The production of β-lactamases by Gram-negative bacteria is

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1 PUBLISHED: 30 JUE 017 VLUME: ARTICLE UMBER: 1710 ETX51 is a broad-spectrum β-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii Thomas F. Durand-Réville 1 *, Satenig Guler 1, Janelle Comita-Prevoir 1,BrendanChen, eil Bifulco, Hoan Huynh, Sushmita Lahiri,AdamB.Shapiro 1, Sarah M. McLeod 1, icole M. Carter 1, Samir H. Moussa 1, Camilo Velez-Vega 1,elsonB.livier, Robert McLaughlin,ingGao, Jason Thresher, Tiffany Palmer, Beth Andrews,RobertA.Giacobbe,JosephV.ewman, David E. Ehmann, Boudewijn de Jonge,John Donnell 1,JohnP.Mueller 1, Rubén A. Tommasi 1 and Alita A. Miller 1 * Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of β-lactamases, enzymes that inactivate β-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new β-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine β-lactamase inhibitors that potently inhibit clinically relevant class A, C and D β-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of β-lactam activity in a broad range of MDR Gramnegative pathogens. ne of the most promising combinations is sulbactam ETX51, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need. The production of β-lactamases by Gram-negative bacteria is among the most important drivers of the antibiotic resistance that currently threatens modern medicine. These enzymes, which hydrolyse β-lactam antibiotics, are classified into four Ambler classes (A D) based on amino-acid sequence relationships. Classes A, C and D enzymes use an active site serine for β-lactam hydrolysis, whereas class B consists of metalloenzymes that require divalent zinc ions for activity 1.Traditionalβ-lactamase inhibitors (BLIs) (clavulanic acid, sulbactam and tazobactam) are only active against some class A β-lactamases 1. Avibactam, a novel diazabicyclooctanone (DB) BLI and other non-β-lactam BLIs currently in late-stage development (relebactam, vaborbactam) inhibit many clinically relevant class A and C β-lactamases, as well as a limited number of class D enzymes such as XA- (ref. 3). Avibactam has recently been approved for use in combination with ceftazidime for the treatment of serious Gram-negative infections. This combination is a welcome addition to hospital formularies to combat bacteria that produce extendedspectrum β-lactamases (ESBLs), including some (but not all) strains of Pseudomonas aeruginosa. However, neither avibactam nor any of these new BLIs in late-stage development are effective against Acinetobacter baumannii infections, due in part to the wide variety of class D β-lactamases expressed by this organism 5,. A. baumannii, one of the ESKAPE pathogens 7, is an important cause of severe infections, particularly in immunocompromised patients, and has been listed by the World Health rganization first on their global priority list of multidrug-resistant (MDR) pathogens currently threatening human health ( int/medicines/publications/wh-ppl-short_summary_5feb-et_ M_WH.pdf). In the USA, 3% of A. baumannii isolates are MDR and many are extensively drug resistant (XDR). Although ESBLs and carbapenemases are among the most threatening resistance mechanisms in A. baumannii and P. aeruginosa, resistance to other last-resort agents, such as aminoglycosides, tigecycline and colistin, is also on the rise 9. The potential for pan-drug resistance due to combinations of all these resistance mechanisms obviously needs to be addressed immediately. Here, we report the results of our successful efforts to modify the DB scaffold to extend its spectrum of activity to include coverage of a broad range of class D enzymes while further improving potent class A and C β-lactamase inhibition. This optimization concomitantly led to enhanced antibacterial activity due to binding of penicillin-binding proteins (PBPs), the targets of β-lactams. The best combination of these attributes was manifested in ETX51, whose intrinsic antibacterial activity versus Enterobacteriaceae, in 1 Entasis Therapeutics, 35 Gatehouse Drive, Waltham, Massachusetts 051, USA. AstraZeneca, 35 Gatehouse Drive, Waltham, Massachusetts 051, USA. Present addresses: Blueprint Medicines, 3 Sidney Street, Cambridge, Massachusetts 0139, USA (.B.); Alkermes, 5 Winter Street, Waltham, Massachusetts 051, USA (H.H.); Macrolide Pharmaceuticals, 0 Arsenal Street, Watertown, Massachusetts 07, USA (S.L.); Kaleido Biosciences, 7 Moulton Street, Cambridge, Massachusetts 013, USA (J.T.); Moderna Therapeutics, 30 Bent Street, Cambridge, Massachusetts 011, USA (T.P.); WarpDriveBio, 00 Technology Square, Cambridge, Massachusetts 0139, USA (R.A.G.); Tetraphase Pharmaceuticals, 0 Arsenal Street, Watertown, Massachusetts 07, USA (J.V..); Shire, 300 Shire Way, Lexington, Massachusetts 01, USA (D.E.E.); Pfizer, 10 Main Street, Cambridge, Massachusetts 0139 (B.D.J.). * t.durand-reville@entasistx.com; alita.miller@entasistx.com ATURE MICRBILGY, 1710 (017) DI: /nmicrobiol Macmillan Publishers Limited, part of Springer ature. All rights reserved.

2 ATURE MICRBILGY Table 1 Structures and in vitro activity of BLI compounds. H H C C3 C H H H H H 3 H H H S 3 a S 3 a S 3 a S 3 a S 3 S 3 a S 3 a S 3 S 3 a Avibactam ETX51 7 β-lactamase IC 50 (μm) Piperacillin MIC (mg l 1 )±mgl 1 BLI BLI compound Class A KPC- Class C AmpC Class D XA- Parent ampc poxb Class A KPC- Class C AmpC Class D XA- Hydrolysis, rate constant k (h 1 ) one > > > Avibactam > > > T T T T T 5 ETX T T T T T T T T T > T IC 50 values of avibactam and diazabicyclooctenone analogues for representative class A, C and D β-lactamases were measured after 5 min, following a 5 min preincubation of BLI compound with enzyme (n ). MICs in combination with piperacillin for isogenic P. aeruginosa strains overexpressing individual β-lactamase enzymes were measured for BLI compounds (n ). The piperacillin MIC of the parent strain (whose endogenous β-lactamase genes ampc and poxb are deleted) is mg l 1. When the parent strain is transformed with any of the plasmids that drive constitutive expression of the β-lactamase of interest, the piperacillin is hydrolysed and its MIC increases to > mg l 1. Hydrolytic stability was evaluated by measuring the hydrolysis rate constant at 70 C in phosphate buffer (ph 7.) (n = 1). T, not tested. addition to the most broadly potent spectrum of β-lactamase inhibition described to date, translates to effective activity versus all Gram-negative ESKAPE pathogens, including carbapenem-resistant Enterobacteriaceae (CRE), MDR P. aeruginosa and A. baumannii. Its excellent preclinical safety results, as described herein, also strongly supports further advancement towards clinical testing. Results Rational design of an extended-spectrum inhibitor of class A, C and D β-lactamases. To discover the next-generation BLI that would be effective against hard-to-treat MDR P. aeruginosa and A. baumannii, we sought to design DBs with broadened class D coverage and enhanced Gram-negative cell permeation, while maintaining or improving class A and C inhibition. Using X-ray crystal structures of class D enzymes, we focused on structural modifications around size and polarity that would affect both affinity for a broad set of β-lactamases and bacterial uptake. Chemical reactivity was also taken into account in our design hypotheses, as the DBs are covalent inhibitors and must react with the active site serine of these enzymes. We also measured the reactivity against PBPs, as it has previously been shown that DBs can have this additional mode of action (MA) 10,11. This dual MA is attractive for its potential to both improve bacterial cidality and limit the emergence of bacterial resistance. Among the serine β-lactamases, the biochemically and structurally highly diverse class D oxacillinase (XA) enzymes represent a particularly worrisome challenge as many members hydrolyse carbapenems, which are among the last-resort drugs for hard-to-treat MDR Gram-negative infections 5. The sequence and structural analysis of several XAs revealed a high frequency of aliphatic, hydrophobic and aromatic amino acids around the active site serine 1. A large number of clinically relevant XAs (including XA-3, XA-, XA-51 and XA-5) contain a hydrophobic bridge that spans the active site pocket, allowing the β-lactam substrate to remain in place for enzymatic hydrolysis 13.In contrast, XA- does not have a hydrophobic bridge and its active site contains several more polar residues. Avibactam and all other DBs currently in clinical testing can inhibit the XA- subfamily but not those XAs with more hydrophobic active sites. In contrast to avibactam and all other DBs currently in clinical development, we considered modification of the bicyclic core, although synthetically challenging, as the best approach to modulate the reactivity of the scaffold, which we hypothesized would be required to achieve a broader β-lactamase coverage. We first tested analogues for activity against a panel of representative serine β-lactamase enzymes, followed by a cell-based assay to demonstrate restoration of the minimum inhibitory concentration (MIC) of piperacillin against an isogenic panel of P. aeruginosa strains expressing individual β-lactamases (Table 1). This isogenic panel was particularly useful in correlating enzymatic to cellular potency, thereby indirectly assessing bacterial permeation, one of the most challenging aspects of Gram-negative antibacterial drug discovery 1. We used P. aeruginosa as the background for this step in our screening cascade due to its notoriously low outer membrane permeability. This choice was also prudent because some of our analogues had intrinsic antibacterial activity against Escherichia coli, which would have complicated a direct demonstration of β-lactamase inhibition in this background. Early designs were aimed at introducing a carbon carbon double bond in the piperidine ring to increase ring strain and chemical reactivity. The first analogues (1, ) lacked the carboxamide at C but already showed higher chemical reactivity compared to avibactam, with hydrolysis rate constants of 0.0 and 0.05 h 1 versus 0.0 h 1, respectively (Table 1). Compound 1 showed a similar potency against the class A and C β-lactamases as avibactam, with a modest improvement (. versus 1 µm) against class D XA-. Lipophilic substitutions significantly improved inhibition of XA-, consistent with our class D β-lactamase structural analysis. The C methoxy methyl and C -propene groups on compound significantly decreased the XA- IC 50 (half maximal inhibitory concentration) compared to avibactam and compound 1 by 1,100- and 10-fold, respectively. This considerable improvement in enzymatic potency did not translate into activity in bacterial cells, however, as compound failed to restore piperacillin activity in the P. aeruginosa isogenic panel, probably due to poor permeation and/or high efflux from the bacteria. Innovative chemistry (Supplementary Method 1 and ref. 15) was used to re-install the carboxamide functionality, and a series of analogues bearing a C carboxamide group (compounds 3 ) were evaluated in vitro. Further increased reactivity was observed, together with higher potency against class A and C β-lactamases. The introduction of polar moieties (either neutral or charged) on the double bond (compounds and ) was detrimental to XA- inhibition. The positively charged side chain of compound ATURE MICRBILGY, 1710 (017) DI: /nmicrobiol Macmillan Publishers Limited, part of Springer ature. All rights reserved.

3 ARTICLES ATURE MICRBILGY Table ETX51 restores β-lactam sensitivity in A. baumannii overexpressing individual class A, C or D β-lactamases. Piperacillin +AVI > > β-lactamase one > > 3 > > one TEM-1 KPC- ADC-30 XA-3 XA- +ETX51 MIC (mg l 1) Meropenem +AVI Alone 3 1 +ETX Alone Sulbactam +AVI 1 +ETX51 AVI, avibactam. MICs for indicated β-lactams against an isogenic panel of A. baumannii strains overexpressing individual β-lactamase enzymes were compared to their MICs in combination with mg l 1 avibactam or ETX51 (n ). a b Met3 7. Tyr11 c Met3 Met3.7.3 Tyr Tyr Ser1 Ser1 Ser1 e d f Tyr11 Tyr11 Tyr11 Met3 Met3 Ser1 Met3 Ser1 Ser1 Figure 1 Time-averaged solution structures of avibactam, 3 and 5 covalently bound to Ser1 of XA-, from MD simulations. a, Model of avibactam XA- (light blue) overlaid onto its corresponding crystal (pink, PDB code WM9). b, Model of 3 XA- (light blue) overlaid onto its corresponding crystal (pink). Also shown is a second copy of the ligand, which was found to be non-covalently bound to the protein and the ligand (Supplementary Fig. 1). c, Model of 5 XA-. d f, Structure side views of avibactam XA- (d), 3 XA- (e), 5 XA- (f) with high water occupancy obtained from MD simulations. Dark blue areas represent regions with at least approximately four times higher water occupancy relative to bulk water. nly the relevant residues Met3 and Tyr11, and the ligand covalently bound to Ser1, are shown. Dashed lines correspond to hydrogen bonds, and distances are in angstroms. xygen, nitrogen, hydrogen and sulfur are in red, blue, white and gold, respectively. Some atomic groups appear distorted (for example, the sulfate, in all cases). This distortion is the result of averaging atomic coordinates over MD frames and is in itself a useful way of denoting higher mobility throughout the simulation in a single snapshot. was also detrimental to AmpC inhibition (IC50 = 0.7 µm). Several substituted amides and amide isosteres were subsequently synthesized to explore the structure activity relationship (SAR) around the C position. Whereas neutral side chains led to improvement in class D enzymatic activity, a positive charge was once again detrimental to binding to XA- (IC50 of compound 7 = 70 µm). The addition of a C nitrile (compound ) failed to deliver potent class A and C inhibition despite a low XA- IC50 of µm. Ultimately, the greatest biochemical and antibacterial potencies were achieved with small alkyl groups at the C3/C positions in the presence of the C carboxamide. All those features were successfully combined in compound 5, a rationally designed BLI with the broadest spectrum reported to date against serine β-lactamases. Compound 5 was subsequently nominated as our preclinical lead candidate, ETX51. To confirm that the broad-spectrum inhibition of class A, C and D β-lactamases by ETX51 observed in vitro could translate into restoration of β-lactam activity in A. baumannii whole cells, we tested its activity in combination with piperacillin, meropenem or sulbactam against an isogenic set of A. baumannii strains, each of which overexpressed an individual, representative β-lactamase. As shown in Table, β-lactamase overexpression correlated to reduced β-lactam activity against these strains, which was restored by avibactam against class A and C enzymes, as expected. In contrast, ETX51 fully restored β-lactam activity against not only class A and C, but also class D-expressing strains. Co-crystal structure with XA-. As part of our rational design strategy, we attempted to solve crystal structures of our analogues bound to XA-. Although our efforts to co-crystallize ETX51 were not successful, we were nevertheless able to obtain a co-crystal structure of compound 3 with XA- (PDB code 5VFD, ATURE MICRBILGY, 1710 (017) DI: /nmicrobiol Macmillan Publishers Limited, part of Springer ature. All rights reserved. 3

4 resolution 1.93 Å). Supplementary Table 1 shows the corresponding crystallographic data for this structure. Supplementary Fig. 1 shows an omit Fo-Fc electron density map of compound 3. Two copies of the ligand were found in the binding site; one is covalently attached to Ser1, as expected by its mechanism of action, while the second is non-covalently bound to Met11 via its sulfate and to the first ligand via its carboxamide. The carboxamide of the covalently bound ligand is perpendicular to the one observed in the avibactam structure (PDB code WM9, resolution. Å), resulting in a direct hydrogen bond between the carboxamide H and the backbone carbonyl of Trp1. The sulfate of the covalently bound ligand displays interactions with Lys1, Ser19 and Arg1, as in the avibactam crystal. The hydrophobic bridge formed by Tyr11 and Met3, which has been reported previously in published structures 7, remained intact upon binding compound 3. Moreover, this hydrophobic interaction appears to be tighter than that observed in the avibactam crystal, based on normalized temperature factor differences between both structures for these residues (Supplementary Table ). The mechanistically critical Lys is also observed in its carboxylated form 1, in contrast to the avibactam structure. Molecular modelling of XA- in complex with avibactam, compound 3 and compound 5. To complement the structural information provided by the co-crystal structure of compound 3, we carried out molecular dynamics (MD) simulations of compounds 3, 5 and avibactam, in complex with XA-. MD supports the hypothesis that the hydrophobic bridge between Met3 and Tyr11 is less stable when avibactam is bound, compared to when compound 3 or compound 5 is bound. Figure 1 shows time-averaged structures of avibactam (Fig. 1a), compound 3 (Fig. 1b) and compound 5 (Fig. 1c), covalently bound to Ser1 of XA-, as well as the key residues Tyr11 and Met3. The corresponding crystal structure has been overlaid onto the models of compound 3 and avibactam. Figure 1a illustrates that the MD time-averaged distance of the hydrophobic bridge (MD model in light blue) is twice as large (7. Å) as that of its reference crystal (3.9 Å) when avibactam is bound. Conversely, short distances between these residues are maintained for both compound 3 (.7 Å, Fig. 1b) and compound 5 (.3 Å, Fig. 1c). Presumably, the presence of a methyl at C (as in compound 3) or at C3 (as in compound 5) promotes the formation of a hydrophobic cluster with strengthened van der Waals energy. Moreover, these tight interactions could also block access of the solvent to the ligands sulfate and concurrently to the covalent reaction site, preventing deacylation. Figure 1 also illustrates how solvent populates this channel when avibactam is bound (Fig. 1d, red oval), something not observed in MD simulations of compound 3 (Fig. 1e) and compound 5 (Fig. 1f). To assess how the actual covalent reaction step is affected by including a double bond in the piperidine core (for example, compounds 3 and 5), we performed quantum mechanical calculations for avibactam and compound 5. ur analysis suggests that compound 5 is more reactive than avibactam by.7 kcal mol 1. This energy should be enough to account for a dominant portion of the greater than 100-fold difference in k inact /K i between these two compounds. verall, crystallography and molecular modelling suggest that the combination of increased chemical reactivity and tighter β-lactamase binding is the likely reason for the improved potency and broader-spectrum class D activity of this unique series of diazabicyclooctenone BLIs. β-lactamase and PBP inhibition. Table 3 shows the second-order rate constant (k inact /K i ) for acylation by ETX51 of a set of eight serine β-lactamases representing classes A, C and D. ETX51 Table 3 In vitro characterization of ETX51. ATURE MICRBILGY Class or species Enzyme k inact /K i (M 1 s 1 )* Class A CTX-M-15 7 (±) 10 TEM-1 1. (±0.) 10 7 KPC- 9.3 (±0.) 10 5 SHV-5. (±) 10 Class C AmpC 9 (±5) 10 5 P99.3 (±0.) 10 Class D XA-10 9 (±) 10 3 XA (±0.) 10 3 XA- 9 (±) 10 3 XA- (±) 10 5 A. baumannii PBP1a 1 (±1) 10 1 PBP 1. (±0.) 10 3 PBP ± 0.0 P. aeruginosa PBP1a 1 ± 7 PBP.3 ± 0. PBP3 (±1) 10 1 E. coli PBP1a 1 (±) 10 1 PBP 1.7 (±0.3) 10 PBP3.3 ± 0. *Data expressed as mean ± s.d.; n = 3, except n = for TEM-1, AmpC and E. coli PBP1a. Acylation by ETX51 of representative class A, C and D β-lactamases or Gram-negative bacterial PBPs. potently inhibited all three classes, with k inact /K i on the order of M 1 s 1 for class A and C enzymes and M 1 s 1 for class D enzymes. These rate constants were greater than those of avibactam by a factor of 100 for class A and C enzymes and 1,000 for class D enzymes 1. Given the evolutionary and mechanistic relationship between serine β-lactamases and PBPs (ref. 17), as well as properties of several recently described DBs (refs 10, 1), we also measured the second-order acylation rate constants by ETX51 of PBP1a, PBP and PBP3 of A. baumannii, P. aeruginosa and E. coli (Table 3). ETX51 showed selective inhibition of PBP in E. coli and A. baumannii, with weaker inhibition of PBP1a and relatively little inhibition of PBP3. Inhibition of all three P. aeruginosa PBPs was comparatively modest. Antibacterial activity and restoration of carbapenem activity in CRE strains. ETX51 exhibited antibacterial activity against Enterobacteriaceae including E. coli, Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia and Citrobacter species (Supplementary Table 3). f particular note was the activity of ETX51 alone against the most threatening MDR pathogens within this family, carbapenem-resistant (CRE) Enterobacteriaceae, including class B metallo-β-lactamase-positive strains (Fig. a and Supplementary Table ). ETX51 was also active against colistin-resistant E. coli isolates bearing the mcr-1 plasmid (Fig. b). The added benefit of combining intrinsic antibacterial activity with increased BLI potency is evident upon comparison of the ability of ETX51 versus relebactam, which lacks intrinsic antibacterial activity, to restore imipenem activity against these strains. The imipenem MIC 90 for 3 CRE strains decreased by 51-fold upon addition of ETX51 (from to 0.15 mg l 1 ), whereas addition of relebactam resulted in only a twofold drop (to 3 mg l 1 ) (Supplementary Table ). In contrast, ETX51 had minimal antibacterial activity alone versus A. baumannii and P. aeruginosa (Table ). The MA of antibiotics can be characterized by treatmentinduced changes in bacterial morphology 19 (Fig. c e). When treated with ETX51, both E. coli and A. baumannii cells became round, consistent with PBP-mediated inhibition (Fig. c,d), whereas ETX51-treated P. aeruginosa cells became elongated with distinct bulges (Fig. e). These results, together with the acylation data, support the hypothesis that PBP inhibition is the ATURE MICRBILGY, 1710 (017) DI: /nmicrobiol Macmillan Publishers Limited, part of Springer ature. All rights reserved.

5 ATURE MICRBILGY ARTICLES a umber of strains MIC (mg l 1 ) ETX51 alone IPM alone IPM + REL IPM + ETX51 b umber of strains 0 ETX51 MIC (mg l 1 ) Colistin c d E. coil no drug ETX51 A. baumannii no drug ETX51 Aztreonam Mecillinam Sulbactam Sulbactam ETX51 e P. aeruginosa no drug ETX51 Figure Spectrum and mechanism of ETX51 intrinsic antibacterial activity, which enhances its ability to restore carbapenem activity against CRE strains. a, Antibacterial activity of ETX51 and imipenem (IPM) alone or in the presence of mg l 1 ETX51 or relebactam (REL) against 3 CRE strains. The species, β-lactamase content and susceptibility of each strain are described in Supplementary Table. b, Antibacterial activity of ETX51 and colistin against 10 mcr-1 + strains of E. coli. c e, MicroscopyofE. coli (c), A. baumannii (d) andp. aeruginosa (e) with and without antibiotics. Selective PBP inhibition by mecillinam (a PBP-specific β-lactam) causes the cells to become round instead of rod-like due to inhibition of cell elongation, whereas selective PBP3 inhibition by aztreonam causes the cells to become highly elongated due to inhibition of septation during replication (c). Antibiotics were dosed at MIC, except for ETX51 versus P. aeruginosa, which was dosed at.5 MIC, and sulbactam ETX51, which were dosed at MIC for sulbactam and mg l 1 for ETX51. Images shown are representative of at least three different experiments. Scale bars, 5 µm. probable mechanism behind the intrinsic antibacterial activity of ETX51 versus Enterobacteriaceae. They also demonstrate that PBP inhibition by ETX51 results in phenotypic changes in less susceptible bacterial cells. Restoration of β-lactam antibacterial activity across Gramnegative pathogens. We determined the extent to which addition of mg l 1 ETX51 improved the activity of representative β-lactams against approximately 00 global, randomly selected, recent Gram-negative clinical isolates. As expected, all β-lactam- ETX51 combinations resulted in very potent activity against Enterobacteriaceae (MIC mg l 1, Supplementary Table 5) due to the dual MA of ETX51, as described above. ETX51 also improved the activity of all β-lactam partners tested against both P. aeruginosa and A. baumannii (Table ). The most potent combination against P. aeruginosa was imipenem ETX51, with an MIC 90 corresponding to the breakpoint 0 for imipenem susceptibility, whereas the most potent combination against A. baumannii was sulbactam ETX51. This correlates well with the degree to which ETX51 restored sulbactam activity against the A. baumannii isogenic β-lactamase panel described above (Table ). Antibacterial activity of sulbactam combined with ETX51 against A. baumannii. Although primarily known as a class A BLI, sulbactam also has intrinsic antibacterial activity against Acinetobacter spp., which is linked to its inhibition of PBP3 (ref. 1). However, its degradation by certain emerging β-lactamases, such as TEM-1, ADC-30 and a number of XAs, has eroded its clinical utility for Acinetobacter infections,3.whentestedalone, sulbactam was weakly active against A. baumannii clinical isolates, but when combined with ETX51, its MIC 90 improved 1-fold to mgl 1 (Table ). The frequency of spontaneous resistance to this combination was very low ( at MIC) against four distinct clinical isolates. To assess whether PBP inhibition by ETX51 contributes to the potentiation of the antibacterial activity of sulbactam, we evaluated the morphological effect of the combination on sulbactam-sensitive A. baumannii. These cells became filamentous ATURE MICRBILGY, 1710 (017) DI: /nmicrobiol Macmillan Publishers Limited, part of Springer ature. All rights reserved.

6 ATURE MICRBILGY Table Restoration of β-lactam activity by ETX51 in P. aeruginosa and A. baumannii. MIC P. aeruginosa (n =0) A. baumannii (n =195) MIC 50 MIC 90 Susceptible MIC 90 Susceptible β-lactam* (mg l 1 ) (mg l 1 ) Range breakpoint 50 (mg l 1 ) (mg l 1 ) Range breakpoint IPM Alone 1 > > 0.0 > +ETX > > MEM Alone > 1 > 0.0 > +ETX > > ATM Alone > > > one +ETX > 0.0 > CAZ Alone > 1 > > 1 > +ETX > > SUL Alone one 1 1 +ETX Each MIC was performed as a single test. *IPM, imipenem; MEM, meropenem; ATM, aztreonam; CAZ, ceftazidime; SUL, sulbactam. ETX51, which has minimal to no activity against P. aeruginosa or A. baumannii, was tested at mg l 1. The MIC (mg l 1 ) that correlates with clinical efficacy as defined by the Clinical Laboratory Standards Institute (CLSI). Based on ampicillin sulbactam (:1) susceptible breakpoint of mg l 1 / mg l 1. a log c.f.u. per g tissue PRE Vehicle.5:0.5 5:1.5 10:.5 0:5 30:7.5 0:10 Sulbactam:ETX51 (mg kg 1 ) 0:0 b log c.f.u. per g tissue PRE Vehicle.5:0.5 5:1.5 10:.5 0:5 30:7.5 0:10 0:0 Sulbactam:ETX51 (mg kg 1 ) PRE Vehicle SUL alone (15 mg kg 1 ) 15:1 15:.5 15:5 15:0 15:30 15:50 Sulbactam:ETX51 (mg kg 1 ) Figure 3 Sulbactam ETX51 in vivo efficacy in MDR A. baumannii infection mouse models. a c, Dose response of a :1 ratio of sulbactam to ETX51 (in mg kg 1, subcutaneous dosing) against MDR A. baumannii in thigh (a) and lung (b) models of infection. Titration of ETX51 with a set dose of 15 mg kg 1 of sulbactam (c) was tested in a thigh infection model. The strain used for these studies is ARC3 (XA- +,XA-7 +, TEM-1 +, ADC-30 + ; sulbactam MIC 3 mg l 1, sulbactam MIC in the presence of mg l 1 ETX51 = mg l 1 ). The y axis corresponds to the log of the bacterial c.f.u. recovered per g of tissue. The x axis indicates the amounts of sulbactam ETX51 tested (mg kg 1 ). PRE, pretreatment. P < 0.05 across all dose arms using one-way AVA. Error bars show standard deviation from five mice per treatment group. c log c.f.u. per g tissue when treated with sulbactam alone 1 (Fig. d) and spherical when exposed to ETX51 (Fig. d). When treated with a combination of sulbactam and ETX51, the cells became even larger spheres than when treated with ETX51 alone (Fig. d). These results support the hypothesis that the MA of the combination includes complementary inhibition of PBP by ETX51 and PBP3 by sulbactam. β-lactamase content in MDR A. baumannii. The in vitro activity of sulbactam ETX51 was determined against 1,131 A. baumannii clinical isolates from globally diverse, hospitalized patients in 01. Sulbactam ETX51 was 1-fold more active than sulbactam alone (MIC 90 values of and mg l 1, respectively). This increased potency was maintained against all subsets of resistant phenotypes including 731 meropenem-resistant, 5 colistin-resistant and 77 MDR isolates 5. To better understand the complexity of β-lactamase content in contemporary MDR A. baumannii, we sequenced the genomes of non-clonal, globally diverse clinical isolates from 00 to 01 (Supplementary Datafile 1). ur analysis showed that all strains contained two to five distinct β-lactamase genes. In addition to an endogenous class C adc (Acinetobacter-derived cephalosporinase), each strain encoded at least one and up to three class D β-lactamases. f the endogenous adc genes, % were variants with extended-spectrum β-lactamase activity. Furthermore, 5% of the isolates encoded class A in addition to class C and D β-lactamase genes. This analysis confirmed that any effective A. baumanniitargeting BLI must demonstrate potent, broad activity against all three classes of serine β-lactamases. The carbapenem MIC 90 against this collection of strains was 1 mg l 1, while the sulbactam ETX51 MIC 90 was mg l 1 (Supplementary Datafile 1). Drug metabolism, pharmacokinetic profile and in vivo efficacy of sulbactam ETX51. ETX51 demonstrates similar physicochemical, drug metabolism and pharmacokinetic (DMPK) properties as other representatives of the DB series 7. ETX51 is suitable for intravenous administration, with stability for hat room temperature and solubility 00 mg ml 1 in water. The pharmacokinetic parameters of ETX51 in mice are summarized in Supplementary Table. Efficacy studies of sulbactam ETX51 in thigh and lung murine infection models showed a dosedependent reduction in MDR A. baumannii bacterial counts (Fig. 3a,b). Combination PK of sulbactam ETX51 was completed over the dose range of the efficacy studies and used to estimate exposures for all doses. Bactericidal activity of sulbactam ETX51 greater than one-log kill was achieved when sulbactam concentrations exceeded the combination MIC of mg l 1 for 50% of the dosing interval. There was no activity associated with sulbactam administered alone dosed every three hours at 15 mg kg 1 (a dose for which all concentrations are below the sulbactam MIC of 3 mg l 1 ). Addition of ETX51 to sulbactam increased the activity in a dose-dependent manner consistent with the lower ATURE MICRBILGY, 1710 (017) DI: /nmicrobiol Macmillan Publishers Limited, part of Springer ature. All rights reserved.

7 ATURE MICRBILGY combination MIC of mg l 1 (Fig. 3c). Similar efficacy was observed with five other MDR A. baumannii strains. Toxicology and safety pharmacology studies of ETX51. ETX51 was well tolerated in both rat and dog in 1-day repeat dose toxicology studies up to,000 mg kg 1, with no significant clinical findings after intravenous administration. There were no changes in ophthalmology, urinalyses, haematology parameters or organ weight. In cardiovascular safety pharmacology studies, ETX51 demonstrated no effects on qualitative echocardiogram parameters, heart rates or arterial pressures up to doses of,000 mg kg 1. Discussion The antibacterial pipeline remains dangerously sparse for agents with activity against MDR Gram-negative bacteria. Significant efforts have been devoted to the discovery of novel BLIs over past decades, with some recent, encouraging results. The non-β-lactam DB class represented a breakthrough in the field with respect to both mechanism of inhibition and spectrum of activity. In addition to ceftazidime/avibactam (approved) and the imipenem/ relebactam combination in phase 3, two other DBs are in clinical trials (zidebactam and nacubactam) that not only inhibit class A and C β-lactamases but also show some antibacterial activity, targeting PBP in Enterobacteriaceae 3,11,9. Another class of non-β-lactam BLIs are the boronates, the most advanced of which is vaborbactam, a class A BLI being developed in combination with meropenem 30. However, none of these combinations has activity against A. baumannii. Several types of class D BLI have also been described, but none of these potently restores β-lactam activity in P. aeruginosa and A. baumannii. Recently, data were disclosed on WCK3, a DB that inhibits XA-3 and XA-51 as well as class A and C β-lactamases. WCK3 is less active against XA-- or XA-5- expressing strains and is devoid of intrinsic antibacterial activity, features that may explain its weaker activity against A. baumannii in combination with partner agents 31. With the ever growing number and diversity of β-lactamases, novel BLIs with expanded activity are urgently needed. Structurebased drug design, computational chemistry and medicinal chemistry were used here to discover a differentiated series of broad-spectrum diazabicyclooctenone BLIs. ur design hypothesis behind this next-generation BLI was based on a combination of increased chemical reactivity and improved enzymatic binding to obtain the broadest spectrum BLI possible, which also resulted in PBP inhibition. Attention was also given to designing good Gramnegative permeation (especially in P. aeruginosa and A. baumannii) and physicochemical properties suitable for intravenous dosing. Additionally, the DMPK properties had to be compatible with those of potential β-lactam partners, both for dosing and to exploit the multi-target MA. The diazabicyclooctenone class described herein represents a significantly differentiated expansion of the DB chemotype that may enable the development of several effective β-lactam BLI combinations for pathogen-targeted therapies to address areas of high unmet medical need. The attributes of ETX51 as described above culminate in restoration of imipenem activity in MDR CRE and P. aeruginosa and potentiation of sulbactam activity in MDR A. baumannii, all to clinically relevant levels. The sulbactam ETX51 combination notably demonstrated potent antibacterial activity against >1,300 recent clinical isolates of A. baumannii (including the particularly hard to treat carbapenem-resistant and colistin-resistant strains), in vivo efficacy in murine thigh and lung models of MDR A. baumannii infection, and excellent preclinical safety. The remarkable complexity of β-lactamase content in MDR A. baumannii clinical isolates further supports the potential clinical utility of this combination. The antibacterial ARTICLES pipeline is currently dangerously thin for MDR A. baumannii agents. Therefore, sulbactam ETX51 may provide significant benefits to infected patients and the global healthcare system. Fortunately, the clinical utility of this combination can now be realized thanks to the recently passed 1st Century Cures Act, which allows for development of narrow-spectrum agents based on small trials (which were previously not feasible). ETX51 has recently entered phase 1 clinical trials, and the results of its continued development will be reported in due course. Methods Experimental design. The objective of this programme was to discover a novel broad-spectrum serine BLI to be combined with a β-lactam with the ultimate goal to treat patients infected by MDR Gram-negative bacteria. Iterative medicinal chemistry cycles using structure activity relationships, structure-based drug design and computational models led to the discovery of ETX51. In vitro activity and DMPK properties were used to select dosage regimens for in vivo efficacy testing in murine infection models. Detailed understanding of pharmacokinetic/ pharmacodynamic (PK/PD) relationships and results from safety studies informed phase 1 clinical trial design. Synthesis of analogues. The eight new diazabicyclooctenone analogues (1 ) were synthesized and isolated as described in a recent patent 15. The purity of all final compounds was judged by 1 H MR to be >90%. Detailed procedures can be found in Supplementary Method 1. Avibactam was synthesized according to the literature 3. β-lactamase inhibition assays. β-lactamase IC 50 values were measured in a spectrophotometric assay employing nitrocefin with a 5 min pre-incubation of BLI and enzyme as previously described 33. The methods used to purify β-lactamases and determine kinetic parameters are described in Supplementary Methods and 3. Fluorescence anisotropy assays for PBP inhibition. The methods used to purify PBPs are described in Supplementary Method. Second-order acylation rate constants for ETX51 with PBP from P. aeruginosa and A. baumannii and with PBP3 from P. aeruginosa, A. baumannii and E. coli were measured using the BCILLI FL penicillin fluorescence anisotropy assay method; specific conditions for each are described in the following 3,35. The lack of a significant change in BCILLI FL fluorescence anisotropy upon reaction with E. coli PBP1a and PBP necessitated the use of SDS PAGE to measure the reaction kinetics 3. The BCILLI FL (Thermo-Fisher Scientific) concentration was 30 nm in each case, except for EcPBP3, for which the BCILLI FL concentration was 0 nm. The PBP concentrations were 0 nm for P. aeruginosa and A. baumannii PBP1a and PBP3, 300 nm for P. aeruginosa PBP, 00 nm for A. baumannii PBP and 175 nm for EcPBP3. The assay buffer was 0.1 M sodium phosphate with 0.01% Triton X-100. The ph was 7.0 for all PBPs with the exception of P. aeruginosa PBP, for which the ph was.. Serial twofold dilutions of ETX51 were employed, with concentrations ranging from 1,000 to µm for A. baumannii PBPs, 3 to 0.0 µm for P. aeruginosa PBPs and 3,330 to 0 µm for E. coli PBP3. For E. coli PBP1a and PBP, an SDS PAGE-based assay was performed, as follows. Reactions (10 µl) in 0.1 M sodium phosphate (ph 7.0) contained µm BCILLI FL for E. coli PBP1a or µm BCILLI FL for E. coli PBP, 15 nm PBP1a or 0 nm PBP and one of several concentrations of ETX51. Reactions were quenched after,,, 1 min at room temperature with µl of LDS Tru-PAGE sample buffer (Sigma-Aldrich) + 1. µl of 10 upage sample reducing agent (Thermo-Fisher Scientific), heated at 70 C for 10 min and subjected to electrophoresis on 0% acrylamide denaturing TruPAGE gels, one ETX51 concentration per gel, at 10 V with TEA-Tricine running buffer. Gels were fixed with 0% (vol/vol) methanol/10% (vol/vol) acetic acid/50% (vol/vol) water for at least 15 min, then washed with water for at least 1 h. The fluorescence intensity of the PBP band was measured with an Azure Biosystems C00 cooled charge-coupled device (CCD) camera-based gel documentation system with green fluorescence settings, using the same exposure time for all gels, taking care to avoid pixel saturation. Each gel was then stained with 5 ml of colloidal Coomassie Blue (Sigma-Aldrich), destained with 50 ml of water and photographed with the Azure Biosystems C00 system using white-light settings. All Coomassie-stained gels were photographed simultaneously using automatic exposure time selection. The fluorescent and Coomassie-stained band intensities were integrated using AzureSpot software (Azure Biosystems), with subtraction of background from an adjacent portion of the gel within the same lane for each band. The integrated fluorescence band intensities were divided by the integrated Coomassie-stained band intensities to correct for loading differences. These values were then used to calculate the second-order acylation rate constant using the same method as for the fluorescence anisotropy assay. Crystallization and structure determination. Crystallization of XA- with compound 3 was carried out as previously described using the vapour diffusion ATURE MICRBILGY, 1710 (017) DI: /nmicrobiol Macmillan Publishers Limited, part of Springer ature. All rights reserved.

8 technique in a sitting drop set-up. Crystallization was achieved using mg ml 1 of enzyme in 3 mg ml 1 of compound in 0.1 M MES buffer (ph.0) containing. M (H ) S as the precipitant solution. Data were collected at cryogenic temperature using the Rigaku FRE+ generator equipped with the Saturn 9 CCD detector. Data reduction was accomplished with the AutoPRC toolbox (Global Phasing) and phases were calculated by molecular replacement with the program PHASER (University of Cambridge, Department of Haematology) using the native XA- coordinates (PDB code JC7) as the starting model. Stereochemistry libraries were calculated using GRADE (Global Phasing), and a model of compound 3 was fitted into positive electron density with the program RHFIT (Global Phasing). Coot 37 was used to inspect the model, and electron density and BUSTER (Global Phasing) were used for macromolecular refinement calculations. The geometry of the model was analysed with MolProbity (Duke University, Department of Biochemistry), and the stereochemistry of the compound was analysed with MGUL (Cambridge Crystallographic Data Centre). Molecular modelling, MD simulations. The crystal structure of avibactam in complex with XA- (PDB code WM9) was prepared using Schrodinger s Protein Preparation Wizard and post-processed manually to ensure consistency of the rotameric and protonation states across the protein s hydrogen-bonding network. All crystal waters were removed and the protein was then solvated in TIP3P water. Gromacs 5.1. (ref. 3) was used as the MD engine, and post-processing of the trajectories was in part performed using VMD (IH Center for Macromolecular Modeling and Bioinformatics, Theoretical and Computational Biophysics Group) and Placevent 39 software packages. Two 5 ns explicit solvent covalent MD simulations were run for each compound, and each simulation was given a random seed for the initial assignment of atomic velocities, to slightly perturb the system and promote better sampling. A time-averaged solute solvent structure was produced for each run, following previous work 0, and the resulting models were analysed. In all three cases, the two MD runs led to equivalent results. Some solute groups of these structures may look distorted (Fig. 1), given that they are averages of atomic coordinates over numerous MD frames. Distorted regions suggest less structural stability for that particular group of atoms. The time-averaged solvent structures also shown in Fig. 1 represent solvation that has at least four times higher occupancy than bulk water. Molecular modelling, quantum mechanical calculations. A simplified model was used to estimate the difference in reactivity between avibactam and compound 5. Jaguar software (Schrödinger) was used for this purpose. The solution-phase energy (density functional theory at the M0X/-31G** level, Poisson Boltzmann finite element solvation method) was independently evaluated for each compound and for a methoxide ion representing the reactive group of Ser1. Analogous energy calculations were then performed for systems comprising both the compound and the methoxide, at an optimized distance and Dunitz angle for nucleophilic attack. The solution-phase energy difference between the joint system (compound methoxide) and its isolated counterparts was considered representative of the reactivity of each compound. This approach was validated previously by comparing calculated reactivity energies with hydrolytic stability measurements for (1) a set of 13 in-house analogues with the same scaffold as compounds 3 and 5 (r = 0.1) and () a set of six lactam rings reported in the literature 1 (r = 0.). Antibacterial susceptibility testing. Piperacillin was purchased from Sigma. Aztreonam, ceftazidime, sulbactam, imipenem and meropenem were purchased from USP. The MICs of these compounds, alone or in combination with BLIs, against each organism were determined using the Clinical and Laboratory Standards Institute (CLSI) guidelines broth microdilution methodology. Unless otherwise indicated, each reported MIC is from a single test. Bacterial strains with an ARC designation are part of the microbiological culture collection at Entasis Therapeutics and were used to generate data for Table 1, Supplementary Table, Fig. 3 and Supplementary Datafile 1. The mcr-1 + E. coli strains were a gift from P. ordmann and L. Poirel at the University of Fribourg, Switzerland. The parent strain for the isogenic P. aeruginosa panel of β-lactamases is ARC33 (PA1 ampc poxb ). This isogenic panel consists of ARC33 transformed with pbbr1mcs-derived expression vectors 3, with each strain constitutively expressing an individual β-lactamase as indicated. The parent strain for the A. baumannii panel of β-lactamases is ATCC This isogenic panel consists of ATCC1797 transformed with pwh1-derived expression vectors, with each strain constitutively expressing an individual β-lactamase as indicated. pwh1 (ref. ) was a gift from P. Dunman at the University of Rochester. Susceptibility testing on larger collections of isolates (Table, Supplementary Table 5; and 1,131 A. baumannii strains from 01) was conducted at IHMA (Schaumburg, IL). For these larger sets, the indicated MIC 50 and MIC 90 values correspond to the concentrations at which antibacterial activity is observed for 50 and 90% of the strains under consideration, respectively. Hydrolytic stability. Solutions of BLI compounds were monitored to characterize their hydrolytic stability in ph 7. phosphate-buffered saline with EDTA at 70 C for > h. Stock solutions (10 mm) of each compound in DMS were prepared, and ATURE MICRBILGY 5 µl of each stock was diluted with 995 µl buffer to make 50 µm sample solutions. Each solution was followed by ultra performance liquid chromatography/tandem mass spectroscopy (Waters) for an extended time period of > h at 70 C, and firstorder kinetics was used in deriving rate constants (K). Microscopy. Laboratory strains E. coli (ATCC59), A. baumannii (ATCC1797) and P. aeruginosa (PA1) were grown in 3 ml Mueller Hinton Broth II (MHBII), overnight at 35 C with shaking, subcultured 1/100 in MHBII and incubated at 35 C with shaking to exponential phase (D 00 of 0.3). These cultures were diluted 15-fold in MHBII with either no drug, 1/ MIC or.5 MIC of the compound of interest and incubated at 35 C with shaking for 3 h. A 5 µl volume was spotted onto a Mueller Hinton (MH) agar pad prepared on a microscope slide with a coverslip. Bacteria were visualized at 00 under oil with an lympus BX53 microscope, using cellsens Standard 1.1 software for image capture. The morphology of PA1 at 1/ MIC of ETX51 was similar to the no drug control. Whole-genome sequencing. Whole-genome sequencing was performed as described previously 1. Murine models of A. baumannii infection. All animal procedures were carried out according to AstraZeneca s Institutional Animal Care and Use Committee (IACUC) approved protocol. The BSL animal facilities at AstraZeneca were fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC) with policies in place as prescribed by the Guide for Care and Use of Laboratory Animals (Department of Health and Human Services). In vivo neutropenic infection models were conducted in female Crl:CD-1 mice as previously described 5,. Eight- to ten-week-old female Crl:CD-1 mice were rendered neutropenic with intraperitoneal injections of cyclophosphamide at 150 mg kg 1 on day and 100 mg kg 1 on day 1. A. baumannii cultures were incubated overnight in tryptic soy broth (TSB) at 37 C with shaking. The resulting culture was diluted 1:10 in TSB and diluted in saline (thigh model) or 1% agar (lung model) to obtain a target inoculum of 1 10 colony-forming units (c.f.u.) per thigh via intramuscular injection or c.f.u. per lung via intratracheal administration. The number of animals selected per treatment arm was determined using the Resource Equation 7. Mice were randomized following inoculation and then assigned to control or treatment groups. At h post infection, one group of five mice was euthanized to determine the viable bacterial counts in the infected tissues at start of treatment. The remaining groups of mice (five per treatment group) were administered either test compound, control compound or vehicle. Efficacy was determined h after start of treatment. Mice were euthanized by C asphyxiation and cervical dislocation and the infected tissues were aseptically removed, weighed and transferred to tubes containing 1 ml of saline for homogenization. Homogenates (100 µl) were serially diluted in saline and plated onto blood agar. Treatment groups within both thigh and lung infection models consisted of a :1 fixed ratio of sulbactam ETX51 spanning doses of.5/0.5 to 0/0 mg kg 1 administered subcutaneously every three hours. An additional thigh study using a fixed dose of sulbactam at 15 mg kg 1 combined with a dose titration of ETX51 from 0 to 50 mg kg 1 was also completed to further elucidate the contribution of activity attributed to ETX51. Solution doses were prepared in saline and administered at a dose volume of 10 ml kg 1. o blinding was included in this study. Pharmacokinetic studies. Separate groups of mice used for pharmacokinetic determinations were infected with A. baumannii. Animals were assigned to dosing groups of 3 mg kg 1 ETX51 alone and 0/5, 0/10 and 0/0 mg kg 1 of sulbactam/etx51 (nine animals per dose level). Dosing was initiated two hours after infection and whole blood samples were obtained via submandibular bleeds at 0.0,,, 1,, 3,, and h post-dose using three animals per time point and three time points obtained per animal. Whole blood was sampled into microcontainer tubes containing K EDTA (Beckton Dickinson). Plasma was separated by centrifugation for five minutes at,000 RCF and stored at 0 C until analysis. Bioanalytical preparation of the plasma and LC MS/MS instrument parameters are provided in Supplementary Method. Pharmacokinetic analysis. on-compartmental analysis was applied to the mean plasma ETX51 concentration versus time profiles for all dose levels using Phoenix WinonLin v.., and parameter estimates were determined for C max, T max, AUC 0-tlast, AUC 0 and T 1/ (half-life). Statistical analysis. For in vivo studies, viable counts determined for control and treatment groups were assessed statistically using a one-way analysis of variance (AVA), with P < 0.05 used as criteria for statistical significance across all groups. Data availability. The data that support the findings of this study are available from the corresponding authors upon request. The PDB code for the co-crystal structure of compound 3 with XA- (resolution 1.93 Å) is 5VFD. Received 9 ovember 01; accepted 5 May 017; published 30 June 017 ATURE MICRBILGY, 1710 (017) DI: /nmicrobiol Macmillan Publishers Limited, part of Springer ature. All rights reserved.