# A Mechanism Based Approach to Metallo beta-lactamase Inhibition

> **NIH NIH R01** · CASE WESTERN RESERVE UNIVERSITY · 2022 · $465,048

## Abstract

Gram-negative pathogens producing metallo-β-lactamases, MBLs, seriously threaten the public health. MBLs
are the most worrisome carbapenemases, inactivating the “last resort” carbapenems and most β-lactams, and
resist all commercially available β-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- β-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), Δ4-
Thiazolidines (Δ4-TZs), and Δ4-Oxazolidines (Δ4-OXZs)] or carbapenem mimics [Δ4-Bisthiazolidines (Δ4-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 β-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 β-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 ...

## Key facts

- **NIH application ID:** 10401424
- **Project number:** 5R01AI100560-10
- **Recipient organization:** CASE WESTERN RESERVE UNIVERSITY
- **Principal Investigator:** ROBERT A. BONOMO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $465,048
- **Award type:** 5
- **Project period:** 2012-02-07 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10401424

## Citation

> US National Institutes of Health, RePORTER application 10401424, A Mechanism Based Approach to Metallo beta-lactamase Inhibition (5R01AI100560-10). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10401424. Licensed CC0.

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