/04/15 · BWC0977, a novel...
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BWC0977, a novel dual target topoisomerase inhibitor: antimicrobial potency, spectrum and mechanism of action.S. Hameed, S. Sharma, R. Nandishaiah, N. Katagihallimath, N. Bharatham, A. Shanbhag, S. Rajagopal, P. Bhowmik, P. Amar, R. Narjari, V. Ramachandran, S. Datta & V. Balasubramanian
Bugworks Research India Pvt. Ltd. Bengaluru 560065; email: [email protected]
Killing Kinetics Combination StudiesIntroduction Mode of Inhibition
Target Inhibition across Species
Interaction Residues of Gyrase
Acknowledgement
References
Effective against Ciprofloxacin Resistant Clinical Isolates
Post Antibiotic Effect
Resistant Mutant Generation
SOS Response to DNA Damage
• We have identified an antibacterial preclinical candidateBWC0977 that is equipotent against targets - Gyrase andTopoisomerase IV. It exhibits broad spectrum antibacterial activityagainst the ESKAPE pathogens.
• BWC0977 inhibits gyrase by stabilizing single strand breaks in theDNA unlike Ciprofloxacin that stabilizes double strand breaks.
• Computational studies predicted that the residues M120, D82 andR121 of GyrA are important for interacting with BWC0977. Theupward shift in inhibition of supercoiling activity of thecorresponding mutant enzymes confirms these interactions.
• BWC0977 triggers an SOS response in bacterial cells similar toCiprofloxacin through the induction of recA, recN, sulA and lexApromoters1,2.
• BWC0977 is bactericidal, shows time dependent kill and a postantibiotic effect of 1.5 to 2 hrs.
• Efforts to raise resistant mutants against BWC0977 in Escherichiacoli and other Gram-negative organisms by single step methodsyielded no mutants indicating very low resistance frequency.Though mutants obtained by long serial passage experimentsshowed a shift in MIC, sequencing revealed no target mutations.
• Side chains of the M120 residues at the GyrA dimer interface forms strong hydrophobic interactions with the pyrazino-oxazinone ring of BWC0977.
• -NH of pyrazino-oxazinone ring forms H-bond with D82.• R121 interacts with Oxazolidinone ring of BWC0977.
R121
M120D82
R121
M120
DG10 DG10
DC11
DC11
Major Interaction residuesCiprofloxacin : S83L
BWC0977 : M120K & D82N
E. coli gyrase SC, IC50 in µMCompound WT S83L M120K D82N BWC0977 0.004 0.013 92 3.6
Ciprofloxacin 0.183 11.6 0.91 3.1
The shift in IC50 of BWC0977 with M120K and D82N mutant gyrase enzymes confirms their interactions invitro.
BWC0977 inhibits gyrase and topo IV with nanomolar potency across species. Ciprofloxacin shows higher micromolar inhibition on S. aureus gyrase activity.
IC50 (µM)
Compound E. coli P. aeruginosa S. aureus Gyrase Topo IV Gyrase Topo IV Gyrase Topo IVBWC0977 0.004 0.01 0.009 0.071 0.01 0.095
Ciprofloxacin 0.189 16.3 0.244 22.9 25.5 8.5
Cleavage-complex assays were performed with E. coli gyrase using supercoiled plasmid in the absence of ATP to study the mode of inhibition of compounds.
• E. coli SOS-inducible promoters fused to fast-folding green fluorescentprotein (GFP) on a low-copy plasmid in E. coli MG1655 were used asreporter strains2.
• The selected reporter strains were treated with varying concentrations ofcompounds during early growth phase (A600 = 0.2, growing at 37°C ) and theGFP fluorescence was measured at the end of 6 hours of induction.
• RFUs were normalized to the cell growth and fold induction was measuredas a ratio of the treated vs. untreated cultures.
C i p r o f l o x a c i n B W C 0 9 7 7 G e p o t i d a c i n R i f a m p i c i n
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C i p r o f l o x a c i n B W C 0 9 7 7 G e p o t i d a c i n R i f a m p i c i n
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C i p r o f l o x a c i n B W C 0 9 7 7 G e p o t i d a c i n R i f a m p i c i n
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C i p r o f l o x a c i n B W C 0 9 7 7 G e p o t i d a c i n R i f a m p i c i n
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• Single step selection methods to raise resistant mutants yielded no mutants.
• A serial passage MIC protocol was done to obtain mutants in both wildtype and ∆tolC E. coli.
Serial passage cycles (Days)
MIC
(µg/
mL)
0 5 10 15 20 25 30 35 400.0001
0.001
0.01
0.1
1
10
E.coli WTE. coli DtolC
After removal ofdrug pressure
A 100-fold shift in MIC was observed in the wildtype E. coli at the end of 35days while 10-fold shift was observed in ∆tolC. Individual colonies isolatedfrom the wildtype culture were grown in the absence of drug pressure. TheMIC of these stable mutants were 0.5µg/ml. The gyrA and parC genes of thesemutants were sequenced. No mutations were identified in the target genes.
BWC0977 was bactericidal in quinolone resistant clinical strains
in a time dependent pattern.
BWC0977 post antibiotic effect is similar to Ciprofloxacin.
PAE in hours
Concentration BWC0977 Ciprofloxacin1X MIC 0.28 0.82
2X MIC 1.35 1.1
4X MIC 1.6 1.6
8X MIC 2.5 >2.5
Antibiotic FIC (A)antibiotic
FIC (B) BWC0977
FIC index (A+B)
Interaction
Amoxicillin 0.75 0.5 1.25 IndifferenceAmpicillin 0.5 0.5 1 IndifferenceAzithromycin 0.5 0.5 1 IndifferenceAztreonam 0.5 0.5 1 IndifferenceCefotaxime 0.25 0.5 0.75 AdditiveCefepime 0.125 0.5 0.625 AdditiveChloramphenicol 0.5 0.25 0.75 AdditiveCiprofloxacin 0.125 0.5 0.625 AdditiveClarithromycin 0.5 0.25 0.75 AdditiveClindamycin 0.5 0.25 0.75 AdditiveErythromycin 0.75 0.5 1.25 IndifferenceKanamycin 0.25 0.5 0.75 AdditiveLevofloxacin 0.25 0.5 0.75 AdditiveMeropenem 0.5 0.5 1 IndifferenceMetronidazole 0.75 0.5 1.25 IndifferenceMoxifloxacin 0.5 0.5 1 IndifferenceNitrofurantoin 0.75 0.5 1.25 IndifferencePolymyxin 0.5 0.25 0.75 AdditiveStreptomycin 0.25 0.5 0.75 AdditiveVancomycin 0.5 0.25 0.75 Additive
BWC0977 exhibited additive and indifferent interactions. No antagonism was observed with any class of antibiotics.
This study is supported by BIRAC, DBT, Govt. of India (BT/BIPP/0803/30/14, C-CAMP/BIG/98B) and CARB-X (6 IDSEP160030-01-02).We thank our partners Syngene International, St. John’s Research Institute andNarayana Health.
1. Zaslaver A. et al, 2006. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli.
2. Scott D. mills et al, 2014. A Novel High-Throughput Cell-Based Assay Aimed at Identifying Inhibitors of DNA Metabolism in Bacteria.
3. PCT WO/2018/225097
No cross-resistance observed between fluroquinolones and BWC0977.
E. coli SEC006
Time(hrs)
Log 1
0 CFU
0 2 4 6 8 102
3
4
5
6
7
8
9
0.015
0.15
1.250.60.3
0.070.03
CC
µg/mL
E. coli SEC015
Time(hrs)
Log 1
0 CFU
0 2 4 6 8 102
3
4
5
6
7
8
9
0.6
52.51.25
0.30.150.07CC
µg/mL
A. baumannii SAB002
Time(hrs)
Log 1
0 CFU
0 2 4 6 8 102
3
4
5
6
7
8
9
2.5
20105
1.250.60.3CC
µg/mL
A. baumannii SAB006
Time(hrs)
Log 1
0 CFU
0 2 4 6 8 102
3
4
5
6
7
8
9
0.3
2.51.250.6
0.150.070.03CC
µg/mL
K. pneumoniae SKB067
Time(hrs)
Log 1
0 CFU
0 2 4 6 8 102
3
4
5
6
7
8
9
5
402010
2.51.250.62CC
µg/mL
K. pneumoniae SKB077
Time(hrs)
Log 1
0 CFU
0 2 4 6 8 102
3
4
5
6
7
8
9
0.6
52.51.25
0.30.150.07CC
µg/mL
P. aeruginosa SPA018
Time(hrs)
Log 1
0 CFU
0 2 4 6 8 102
3
4
5
6
7
8
9
5
402010
2.51.250.62CC
µg/mL
P. aeruginosa SPA032
Time(hrs)
Log 1
0 CFU
0 2 4 6 8 102
3
4
5
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7
8
9
5
402010
2.51.25CC
µg/mL
Clinical isolate MIC (µg/mL) Emax (log10 CFU at 4X MIC)SEC006 0.015 2SEC015 0.15 1.9SKB067 1.25 2.8SKB077 0.15 3.1SAC002 1.25 2.3SAC006 0.15 2.4SPS018 1.25 3SPS032 5 4.2
MIC values in µg/mLBWC0977 - 0.015
Ciprofloxacin - 0.01
R e c o v e r y t i m e ( h r s )
Lo
g1
0 C
FU
0 1 2 3 4 5
3
4
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7
8
B W C 0 9 7 7 - 8 X
B W C 0 9 7 7 - 4 X
B W C 0 9 7 7 - 2 X
N o d r u g
C i p r o f l o x a c i n - 8 X
C i p r o f l o x a c i n - 4 X
C i p r o f l o x a c i n - 2 X
C i p r o f l o x a c i n - 1 X
B W C 0 9 7 7 - 1 X
BWC0977 induces SOS response similar to Ciprofloxacin and Gepotidacin.
