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