Addressing the evolving challenge of β-lactamase mediated antimicrobial resistance: ETX2514, a next-generation BLI with potent broadspectrum 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 Class B are metalloenzymes that require zinc at the active site Class A, C, and D have a serine at the active site and require water in the active site for β-lactam hydrolysis Drawz & Bonomo, (2010) Clin. Microbiol. Rev. 23: 160-201 3
β-lactamases evolve after use of β-lactam antibiotics Class A Class B Class C Class D 1960s 1970s 1980s 1990s 2000s 2010s 2020s 1 st Gen Cephalosporins Cefalothin Cephalexin Cefazolin 4
β-lactamases evolve after use of β-lactam antibiotics: 1960-1970 TEM-1, SHV-1 Class A Class B Class C Class D 1960s 1970s 1980s 1990s 2000s 2010s 2020s 1 st Gen Cephalosporins 2 nd Gen Cephalosporins Cephamycins Cefaclor Cefotetan β-lactam drugs Cefoxitin 5
β-lactamases evolve after use of β-lactam antibiotics: 1970-1980 TEM-1, SHV-1 AmpC overexpression Class A Class B Class C Class D 1960s 1970s 1980s 1990s 2000s 2010s 2020s 1 st Gen Cephalosporins 2 nd Gen Cephalosporins Cephamycins 3 rd Gen Cephalosporins Monobactam 1 st Gen BL/BLI combinations Cefotaxime Ceftazidime β-lactam drugs Aztreonam Amoxicillin/Clavulanate, Ampicillin/Sulbactam 6
β-lactamases evolve after use of β-lactam antibiotics: 1980-1990 TEM-1, SHV-1 AmpC overexpression ESBL TEM, SHV ESBL CTX-M ESBL OXA Class A Class B Class C Class D 1960s 1970s Plasmid AmpC 1980s 1990s 2000s 2010s 2020s 1 st Gen Cephalosporins 2 nd Gen Cephalosporins Cephamycins 3 rd Gen Cephalosporins Monobactam 1 st Gen BL/BLI combinations Carbapenems 2 nd Gen BL/BLI Imipenem Meropenem β-lactam drugs Piperacillin/tazobactam 7
β-lactamases evolve after use of β-lactam antibiotics: 1990-2000 TEM-1, SHV-1 AmpC overexpression ESBL TEM, SHV ESBL CTX-M ESBL OXA Plasmid AmpC KPC carbapenemase VIM OXA carbapenemase NDM Class A Class B Class C Class D 1960s 1970s 1980s 1990s 2000s 2010s 2020s 1 st Gen Cephalosporins β-lactam drugs 2 nd Gen Cephalosporins Cephamycins 3 rd Gen Cephalosporins Monobactam 1 st Gen BL/BLI combinations Carbapenems 2 nd Gen BL/BLI No new β-lactams for Gramnegatives Therapy limited to colistin or tigecycline 8
Older β-lactamase inhibitors only work against a few classes of β-lactamases TEM-1, SHV-1 AmpC overexpression ESBL TEM, SHV ESBL CTX-M ESBL OXA Plasmid AmpC KPC carbapenemase VIM OXA carbapenemase NDM Class A Class B Class C Class D 1960s 1970s 1980s 1990s 2000s 2010s 2020s Inhibited by Clavulanic Acid and Sulbactam Amoxicillin-clavulanate Ticarcillin-clavulanate Ampicillin-sulbactam clavulanic acid 9
Older β-lactamase inhibitors only work against a few classes of β-lactamases TEM-1, SHV-1 AmpC overexpression ESBL TEM, SHV ESBL CTX-M ESBL OXA Plasmid AmpC KPC carbapenemase VIM OXA carbapenemase NDM Class A Class B Class C Class D 1960s 1970s 1980s 1990s 2000s 2010s 2020s Inhibited by Tazobactam Piperacillin-tazobactam Ceftolozane-tazobactam (Zerbaxa) tazobactam 10
Avibactam and other DABCO*s have broader spectra of inhibition than older β-lactamase inhibitors TEM-1, SHV-1 AmpC overexpression ESBL TEM, SHV ESBL CTX-M ESBL OXA Plasmid AmpC KPC carbapenemase VIM OXA carbapenemase NDM Class A Class B Class C Class D 1960s 1970s 1980s 1990s 2000s 2010s 2020s Inhibited by Avibactam 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) *di-aza-bicyclo-octanone 11
Very limited coverage of Class D -lactamases by avibactam Class A Class B Class C Class D Inhibited by Avibactam ESBL TEM, SHV TEM-1, SHV-1 ESBL CTX-M AmpC overexpression ESBL OXA Plasmid AmpC OXA carbapenemase KPC carbapenemase VIM Addressed by ATM-AVI in Enterobacteriaceae NDM 1960s 1970s 1980s 1990s 2000s 2010s 2020s Gaps in Coverage 12
Multi-drug resistant Acinetobacter baumannii Gram-negative bacteria that causes infections in critically ill patients, with mortality rates as high as 43% 1 CDC Unmet Need Threat Level: Serious 2 63% of A. baumannii isolates are considered multidrug 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 -lactamases 3,4 A. baumannii 1. Am. J. Respir. Crit. Care Med. 2011.1409; Int. J. Antimicrob. Agents 2009.575 2. CDC. 2013. Antibiotic Resistant Threats in the US. pg. 58-60 3. M.M. Ehlers, et. al. 2012. InTech, DOI: 10.5772/30379 4. Potron, et al. 2015. Int. J. Antimicrob. Agents 45:568 13
Complexity of β-lactamase content in A. baumannii Whole-genome sequencing of 84 recent MDR A. baumannii strains provides insight into what is required for a successful next generation BL/BLI therapy Class N % Most prevalent variant(s) A 45 53.6 TEM-1 (41/45) B 1 1.3 IMP-1 Extended spectrum C* 71 84.5 ADC-30 (18/84) ADC-73 (18/84) Multiple (70/84 encode two or more, (46/70 D 84 100 were OXA-23+OXA-51- like) *all strains contain chromosomal adc gene Inhibition of Classes A, C and D Required for Robust BLI Activity in A. baumannii 14
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? 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 15
Discovery of ETX2514, a novel broad-spectrum serine BLI Using a combination of innovative chemistry and structure-based design avibactam di-aza-bicyclo-octenone 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. 16
ETX2514 exhibits excellent β-lactamase inhibition across classes A, C and D IC 50 after 5 min incubation (in µm) Class A Class C Class D Compound Name9.2 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 17
Exceptional enzymatic spectrum translates into excellent activity across an isogenic panel of P. aeruginosa strains IC 50 (in µm) Compound Name E. cloacae TEM-1 Class A Class C Class D K. pneumoniae E. cloacae E. cloacae P. aeruginosa P. aeruginosa A. baumannii CTX-M-15 KPC-2 P99 AmpC OXA-10 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 MIC (in mg/l) Compound Name Piperacillin alone Piperacillin +Avibactam Piperacillin +ETX2514 Vector alone P. aeruginosa isogenic strains bearing corresponding -lactamases TEM-1 CTX-M-15 KPC-2 P99 AmpC OXA-10 OXA-24/40 OXA-48 4 >1024 512 256 64 128 256 256 128 4 8 4 8 4 16 128 128 8 4 4 4 4 4 4 4 8 4 No BLI BLI added at 4 mg/ml 18
Aztreonam Inhibition of PBP2 by ETX2514 results in intrinsic antibacterial activity vs. Enterbacteriaceae Pathogen ETX2514 k inact /K i in M -1 s -1 PBP1a PBP2 PBP3 A. baumannii 180 Control 1,800 3 P. aeruginosa 12 24 57 E. coli 120 17,000 2 Mecillinam Morphology of antibiotic-treated E. coli Control Control Aztreonam Aztreonam (PBP3-selective inhibitor) Mecillinam (PBP2-selective Mecillinam inhibitor) ETX2514 ETX2514 Linneas Bioscience
ETX2514 restores β-lactam activity vs. multiple gram-negative pathogens Excellent activity vs E. coli & K. pneumoniae with all β-lactams tested Restores imipenem to MIC 90 of 2 mg/l vs P. aeruginosa Restores sulbactam to MIC 90 of 4 mg/l vs A. baumannii MIC 90 across recent clinical isolates (+/- ETX2514 at 4 mg/l) Compound Imipenem Meropenem Aztreonam Ceftazidime Sulbactam E. coli n = 202 K. pneumoniae n = 198 P. aeruginosa n = 202 A. baumannii n = 195 alone 0.25 1 16 >64 + ETX2514 0.06 0.12 2 16 alone 0.06 0.06 16 >64 + ETX2514 0.06 0.06 8 16 alone 32 32 64 >64 + ETX2514 0.06 0.06 32 >64 alone 16 >64 >64 >64 + ETX2514 0.06 0.06 8 32 alone 64* >64 >64 64 + ETX2514 0.06* 0.12 >64 4 ETX2514 alone 1 8 >64 >64 *n = 21 strains n = 20 strains 20
Intrinsic activity of sulbactam vs. A. baumannii Attributed to inhibition of PBP3 untreated + sulbactam 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 Penwell et al.(2015) AAC 59:1680-89 21
Sulbactam:ETX2514: A novel combination against MDR A. baumannii Sulbactam:ETX2514 * maintains excellent activity over time MIC distributions for globally diverse A. baumannii clinical strains MIC (mg/l) 0.06 0.12 0.25 0.5 1 2 4 8 16 32 >64 2011 N=195 2012 N=209 2013 N=207 2014 N=1131 Cumul % susceptible Cumul % susceptible Cumul % susceptible Cumul % susceptible 1 3.1 13.8 41.5 65.6 89.7 96.9 97.9 99.5 100 100 0 0.5 2.9 20.1 46.9 79 98.6 100 100 100 100 0 0 4.3 15.9 43.4 73.8 96.5 97.5 99 99 100 1 1.6 7.8 27.9 63.7 88.9 99.6 99.6 99.7 100 100 *held at 4 mg/l 22
% s u s c e p t i b l e s t r a i n s Sulbactam:ETX2514 activity remains unchanged in carbapenemresistant, colistin-resistant and MDR A. baumannii strains 5 0 4 0 3 0 2 0 1 0 0 0. 2 5 0. 5 1 2 4 8 1 6 3 2 6 4 1 2 8 M I C ( m g / L ) 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 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. 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 ) 23
Sulbactam:ETX2514 is active against A. baumannii encoding multiple β-lactamases Summary of MICs (mg/l) drug N range MIC 50 MIC 90 imipenem 84 0.125 - >128 64 128 SUL- ETX2514 84 0.25-16 2 4 24
Morphology of A. baumannii in the presence of sulbactam:etx2514 suggests multi-target effects No Drug ETX2514 SUL 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. 25
Frequency of spontaneous resistance to sulbactam-etx2514 is very low against clinical isolates of A. baumannii Strain β-lactamase content FOR at 4X MIC Variant Protein affected SUL- ETX2514 MIC (mg/l) SUL MEM CAZ Parent -- 1/4 4 0.5 4 ARC2058 ARC2681 ARC2782 ADC-99-like; OXA-95 ADC-42-like; TEM- 1; OXA-40; OXA- 132 ADC-79; TEM-1; PER-1; OXA-23; OXA-66 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 7.6 x 10 Parent -- 2/4 8 32 256 4X-1 PBP3 [S390T] >64/4 >64 32 128 <9.0 x 10 Parent -- 0.5/4 32 16 >512 2X-1 PBP3 [T511A] 4/4 64 16 >512 trna synthetase mutants are associated with the stringent response and are commonly seen with PBP2 inhibitors 1 Mutations in PBP3 affected the MIC of SUL-ETX2514 and sulbactam alone Resistant mutants suggest sulbactam-etx2514 works by inhibiting both PBP2 and PBP3 1 Vinella et al. (1992) EMBO J. 11:1493-1501 26
Log(CFU/g) Log(CFU/g) Sulbactam:ETX2514 exhibits excellent in vivo activity Greater than 2-log kill achieved in both neutropenic mouse thigh and lung models of A. baumannii infections Sulbactam/ETX2514 dose response (SC, 4/1 ratio) MDR A. baumannii ARC3486 (OXA-72, OXA-66, TEM-1, AmpC) MIC(sulbactam) 32 mg/l, MIC(sulbactam/ETX2514) = 0.5 mg/l 10 Thigh 10 Lung 9 9 9.40 8 7 6 6.36 8.03 8.02 6.72 Stasis 8 7 6 7.40 8.40 8.03 6.63 6.19 Stasis 5 4 3 4.39 4.24 3.97 4.01 4.07 5 4 3 4.85 4.61 4.19 2 Pretreatment Pretreatment Vehicle 2.5 / 0.625 5 / 1.25 10 / 2.5 20 / 5 30 / 7.5 40 / 10 80 / 20 sulbactam/etx2514 (mg/kg) q3h 2 Vehicle 2.5 / 0.625 5 / 1.25 10 / 2.5 20 / 5 30 / 7.5 40 / 10 80 / 20 sulbactam/etx2514 (mg/kg) q3h Similar results obtained for 5 additional clinical isolates 27
PK and safety of sulbactam:etx2514 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 28
Summary and Conclusions 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 MIC 90 = 4 mg/l (N = 1742 clinical isolates) and excellent in vivo activity. Currently in Phase I testing 29
Acknowledgements AstraZeneca Antibacterial Discovery IHMA, Inc. Linneas 30