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Background Drug-resistant tuberculosis (DR TB) is a deadly, difficult-to-treat infection, and new treatment strategies are needed. Despite the wide success of β-lactams (BLs), DR TB guidelines only include meropenem (MEM) and imipenem (IPM), given with clavulanate (CLA). BlaC, the Mtb β-lactamase, hydrolyzes CRO less efficiently than other cephems and β-lactamase inhibitors improve the in vitro susceptibility of Mtb to CRO. Surprisingly, CRO has not been evaluated in DR TB clinical studies. Moreover, the mechanisms by which CRO disrupts Mtb PG synthesis are not well characterized. CRO inhibits LdtMt1­, but activity against LdtMt2, an important Mtb PG synthase, is unknown. To explore this knowledge gap, we examined CRO inhibition of LdtMt2. In addition, we investigated if combining CRO with MEM or IPM would lower minimum inhibitory concentrations (MICs) more than each agent alone. Methods A panel of Mtb isolates was selected for susceptibility testing with a broth microdilution method. Timed electrospray ionization-mass spectrometry (ESI-MS) and inhibition kinetic assays were performed. Results CRO MICs ranged 0.25 to 16 µg/mL and lowered ≤ 0.06 to 2 µg/mL with CLA (Table 1). Fractional inhibitory concentration indices for CRO + MEM or IPM was < 0.5 for six isolates, suggesting synergy. ESI-MS captured CRO-LdtMt2­ acyl-complexes at timepoints 5 to 120 min, and a 158 Da fragment loss was observed; MEM and IPM were unchanged (Table 2, Figure 1). When LdtMt2 was co-incubated with MEM and CRO together, only MEM complexes were captured. Interestingly, Kiapp­ with CRO (0.07 ± 0.01 µM) was comparable to that with MEM (0.09 ± 0.01 µM). Figure 1Mass spectrometry chromatograms with LdtMt2 (A) alone incubated with ceftriaxone at (B) 5 min, (C) 15 min, (D) 30 min, (E) 120 min. [Ldt¬Mt2] = 13.2 µM and [ceftriaxone] = 264 µM. Changes in MW are also listed in Table 1. (F) shows the structure of ceftriaxone with the proposed leaving group in red, leaving the remaining bound structure accounting for the observed change in MW. Conclusion CRO was effective in lowering MICs with MEM, IPM, and CLA for our Mtb isolates. Based upon ESI-MS, we found that CRO forms a stable complex with LdtMt2 and the R2 side chain is eliminated, while kinetic observations support inhibition of LdtMt2 by CRO (Figure 2). Previous work found MEM and IPM also inhibit multiple other PG synthases (e.g., PonA1, LdtMt1, LdtMt3). We hypothesize that CRO + MEM/IPM inhibits the growth of Mtb by the combined inactivation of multiple cell wall enzymes. Our observations support the further exploration of the notion of “target redundancy” as an approach to treat multidrug-resistant mycobacteria with BLs. Figure 2.Ceftriaxone-LdtMt2 adduct formation leading to release of the R2 side group Disclosures Krisztina Papp-Wallace, Ph.D, Merck: Grant/Research Support|Venatorx: Grant/Research Support|Wockhardt: Advisor/Consultant Robert A. Bonomo, MD, NIH VA: Grant/Research Support|VenatoRx Merck Wockhardt Cystic Fibrosis Foundation: Grant/Research Support.

Background Antimicrobial resistance is a major global health threat. Pseudomonas aeruginosa is a leading cause of nosocomial infections and a key opportunistic pathogen in cystic fibrosis. Multidrug resistant strains are classified as a “serious threat” by the CDC. Pseudomonas-derived cephalosporinase (PDC) is largely responsible for β-lactam antibiotic resistance in P. aeruginosa. Single amino acid substitutions in the essential Ω-loop region (e.g. Y221H by structural alignment-based numbering of class C β-lactamases) have been shown to enhance hydrolysis of ceftazidime (CAZ) and ceftolozane (TOL), limiting therapeutic options for P. aeruginosa. Methods We undertook detailed studies to explore the mechanisms by which Y221H enhances CAZ and TOL MICs. MIC measurements were performed per CLSI guidelines using MH Agar. Thermal stability was determined by circular dichroism. Enzyme kinetic properties were determined using spectrophotometric techniques. Molecular dynamics techniques were used to predict structural changes. Results E. coli expressing blaPDC-3-Y221H is less susceptible to CAZ (MIC 0.5 mg/L WT → 8 mg/L Y221H) and TOL (MIC 2 mg/L WT → 16 mg/L Y221H). Using steady-state kinetic analysis, Y221H was found to hydrolyze CAZ with a KM = 585 µM, a kcat = 3.4 sec-1, and kcat/KM = 0.0058 µM-1s-1. With cephalothin, a good PDC substrate, we observed KM = 26.6 µM, kcat = 70.1 s-1, and kcat/KM = 2.6 µM-1 s-1 for Y221H. Using Electrospray ionization mass spectrometry (ESI-MS), CAZ was detected covalently bound to WT, but not Y221H when incubated at 1000-fold molar excess. Avibactam (AVI) inhibited Y223H (Ki = 70 nM vs. 19 nM for WT). Y221H thermal stability decreased by 5°C (Tm = 47°C vs 52°C WT). AVI at 10-fold molar excess does not increase Tm in Y221H or WT. WT-MetaDynamics (WT MDS) predicts the opening of a hidden pocket by repositioning residue 221 (Figure 1).). Figure 1: (Left) We carried out enhanced sampling metadynamics simulations to generate free-energy landscapes as a function of the dihedral angles of residue 221. This identifies the differences in the dynamics of the tyrosyl side chains in the wild type Y221 and the imidazole ring of the H221 variant. (Right) The rotation of the side chain in H221 opens a cryptic pocket (green mesh), which is occluded in the wild type. The Ω-loop is colored red. Conclusion PDC-3 Y221H increases CAZ & TOL MICs and alters catalytic activity, primarily by a change in kcat. Our modelling analyses suggest altered conformational flexibility and structure-function relationships in the Ω-loop. These results help to advance our understanding of PDC and will inform development of novel antibiotics and inhibitors. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Background Multidrug-resistant (MDR) A. baumannii presents a critical need for innovative antibacterial development. We have identified a new series of γ-lactam (oxopyrazole) antibiotics that target penicillin binding proteins (PBPs) and incorporate a siderophore moiety to facilitate periplasmic uptake. YU253911, an advanced iteration of this class shows potent in vitro activity against clinically relevant Gram-negative organisms including Acinetobacter spp. Methods Minimum inhibitory concentrations (MICs) for YU253911 were determined using broth microdilution against a 198-member panel of clinical isolates of Acinetobacter spp. Resistant strains were further evaluated for susceptibility to YU253911 in combination with sulbactam. The antibiotic’s target protein was evaluated by binding studies with Bocillin™, a fluorescent penicillin analogue, and modeled in the PBP active site. YU253911 was evaluated in vivo in a mouse soft tissue infection model. Results MIC testing for YU253911 revealed an MIC50 of 0.5 μg/mL and an MIC90 of 16 μg/mL, which compared favorably to all tested β-lactam antibiotics including penicillins, cephalosporins, monobactams and carbapenems (MIC50 = 2 to > 16 μg/mL). Combination with sulbactam augmented the activity of the agent. There was no apparent correlation between YU253911-resistance and the presence of specific β-lactamase genes, and incubation with representative β-lactamase proteins (KPC-2, OXA-23, OXA-24, PER-2, PDC-3, NDM-1, VIM-2, and IMP-1) showed negligible hydrolysis of the agent. YU253911 showed promising preclinical pharmacokinetics in mice with a 15 h half-life from intravenous administration and demonstrated a dose-dependent reduction in colony forming units from 50 and 100 mg/kg q6h dosing in a mouse thigh infection model using P. aeruginosa. Conclusion YU253911, a new generation γ-lactam antibiotic effective against MDR A. baumannii demonstrated promising in in vitro potency and favorable pharmacokinetics which correlated with in vivo efficacy. Disclosures Krisztina M. Papp-Wallce, PhD, Entasis (Grant/Research Support)Merck (Grant/Research Support)Venatorx (Grant/Research Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Background Nacubactam, formerly RG6080 and OP0595 (Figure 1A), is a bridged diazabicyclooctane (DBO) that inactivates class A and class C β-lactamases. Unlike avibactam, the DBO that is approved for use in combination with ceftazidime, nacubactam also inhibits penicillin binding proteins (i.e., PBP2) in Enterobacteriaceae. We set out to determine the effectiveness of meropenem-nacubactam against Klebsiella pneumoniae clinical strains and to elucidate the structure–function relationships. Methods Minimal inhibitory concentration (MIC) measurements using broth microdilution according to Clinical and Laboratory Standards Institute for meropenem (MERO) ± nacubactam (fixed concentration of 4 mg/L or fixed 1:1 ratio) was performed on 50 clinical K. pneumoniae strains (6 having OXA-48-like β-lactamases and 44 harboring KPC-2 or KPC-3) and 47 isogenic Escherichia coli strains harboring bla genes encoding K. pneumoniae carbapenemase (KPC) variants with single amino acid substitutions in residues that are involved in catalysis. IC50s for selected KPC-2 variants were determined on periplasmic extracts with varying concentrations of nacubactam using nitrocefin as a reporter substrate. Results The MERO combinations with either 4 mg/L or a 1:1 ratio of nacubactam effectively lowered the MERO MICs of K. pneumoniae strains (Figure 1B). Similarly, all E. coli strains expressing blaKPC-2 variants were susceptible to the MERO-nacubactam combinations based on the breakpoint of MERO. The strains harboring K73R, S130G, and K234R had slightly elevated MERO-nacubactam MICs relative to wild type but did not have corresponding increases in MERO MICs. Strains with pBC SK-KPC2, K73R or S130G had 0.015 mg/L MERO MICs. The pBR322-K234R strain had a twofold lower MERO MIC than pBR322-KPC-2 (Figure 1C). The IC50 of cell extracts containing the K234R variant is 781 µM, which is 12-fold higher than that for KPC-2 (66 µM) (Figure 1C). Extracts containing the S130G variant were not inhibited by nacubactam (IC50 > 2.6 mM). Conclusion Meropenem-nacubactam is an effective β-lactam β-lactamase inhibitor combination for Enterobacteriaceae with KPC or OXA-48 β-lactamases. The single amino acid substitutions K73R, S130G, and K234R in KPC-2 affect the inactivation mechanism. Disclosures M. R. Jacobs, F. Hoffmann-La Roche Ltd.: Grant Investigator, Research grant. K. M. Papp-Wallace, F. Hoffmann-La Roche Ltd.: Grant Investigator, Research grant. R. A. Bonomo, F. Hoffmann-La Roche Ltd.: Grant Investigator, Research grant.

Background AAI101 is a novel β-lactamase inhibitor (BLI), active against ESBLs and other β-lactamases. AAI101 combined with cefepime (FEP) is in Phase 2 clinical trials. The objective of this study was to determine differences between AAI101 and tazobactam in their inhibition of selected β-lactamases of clinical relevance. Methods Isogenic E. coli strains expressing single clinically relevant β-lactamases were tested for susceptibility (broth microdilution MIC) to FEP, FEP/AAI101 and piperacillin-tazobactam (P/T). Periplasmic β-lactamase extracts from selected strains then were used to determine IC50s for AAI101 and for tazobactam. β-Lactamases with low IC50s for AAI101 were purified, and steady-state inactivation kinetics determined for AAI101 and for tazobactam. Results AAI101 restored activity of FEP against E. coli strains producing defined β-lactamases, and FEP/AAI101 was more potent than P/T (Table). Table. MIC values [mg/L] β-Lactamase FEP FEP/ AAI101 P/T E. coli DH10B (host) ≤0.06 ≤0.06 4 SHV-1 2 0.25 >256 SHV-5 8 ≤0.06 256 SHV-7 (I8F, R43S, G238S, E240K) 8 ≤0.06 32 SHV-26 (A187T) 0.25 ≤0.06 >256 SHV-30 (I8F, R43S, G238S) 2 0.12 32 SHV-102 (G238A) 16 0.12 >256 SHV-106 (I8F, G238S) 4 ≤0.06 16 SHV-129 (G238S, E240K, R275L, N276D) 16 ≤0.06 128 SHV-161 (R43S) 0.5 ≤0.06 >256 TEM-10 (R164S, E240K) 4 ≤0.06 4 TEM-26 (E104K, R164S) 0.5 ≤0.06 2 TEM-30 (R244S) ≤0.06 ≤0.06 256 CTX-M-14 8 0.06 2 CTX-M-15 32 0.06 2 KPC-2 4 0.12 256 KPC-3 4 ≤0.06 256 OXA-1 2 0.12 256 OXA-48 0.12 0.12 256 CMY-2 0.25 ≤0.06 4 AAI101 had low IC50s (≤0.52 µM) towards periplasmic extracts of class A β-lactamases tested. Linear inhibition of SHV-1 (original spectrum β-lactamase, OSBL) by AAI101 was observed, whereas inhibition of SHV-1 by tazobactam plateaued at lower BLI concentrations. Similar inhibitory patterns were observed for KPC-2, accompanied by a marked increase in potency of AAI101 vs. tazobactam (Figure). Conclusion Addition of AAI101 enhances cefepime activity vs. a selected array of β-lactamases expressed in E. coli in an isogenic Background. The inhibitory kinetics of β-lactamases by AAI101 compared with those of tazobactam indicate different mechanisms of β-lactamase inhibition. Disclosures K. M. Papp-Wallace, Entasis: Grant Investigator, Research grant Allecra: Grant Investigator, Research grant Merck: Grant Investigator, Research grant; Roche: Grant Investigator, Research grant Allergan: Grant Investigator, Research grant M. R. Jacobs, Allecra: Grant Investigator, Research grant Roche: Grant Investigator, Research grant Shionogi: Grant Investigator, Research grant; R. A. Bonomo, Entasis: Grant Investigator, Research grant Allecra: Grant Investigator, Research grant Wockhardt: Grant Investigator, Research grant Merck: Grant Investigator, Research grant Roche: Grant Investigator, Research grant GSK: Grant Investigator, Research grant Allergan: Grant Investigator, Research grant Shionogi: Grant Investigator, Research grant