The Future of Β-Lactamase Inhibitors

The future of β-lactamase inhibitors

Martin Everett

CSO, ANTABIO

ECCMID 2018. Madrid ESCMID eLibrary © by author β-lactamase inhibitors: a successful strategy

● Inhibit hydrolysis of β-lactam antibiotics by bacterial β-lactamases

● Rescue activity of β-lactam when given in combination

● Extended lifetime of our most important class of antibiotics

ESCMID eLibrary © by author β-lactam action and resistance mechanisms

β-lactam Cross-section of Gram-negative bacterial cell envelope

● β-lactams bind and inhibit peptidoglycan synthases (Penicillin Binding Proteins)

● Disrupt cell wall synthesis and cause cell lysis

● β-lactamase enzymes in periplasm (or secreted) can inactivate β-lactams and confer resistance inactive

● Loss of porin function (or increased efflux) also ESCMID contribute to resistance eLibrary © by author β-lactamase classification

Molecular class Enzyme type Functional group Characteristics

A KPC, ESBLs 2 Penicillins ( cephalosporins, carbapenems)

B NDM, VIM 3 All β-lactams (except monobactams)

C AmpC, CMY 1 Cephalosporins (chromosomal)

D OXAs 2d Oxacillins ( carbapenems)

= Serine Beta-Lactamases (SBLs)

= Metallo Beta-Lactamases (MBLs) AM ESCMID eLibrary © by author Timeline: β-lactams, β-lactamases and β-lactamase inhibitors

Amoxycillin/ Piperacillin/ Cephalothin Clavulanic acid Tazobactam Ceftazidime/ Avibactam Methicillin Cefixime Meropenem/ Penicillin Ampicillin Imipenem Meropenem Vaborbactam

1940 1950 1960 1970 1980 1990 2000 2010

ESBLs NDM-1 First penicillinase TEM-1 (TEM, SHV, CTX) described AmpC Carbapenemases hyperproducers (KPC, OXA, IMP, VIM)

β-lactams β-lactamases ESCMID eLibrary β-lactamase inhibitors © by author Recent β-lactamase inhibitors to enter clinical trials

Name of inhibitor Class Partner Status WHO spectrum (developer) β-lactam CRAB CRPA CRE Avibactam (Pfizer) DBO1 Ceftazidime Approved in US (Avycaz) and EU (Zavicefta) Not MBL

Ceftaroline Ph2 completed (?) Not MBL

Aztreonam Ph2 ongoing Incl MBL Vaborbactam (TMC) Boronate Meropenem Approved in US (Vabomere) Not MBL

Relebactam (Merck) DBO Imipenem2 Ph3 ongoing Not MBL

Zidebactam3 (Wockhardt) DBO Cefepime Ph2 completed Some MBL

AAI101 (Allecra) Sulbactam Cefepime Ph2 completed Not MBL

Nacubactam (Roche) DBO Meropenem Ph1 completed Some MBL

ETX2514 (Entasis) DBO Sulbactam Ph1 completed VNRX-5133 (VenatoRx) Boronate Cefepime Ph1 completed Most MBL AIC-499 (Aicuris) ? ? Ph1 initiated (2017) MBL?

Active 1 DBO, diazabicyclooctane 2 plus cilastin Possibly active 3 PBP2 inhibitor, weak BLI activity ESCMID eLibrary Not or insufficiently active © by author Diazabicyclooctanes (DBOs)

Avibactam (+ aztreonam)

● In development with aztreonam which is poorly hydrolysed by MBLs; hence combination protects the antibiotic from ESBLs, KPCs and MBLs.

● Recent report (Lohan et al. AAC, 2017) suggests NDM-1 can slowly hydrolyse both aztreonam avibactam & avibactam, therefore potential vulnerability to MBL evolution

● Enhancing effect of AVI on ATM activity most important vs NDM strains (Chauzy et al. poster)

Relebactam (+ imipenem/cilastatin)

● Triple combination (includes cilastatin DHP-1 inhibitor)

● Relebactam is more stable to hydrolysis by KPC-2 than avibactam relebactam

● Potentiates activity of imipenem against some CRPA (potential advantage vs vaborbactam/meropenem) ESCMID eLibrary © by author DBOs continued

Zidebactam (+ cefepime)

● Weak inhibitor of SBLs

● Synergisitic activity with cefepime mainly due to high affinity for PBP2

Nacubactam (+ meropenem)

● Some intrinsic antibacterial activity due to PBP 2 inhibition (weaker than

zidebactam) providing some activity against certain MBL producers nacubactam

● May be vulnerable to mutations in PBP2

ETX2514 (+ sulbactam)

● Targeting A. baumanii. Active against SBLs including OXA; does not cover MBLs ETX2514

● Also has intrinsic antibacterial activity through PBP2 inhibition ESCMID eLibrary © by author Boronic acid compounds

Vaborbactam (+ meropenem) - Vabomere

● First in new class of boronate BLIs

● Approved for cUTI

● Mainly inhibits class A β-lactamases. Restores susceptibility to MEM in KPC- vaborbactam producing Enterobacteriaceae but not in MBL producers

VNRX-5133 (+cefepime)

● Dual inhibitor of SBLs and MBLs

● Potent activity against Enterobacteriaceae, including most MBLs (not IMP) VNRX5133

● Improved activity vs PA compared to cefepime alone or meropenem ESCMID eLibrary © by author Sulbactam derivatives

tazobactam AAI101 (+ cefepime)

● Methylated derivative of tazobactam (sulbactam class of BLI)

● Improved inhibitory activity against KPCs and OXAs

● Improved inhibitory activity over cefepime alone or piperacillin/tazobactam

AAI101

ESCMID eLibraryANTABIO PRIVATE AND CONFIDENTIAL © by author Opportunities for β-lactamase inhibitor development

Clear gaps remain - insufficient coverage of:

● A. baumanii and P. aeruginosa isolates Unmet Medical Need ● MBL and OXA enzymes

Challenging to achieve broad spectrum

● Large number/variety of target enzymes

● Multiple species with varied resistance phenotypes/mechanisms

Targeted design strategies

● X-ray structures and NMR can help to a limited extent but access to target is equally important

● Use of ‘in-cell NMR’ and other non-label technologies to assess uptake and inhibition in periplasm, including ESCMID into porin/efflux mutants eLibrary © by author Emerging threats

Multiple enzyme types/variants with diverse epidemiologies and regional variation

● Hidden/unknown reservoirs

● Rapid spread via mobilised genes and plasmid transfer between species

● Co-transmission of other resistance genes

Drivers of resistance (acquisition and evolution)

● Continuing increase in carbapenem usage (first –line in many countries)

● Introduction of new BL/BLI combinations, are likely to select for MBL and OXA β-lactamases which are inadequately covered

Improved surveillance is required to identify emerging ESCMIDthreats and to assess eLibrary medical need © by author The spread of MBLs: a global emergency

109

ECDC survey (Nov 2013 – Apr 2014). % carbapenem non- susceptible K. pneumoniae isolates with NDM or VIM

● Rapid spread of MBLs, especially NDM-1

● Endemic in South and South East Asia

● Established reservoirs in Eastern European countries; increasing in US & Canada

● Threat of pan-resistance due to carriage of multiple resistance genes on MBL plasmids ESCMIDNew agents are needed toeLibrary address unmet medical need © by author Alarming increase in NDM-1 producing CRE

NDM reports in S.America (2017) Romero-Alvarez et al. Int J Infect Dis (2017)

ESCMID eLibraryMap from Lee et al. , Front. Microbiol . , 2016 © by author Alarming increase in NDM-1 producing CRE

24% NDM-1 68% NDM-1 21% NDM-1 30% NDM-1 Tunisia (6/15—3/16) Bulgaria (7/15—4/16) Istanbul (2/14—2/16) Istanbul (1/11—12/14) Recent surveillance studies 7/29 CN-S KP 17/25 CN-S KP 17/75 CPE 13/44 CN-S KP Mansoor et al. 2017 Savov et al. 2017 Cizmeci et al. 2017 Interhospital spread Haciseyitoglu et al. 2017 92% NDM-1 Jalisco (9/14—7/15) 19% NDM-1 48/52 CRE (88% KP) Adana (1/13—12/14) Bocanegra-Ibarias et al. 2017 19/98 CN-S KP Candevir et al. 2017

67% NDM-1 Iraq 37/55 CR KP Hussein et al. 2017

36% NDM-1 Beijing (1/10—12/14) 65/179 CN-S KP (7 to 63% increase) Dong et al. 2017

32% NDM-1 China (6/14—6/15) NDM reports in Nationwide survey: S.America (2017) 354/1105 CRE Zhang et al. 2017 Romero-Alvarez et al. Int J Infect Dis (2017)

30% NDM-1 Reunion (1/11—12/14) 13/43 CPE (40% KP) 48% NDM-1 Holman et al. 2017 S. Africa (1/12—12/15) 469/812 CPE ESCMIDSingh-Moodley et al. 2016 eLibraryMap from Lee et al. , Front. Microbiol . , 2016 © by author SENTRY Longitudinal Survey: 2007-2009 to 2014-2016

R ● Increase in incidence of MEM Enterobacteriaceae

● 1.5  2.7% (81% KPN)

● Increase in prevalence of NDM between 2007-2009 and 2014-2016

● APAC: 15.9  53.6%

● EU: 0  11.8%

● LA: 0  23.5%

● NA 0  1.8%

Source: SENTRY – Marianna Castanheira (JMI)

Rapid spread but also rapid evolution of NDM (now 20 reported variants)

● Transfer via commensal enterics

Unique among β-lactamases

● Lipoprotein tethered to inside of outer membrane

● Provides greater stability to low Zn conditions

NDM variants show increased Zn affinity and greater stability (Bahr et al. AAC, 2018)

● Chelating agents (calprotectin, DPA, AMA) have reduced impact

Need specific inhibitors which cover all major variants and do not ESCMIDdepend on metal chelationeLibrary for their MOA © by author 18 MBLi Program: Metallo-ß-lactamase inhibitors

Compound ANT001 ANT090 ANT330 ANT431

MBL NDM-1 VIM-2 NDM-1 VIM-2 NDM-1 VIM-2 NDM-1 VIM-2

MBL Ki (µM) 3.2 >5.8 7.5 0.035 0.85 0.18 0.37 0.19

Fold-MEM potentiation 8x 4x 4x 64x 128x 64x 128x 64x

 Horsfall, L. E. et al. AAC (2007)  Patent WO2014/198849  Fold decrease ESCMID in meropenem MIC vs E. coli carrying pNDM-1 or pVIMeLibrary-2 in the presence of 30 µg/mL inhibitor © by author Co-crystal structures with VIM-2 reveal active site binding modality

ZincZinc ionion

Superimposition of X-ray structure of ANT330 in complex with VIM-2 (blue) and NDM-1 (brown)/IMP-1(yellow) X-ray structures

● Thiazole N and the carboxylate bind to active site single Zn (II) ● Phenyl ring of the sulphonamide makes strong stacking interaction with Phe side chain ● Docking studies indicate conserved MBL binding interactions ● SpecificESCMID competitive MOA (not a chelating agent)eLibrary © by author MBL9: strong potentiation of MEM against NDM-CRE clinical isolates

Potentiation of meropenem in mouse Cumulative MEM susceptibility of 186 NDM-CRE isolates thigh Kp NDM-1 infection model (yr 2015), in presence of 8 µg/mL MBL9, 4 µg/mL SBLi Data from Evotec All cpds dosed IV, at 30 Data from IHMA mg/kg at 1, 3, 5 and 7 hr, except MEM control (300 mg/kg). CFUs determined at 9 hr. Pt = pre-treatment. V = vehicle. (#) = log reduction compared to V

Kp NDM-1 (NTBC104)

MICMEM = 64 µg/mL MICMEM + ANT (8 µg/mL) = 4 µg/mL

• Potent MBL inhibitor with competitive MOA (NDM-1 Ki <30 nM)

• Selective vs other metallo-enzymes (MMP IC50s > 100 µM)

• Strong potentiation of MEM against NDM-CRE clinical isolates (enhanced by SBLi)

• Efficacious in mouse infection model (Kp NDM-1)

• Non-cytotoxic (HepG2 IC50 > 100 µM), non-genotoxic in Ames test

• High plasma exposure in mice, rats and dogs with significant renal clearance

• Good aqueous solubility (> 20 g/L) and stability

● β-lactamase inhibitors have and will continue to play an important role in the prolongation and enhanced effectiveness of β-lactam antibiotics

● Several β-lactamase inhibitors are in preclinical and clinical development

● There is a need for inhibitors of MBLs, especially NDM-type which are spreading rapidly

● Antabio are developing the first specific MBL inhibitor, targeted at the rapidly emerging global NDM-CRE situation ESCMID eLibrary © by author Many thanks for your attention