Gram-negative pathogens producing metallo-?-lactamases, MBLs, seriously threaten the public health. MBLs are the most worrisome carbapenemases, inactivating the ?last resort? carbapenems and most ?-lactams, and resist all commercially available ?-lactamase inhibitors (BLIs). The main challenges in MBL inhibitor design are understanding the reaction mechanism as it relates to the structural diversity of the 3 distinct subclasses (B1, B2, and B3). In the previous 5-year funding cycle, we achieved important milestones: i) identified the active form of clinically relevant MBLs in the bacterial periplasm; ii) characterized NDM-1 as a membrane-bound protein, establishing how this localization endows NDM-1 with unique stability; iii) demonstrated that MBLs of all 3 subclasses utilize a common mechanism for carbapenem hydrolysis suggesting novel approaches for inhibitor development; iv) designed a series of novel compounds, bisthiazolidines (BTZs), as substrate mimics, comprising a non- ?-lactam ?penicillin core? decorated with metal binding groups; and v) showed that BTZs are non-toxic, effective cross-class MBL inhibitors and identified the structural bases of their inhibitory action. Responding to the clear urgency to find novel therapies, our team will build on these accomplishments to identify, synthesize, evaluate and develop new cross-class MBL inhibitors. Our unique approach is based upon a mechanistic understanding of MBL catalysis which will be utilized to inspire potent inhibitors. To this end, we will synthesize new compounds as mimics of mechanistic intermediates or product mimics [Thiazolidines (TZs), ?4- Thiazolidines (?4-TZs), and ?4-Oxazolidines (?4-OXZs)] or carbapenem mimics [?4-Bisthiazolidines (?4-BTZs) and Bicyclooctanes (BCOs)]. Our second specific aim will evaluate inhibitors for in vitro activity against MBLs of all subclasses. We will next assay the impact of inhibitors in potentiating ?-lactam efficacy against MBL- producing model strains, assess differences between in vitro assays and effect on bacteria, and validate the selected inhibitors against a panel of clinical strains with different MBL alleles.
Our third aim will combine NMR and X-ray crystallography to study the structure of MBL-inhibitor adducts aimed to provide details for inhibitor improvement. We will also pursue mechanistic studies using micro-focusing spectroscopy and crystallography coupled to XFEL (X-ray free electron lasers) to trap transient ?-lactam-bound species in the enzymes NDM-1, L1, and VIM. This ?high-risk, high impact? innovative approach using new technologies will provide information for inhibitor improvement. Lastly, we will assay off-target activity and in vitro toxicity of the synthesized compounds, perform time-kill assays for meropenem-BLI combinations in clinical strains; and use mouse blood stream and lung infection models to assess the in vivo potency of meropenem/MBL-inhibitor combinations. This knowledge will serve to inform the design of therapeutic leads to combat MBL producing bacteria.

Public Health Relevance

Metallo-?-lactamases, MBLs, represent the largest family of carbapenemases that inactivate our most potent antibiotics (carbapenems) and are resistant to commercially available ?-lactamase inhibitors. To overcome this challenge our continuing renewal seeks to design and study the inhibitory abilities of a series of novel compounds [thiazolidines (TZs), oxazolidines (OXZs) and bicyclooctanes (BCOs)] that build upon our success with bisthiazolidines (BTZs). This proposal also aims to explore the fundamental biochemistry underlying the MBL resistance profile, applying new crystallographic tools to characterize mechanistic intermediates as leads for drug design, and test these compounds in animal models.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Xu, Zuoyu
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Case Western Reserve University
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Abboud, Martine I; Kosmopoulou, Magda; Krismanich, Anthony P et al. (2018) Cyclobutanone Mimics of Intermediates in Metallo-?-Lactamase Catalysis. Chemistry 24:5734-5737
van den Akker, Focco; Bonomo, Robert A (2018) Exploring Additional Dimensions of Complexity in Inhibitor Design for Serine ?-Lactamases: Mechanistic and Intra- and Inter-molecular Chemistry Approaches. Front Microbiol 9:622
Caselli, Emilia; Romagnoli, Chiara; Powers, Rachel A et al. (2018) Inhibition of Acinetobacter-Derived Cephalosporinase: Exploring the Carboxylate Recognition Site Using Novel ?-Lactamase Inhibitors. ACS Infect Dis 4:337-348
Blanco, Natalia; Harris, Anthony D; Rock, Clare et al. (2018) Risk Factors and Outcomes Associated with Multidrug-Resistant Acinetobacter baumannii upon Intensive Care Unit Admission. Antimicrob Agents Chemother 62:
Richter, Sandra S; Karichu, James; Otiso, Joshua et al. (2018) Evaluation of Sensititre Broth Microdilution Plate for determining the susceptibility of carbapenem-resistant Klebsiella pneumoniae to polymyxins. Diagn Microbiol Infect Dis 91:89-92
Bouza, Alexandra A; Swanson, Hollister C; Smolen, Kali A et al. (2018) Structure-Based Analysis of Boronic Acids as Inhibitors of Acinetobacter-Derived Cephalosporinase-7, a Unique Class C ?-Lactamase. ACS Infect Dis 4:325-336
Barnes, Melissa D; Bethel, Christopher R; Alsop, Jim et al. (2018) Inactivation of the Pseudomonas-Derived Cephalosporinase-3 (PDC-3) by Relebactam. Antimicrob Agents Chemother 62:
Rosa, Rossana; Rudin, Susan D; Rojas, Laura J et al. (2018) ""Double carbapenem"" and oral fosfomycin for the treatment of complicated urinary tract infections caused by blaNDM -harboring Enterobacteriaceae in kidney transplantation. Transpl Infect Dis 20:
El Chakhtoura, Nadim G; Saade, Elie; Iovleva, Alina et al. (2018) Therapies for multidrug resistant and extensively drug-resistant non-fermenting gram-negative bacteria causing nosocomial infections: a perilous journey toward 'molecularly targeted' therapy. Expert Rev Anti Infect Ther 16:89-110
Nukaga, Michiyoshi; Papp-Wallace, Krisztina M; Hoshino, Tyuji et al. (2018) Probing the Mechanism of Inactivation of the FOX-4 Cephamycinase by Avibactam. Antimicrob Agents Chemother 62:

Showing the most recent 10 out of 147 publications