# Challenges in beta-Lactamase Mediated Resistance

> **NIH NIH R01** · CASE WESTERN RESERVE UNIVERSITY · 2021 · $369,608

## Abstract

β-Lactamases continue to pose a serious threat to patients with bacterial infections. Among the most
understudied are the AmpC cephalosporinases. These class C β-lactamases confer high-level resistance to
penicillins, cephalosporins, and monobactams. Additionally, increased AmpC expression in conjunction with loss
of porins (e.g. increased expression of the Pseudomonas Derived Cephalosporinase (PDC) and loss of OprD in
P. aeruginosa) results in resistance to imipenem, our “last line” agent in the treatment of these serious infections.
 The recent introduction of the novel β-lactam-β-lactamase inhibitor combination, ceftolozane-tazobactam
(TOL/TAZO) offered hope as TOL, a 3'-aminopyrazolium cephalosporin, eludes hydrolysis by the AmpCs and
does not require the presence of OprD for cell entry. Therefore, TOL/TAZO would be an “answer” to
cephalosporin and imipenem-resistant P. aeruginosa infections. Unfortunately, descriptions of resistance to
TOL/TAZO rapidly emerged. As early as 2014, AmpC variants found in P. aeruginosa (e.g. the E247K
substitution in PDC) demonstrated an extended-spectrum AmpC (ESAC) phenotype and were TOL/TAZO
resistant. These findings alerted us to the increasing number of ESACs that are emerging and directed our focus
to overcome this challenge. Our most recent research efforts led us to discover that certain amino acid
substitutions in PDC were responsible for enhanced catalytic efficiency towards TOL and other expanded-
spectrum cephalosporins. Studies in our laboratory with ceftazidime/avibactam raise the concern that even the
newer cephalosporins (e.g., cefiderocol) will likely meet a similar fate unless a better understanding of structure
function relationships in AmpCs is achieved. In this proposal, we will investigate why the Ω-loop and R2 region
of PDC and other AmpCs are “hot-spots” for these substitutions. Furthermore, we propose to study PDC variants
with enhanced hydrolysis of TOL (and other cephalosporins) as a model system representative of other ESACs.
Lastly, we will endeavor to explore an entirely novel approach to AmpC inhibition that relies upon conformational
changes. In Aim 1 we will determine the mechanistic basis and structural evolution of PDC variants located in the
Ω-loop (e.g., at residues V239, G242, E247, and Y249) that confer an ESAC phenotype and resistance to
TOL/TAZO. We believe this phenotype arises due to increased conformational flexibility of the Ω-loop that
promotes TOL hydrolysis. In Aim 2 we will probe structure-function relationships of the PDC variants using
cephalosporin and TOL based boronic acid transition state inhibitors (BATSIs). We believe the PDC variants
have altered acylation and deacylation transition states and these strategically designed compounds will reveal
the mechanistic details of catalysis. In Aim 3 we will identify allosteric sites that are critical for the structure and
function of PDC and variants. We posit that PDC variants possess allosteric sites that modulat...

## Key facts

- **NIH application ID:** 10079455
- **Project number:** 5R01AI063517-13
- **Recipient organization:** CASE WESTERN RESERVE UNIVERSITY
- **Principal Investigator:** ROBERT A. BONOMO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $369,608
- **Award type:** 5
- **Project period:** 2005-02-15 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10079455

## Citation

> US National Institutes of Health, RePORTER application 10079455, Challenges in beta-Lactamase Mediated Resistance (5R01AI063517-13). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10079455. Licensed CC0.

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