Addressing the evolving challenge of β-lactamase mediated antimicrobial resistance:
ETX2514, a next-generation BLI with potent broad-spectrum activity against Class A, C and D enzymes
Alita Miller, PhD
Superbugs & Superdrugs USA, November 14-15, 2016, Iselin, NJ
Overview of presentation
Co-evolution of β–lactams, β–lactamases and their inhibitors
Multidrug resistant Acinetobacter baumannii:
an unmet medical need
ETX2514 In vitro characterization
In vivo efficacy
2
β-lactamases are characterized into four molecular classes
3
Class A, C, and D have a serine at the active site and require water in the active site forβ-lactam hydrolysis
Class B are metalloenzymesthat require zinc at the active site
Drawz & Bonomo, (2010) Clin. Microbiol. Rev. 23: 160-201
β-lactamases evolve after use of β-lactam antibiotics
4
Cefazolin
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
Cephalexin
Cefalothin
β-lactamases evolve after use of β-lactam antibiotics:
1960 - 1970
5
β-lactam drugs
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
2nd Gen CephalosporinsCephamycins
TEM-1, SHV-1
Cefaclor
Cefotetan
Cefoxitin
β-lactamases evolve after use of β-lactam antibiotics:
1970 - 1980
6
β-lactam drugs
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
2nd Gen CephalosporinsCephamycins
TEM-1, SHV-1AmpC overexpression
3rd Gen CephalosporinsMonobactam1st Gen BL/BLI combinations
Amoxicillin/Clavulanate, Ampicillin/Sulbactam
Ceftazidime
Cefotaxime
Aztreonam
β-lactamases evolve after use of β-lactam antibiotics:
1980 - 1990
7
β-lactam drugs
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
2nd Gen CephalosporinsCephamycins
TEM-1, SHV-1AmpC overexpression
3rd Gen CephalosporinsMonobactam1st Gen BL/BLI combinations
ESBL TEM, SHV
ESBL CTX-M
Carbapenems2nd Gen BL/BLI
Imipenem
ESBL OXA
Plasmid AmpC
Meropenem
Piperacillin/tazobactam
β-lactamases evolve after use of β-lactam antibiotics:
1990 - 2000
8
β-lactam drugs
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
2nd Gen CephalosporinsCephamycins
TEM-1, SHV-1AmpC overexpression
3rd Gen CephalosporinsMonobactam1st Gen BL/BLI combinations
ESBL TEM, SHV
ESBL CTX-M
Carbapenems2nd Gen BL/BLI
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM
• No new β-lactams for Gram-negatives
• Therapy limited to colistin or tigecycline
NDM
9
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
TEM-1, SHV-1AmpC overexpression
ESBL TEM, SHV
ESBL CTX-M
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM NDM
Older β-lactamase inhibitors only work against a few classes of β-lactamases
Inhibited by Clavulanic Acid and Sulbactam
Amoxicillin-clavulanateTicarcillin-clavulanateAmpicillin-sulbactam
clavulanic acid
10
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
TEM-1, SHV-1AmpC overexpression
ESBL TEM, SHV
ESBL CTX-M
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM NDM
Older β-lactamase inhibitors only work against a few classes of β-lactamases
Inhibited by Tazobactam
Piperacillin-tazobactamCeftolozane-tazobactam (Zerbaxa)
tazobactam
11
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
TEM-1, SHV-1AmpC overexpression
ESBL TEM, SHV
ESBL CTX-M
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM NDM
Avibactam and other DABCO*s have broader spectra of inhibition than older β-lactamase inhibitors
Inhibited by Avibactam
*di-aza-bicyclo-octanone
• Ceftazidime-avibactam (AvyCaz)• Imipenem-relebactam (Ph III)• Zidebactam, RG6080 (Ph I)• Aztreonam-avibactam (Ph I)
MBL+ Enterobacteriaceae (Class B)(ATM is not degraded by MBLs; AVI inhibits serine BLs)
12
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
TEM-1, SHV-1AmpC overexpression
ESBL TEM, SHV
ESBL CTX-M
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM NDM
Very limited coverage of Class D -lactamases by avibactam
Inhibited by Avibactam
Addressed by ATM-AVI in Enterobacteriaceae
Gaps in Coverage
13
• Gram-negative bacteria that causes infections in critically ill patients, with mortality rates as high as 43%1
• CDC Unmet Need Threat Level: Serious2
• 63% of A. baumannii isolates are considered multi-drug resistant, meaning at least three different classes of antibiotics no longer cure A. baumannii infections including carbapenems, often considered antibiotics of last resort
• Resistance to carbapenems in A. baumannii is associated with increasing prevalence of Class D -lactamases3,4
A. baumannii
1. Am. J. Respir. Crit. Care Med. 2011.1409; Int. J. Antimicrob. Agents 2009.5752. CDC. 2013. Antibiotic Resistant Threats in the US. pg. 58-603. M.M. Ehlers, et. al. 2012. InTech, DOI: 10.5772/303794. Potron, et al. 2015. Int. J. Antimicrob. Agents 45:568
Multi-drug resistant Acinetobacter baumannii
Complexity of β-lactamase content in A. baumannii
14
Whole-genome sequencing of 84 recent MDR A. baumannii strains provides insight into what is required for a successful next generation BL/BLI therapy
Inhibition of Classes A, C and D Required for Robust BLI Activity in A. baumannii
Class N %Most prevalent
variant(s)
A 45 53.6 TEM-1 (41/45)
B 1 1.3 IMP-1
Extended
spectrum C* 71 84.5ADC-30 (18/84)ADC-73 (18/84)
D 84 100
Multiple (70/84 encode two or more, (46/70
were OXA-23+OXA-51-like)
*all strains contain chromosomal adc gene
The ultimate medicinal chemistry challenge
• How to selectively inhibit hundreds of bacterial enzymes?
• How to find the right balance between reactivity and hydrolytic stability?
• How to prepare synthetically challenging, diverse analogs to verify structural hypotheses?
15
• A deep understanding of avibactam’s biology informed design of the next generation BLI
• Crystal structures provided insights to avibactam’s unique interactions with-lactamases
Avibactam-bound structures of CTX-M-15 at 1.1Å,From Lahiri et al (2013) AAC 57: 2496
Discovery of ETX2514, a novel broad-spectrum serine BLI
16
Using a combination of innovative chemistry and structure-based design
avibactam
Active site overlays of avibactam- (in grey, PDB: 4WM9) and an ETX2514 analog- (in green) bound OXA-24 structures. The water molecules are depicted as spheres. The hydrogen bonding network around the ETX2514 analog is shown in dashed lines.di-aza-bicyclo-octenone
ETX2514 exhibits excellent β-lactamase inhibition across classes A, C and D
17
IC50 after 5 min incubation (in µM)
Compound
Name9.2
Class A Class C Class D
E. cloacae
TEM-1
K. pneumoniae
CTX-M-15
E. cloacae
KPC-2
E. cloacae
P99
P. aeruginosa
AmpC
P. aeruginosa
OXA-10
A. baumannii
OXA-24/40
K. pneumoniae
OXA-48
Avibactam 0.011 0.0047 0.19 0.2 0.62 23 16 0.75
ETX2514 0.0012 0.00083 0.0043 0.0013 0.014 0.25 0.2 0.0063
Fold increase in potency 9X 6X 44X 154X 44X 92X 80X 119X
Exceptional enzymatic spectrum translates into excellent activity across an isogenic panel of P. aeruginosa strains
18
IC50 (in µM)
Compound
Name
Class A Class C Class D
E. cloacae
TEM-1
K. pneumoniae
CTX-M-15
E. cloacae
KPC-2
E. cloacae
P99
P. aeruginosa
AmpC
P. aeruginosa
OXA-10
A. baumannii
OXA-24/40
K. pneumoniae
OXA-48
Avibactam 0.011 0.0047 0.19 0.2 0.62 23 16 0.75
ETX2514 0.0012 0.00083 0.0043 0.0013 0.014 0.25 0.2 0.0063
Compound
Name
P. aeruginosa isogenic strains bearing corresponding -lactamases
Vector
aloneTEM-1 CTX-M-15 KPC-2 P99 AmpC OXA-10 OXA-24/40 OXA-48
Piperacillinalone
4 >1024 512 256 64 128 256 256 128
Piperacillin+Avibactam
4 8 4 8 4 16 128 128 8
Piperacillin+ETX2514
4 4 4 4 4 4 4 8 4
MIC (in mg/L)
BLI added at 4 mg/mL
No BLI
Mecillinam(PBP2-selective
inhibitor)
Inhibition of PBP2 by ETX2514 results in intrinsic antibacterial activity vs. Enterbacteriaceae
19
Control
ETX2514
Mecillinam
Aztreonam
ControlAztreonam
(PBP3-selective
inhibitor)
ETX2514
Pathogen
ETX2514 kinact/Ki in M-1s-1
PBP1a PBP2 PBP3
A. baumannii 180 1,800 3
P. aeruginosa 12 24 57
E. coli 120 17,000 2
Control
ETX2514
Mecillinam
Aztreonam
Morphology of antibiotic-treated E. coli
Linneas Bioscience
ETX2514 restores β-lactam activity vs. multiple gram-negative pathogens
20
CompoundE. coli
n = 202
K. pneumoniae
n = 198
P. aeruginosa
n = 202
A. baumannii
n = 195
Imipenemalone 0.25 1 16 >64
+ ETX2514 ≤0.06 0.12 2 16
Meropenemalone ≤0.06 ≤0.06 16 >64
+ ETX2514 ≤0.06 ≤0.06 8 16
Aztreonamalone 32 32 64 >64
+ ETX2514 ≤0.06 ≤0.06 32 >64
Ceftazidimealone 16 >64 >64 >64
+ ETX2514 ≤0.06 ≤0.06 8 32
Sulbactamalone 64* >64¥ >64 64
+ ETX2514 ≤0.06* 0.12¥ >64 4
ETX2514 alone 1 8 >64 >64
*n = 21 strains ¥n = 20 strains
• Excellent activity vs E. coli & K. pneumoniae with all β-lactams tested
• Restores imipenem to MIC90 of 2 mg/L vs P. aeruginosa
• Restores sulbactam to MIC90 of 4 mg/L vs A. baumannii
MIC90 across recent clinical isolates (+/- ETX2514 at 4 mg/L)
Intrinsic activity of sulbactam vs. A. baumannii
• Attributed to inhibition of PBP3
21Penwell et al.(2015) AAC 59:1680-89
+ sulbactamuntreated
• Frequency of resistance is low: 2-4x10-9 at 4X MIC• Resistance maps to residues near active site of PBP3• Resistant mutants are attenuated in fitness
• Sulbactam:ETX2514* maintains excellent activity over time
MIC (mg/L) ≤0.06 0.12 0.25 0.5 1 2 4 8 16 32 >64
2011N=195
Cumul %susceptible
1 3.1 13.8 41.5 65.6 89.7 96.9 97.9 99.5 100 100
2012N=209
Cumul %susceptible
0 0.5 2.9 20.1 46.9 79 98.6 100 100 100 100
2013N=207
Cumul %susceptible
0 0 4.3 15.9 43.4 73.8 96.5 97.5 99 99 100
2014N=1131
Cumul %susceptible
1 1.6 7.8 27.9 63.7 88.9 99.6 99.6 99.7 100 100
Sulbactam:ETX2514: A novel combination against MDR A. baumannii
22
MIC distributions for globally diverse A. baumannii clinical strains
*held at 4 mg/L
Sulbactam:ETX2514 activity remains unchanged in carbapenem-resistant, colistin-resistant and MDR A. baumannii strains
23
0.2
5
0.5 1 2 4 8
16
32
64
12
8
0
1 0
2 0
3 0
4 0
5 0
M I C ( m g / L )
%
su
sc
ep
ti
bl
e
st
ra
in
s
s u l b a c t a m a l o n e v s . a l l ( N = 1 1 3 1 )
s u l b a c t a m : E T X 2 5 1 4 v s . a l l ( N = 1 1 3 1 )
s u l b a c t a m : E T X 2 5 1 4 v s . M E M - R ( N = 7 3 1 )
s u l b a c t a m : E T X 2 5 1 4 v s . C O L - R ( N = 5 6 )
s u l b a c t a m : E T X 2 5 1 4 v s . M D R ( N = 7 7 8 )
Sulbactam:ETX2514 is active against A. baumannii encoding multiple β-lactamases
24
drug N range MIC50 MIC90
imipenem 84 0.125 - >128 64 128
SUL-ETX2514
84 0.25 - 16 2 4
Summary of MICs (mg/L)
Morphology of A. baumannii in the presence of sulbactam:ETX2514 suggests multi-target effects
25
No Drug
SUL
ETX2514
SUL-ETX2514
A. baumannii ATCC 17978 was exposed to 1/2X MIC of drug for 3
hrs at 35° C and examined by light microscopy. Scale bar = 5 mm.
Frequency of spontaneous resistance to sulbactam-ETX2514 is very low against clinical isolates of A. baumannii
26
Strainβ-lactamase
contentFOR at 4X MIC Variant Protein affected
SUL-ETX2514
SUL MEM CAZ
ARC2058ADC-99-like;
OXA-95
Parent -- 1/4 4 0.5 4
2X-1 AspS [Q47P] 16/4 4 16 16
<9.0 x 10-10 2X-2 GltX [M240I] 16/4 4 8 4
2X-3 GltX [R117S] 64/4 4 32 8
2XL-1 PBP3 [V505L] 16/4 16 0.25 4
ARC2681ADC-42-like; TEM-1; OXA-40; OXA-
1327.6 x 10-10
Parent -- 2/4 8 32 256
4X-1 PBP3 [S390T] >64/4 >64 32 128
ARC2782ADC-79; TEM-1; PER-1; OXA-23;
OXA-66<9.0 x 10-10
Parent -- 0.5/4 32 16 >512
2X-1 PBP3 [T511A] 4/4 64 16 >512
MIC (mg/L)
• tRNA synthetase mutants are associated with the stringent response and are commonly seen with PBP2
inhibitors1
• Mutations in PBP3 affected the MIC of SUL-ETX2514 and sulbactam alone
Resistant mutants suggest sulbactam-ETX2514 works by inhibiting both PBP2 and PBP3
1Vinella et al. (1992) EMBO J. 11:1493-1501
Sulbactam:ETX2514 exhibits excellent in vivo activity
27
• Greater than 2-log kill achieved in both neutropenic mouse thigh and lung models of A. baumannii infections
7.40
9.40
8.408.03
6.636.19
4.854.61
4.19
2
3
4
5
6
7
8
9
10
Pre-treatment
Vehicle 2.5 /0.625
5 / 1.25 10 / 2.5 20 / 5 30 / 7.5 40 / 10 80 / 20
Log(
CFU
/g)
Lung
Stasis
sulbactam/ETX2514 (mg/kg) q3h
6.36
8.03 8.02
6.72
4.39 4.243.97 4.01 4.07
2
3
4
5
6
7
8
9
10
Pre-treatment
Vehicle 2.5 /0.625
5 / 1.25 10 / 2.5 20 / 5 30 / 7.5 40 / 10 80 / 20
Log(
CFU
/g)
Thigh
Stasis
sulbactam/ETX2514 (mg/kg) q3h
MDR A. baumannii ARC3486 (OXA-72, OXA-66, TEM-1, AmpC)
MIC(sulbactam) ≥ 32 mg/L, MIC(sulbactam/ETX2514) = 0.5 mg/L
Sulbactam/ETX2514 dose response (SC, 4/1 ratio)
Similar results obtained for 5 additional clinical isolates
PK and safety of sulbactam:ETX2514
28
• Rat and dog PK of ETX2514 showed low to moderate clearance and low volume of distribution translating to a projected half life of 1.1 hr in humans
• Excretion of unchanged drug was the predominant clearance mechanism with relatively low metabolism characterized in vitro and in vivo
• ETX2514 was well-tolerated in both rat and dog 14-day repeat dose toxicology studies up to 2000 mg/kg with no significant clinical findings after intravenous administration
no changes in ophthalmology, urinalyses, hematology parameters or organ weight
• In CV safety pharmacology studies, ETX2514 had no effects on qualitative electrocardiogram parameters, heart rates, or arterial pressures up to 2000 mg/kg
Summary and Conclusions
29
• ETX2514 is a potent inhibitor of a broad-spectrum of Class D β-lactamases while maintaining exquisite potency on Class A and C enzymes.
• ETX2514 potently restores the activity of multiple β-lactams in Gram-negative MDR pathogens.
• Sulbactam:ETX2514 is a novel BL:BLI combination to treat MDR A. baumannii infections, with an MIC90 = 4 mg/L (N = 1742 clinical isolates) and excellent in vivo activity.
• Currently in Phase I testing
Acknowledgements
30
• AstraZeneca Antibacterial Discovery• IHMA, Inc. • Linneas
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