# Core 2 Mechanistic Assay Core

> **NIH NIH P01** · UNIVERSITY OF FLORIDA · 2024 · $555,021

## Abstract

Project Summary/Abstract: Mechanistic Assay Core #2
Carbapenem-resistant Acinetobacter baumannii (CRAB) and Klebsiella pneumoniae (CRKP) were categorized
in the highest threat category (‘Urgent’) according to the U.S. CDC in 2019. While β-lactam antibiotics (BL) have
been used for decades to successfully treat infections by susceptible isolates of AB and KP, resistant isolates
often produce several different types of β-lactamases that can inactivate virtually every BL in monotherapy.
Moreover, efflux pumps and loss of outer membrane porin channels present additional clinically relevant
resistance mechanisms. In 2017, NIAID set forth the Gram-negative toolbox development program (RFA-AI-16-
081) to create novel approaches that generate mechanistic insights to combat these bacterial ‘superbugs’. Our
established team collectively developed a novel mechanistic assay platform as our Gram-negative toolbox
(under R01 AI136803). These assays underpin the present P01. The Mechanistic Assay Core (#2) will provide
the mechanistic foundation to rationally optimize clinically relevant combination dosage regimens of available
and new antibiotics. This core will mutually integrate all Projects and all Cores, and will provide the latest assays
for outer membrane permeability of BL and β-lactamase in inhibitors (BLI). Moreover, it will provide the latest
assays for studying the intracellular drug accumulation and washout kinetics of non-PBP-binding partner
antibiotics and for quantifying intracellular energy metabolite concentrations (e.g. ATP). The latter will be
particularly relevant for mechanistic studies to characterize non-replicating persisters (NRP), along with
automated confocal microscopy and flow cytometry (with sorting) of bacterial strains labelled with fluorescent
protein(s) to determine the rate of protein synthesis. Flow cytometry (with and without sorting) and confocal
microscopy will be used to efficiently identify PBP occupancy patterns that yield extensive morphology changes
for combinations of BL, BLI, and non-β-lactam-PBP-binders in Project #1. This approach has proven highly
beneficial in R01 AI136803. Moreover, the latest proteomic assays will provide important insights into the
expression of proteins involved in antibiotic action (e.g. PBP) and resistance (e.g. β-lactamases, efflux pumps
and outer membrane porin channels). These resistance mechanism studies will be further enhanced by whole-
genome sequencing and other molecular techniques, which are particularly relevant for evaluating the ability of
optimized dosage regimens to suppress resistance in Projects #2 and #3. For the latter projects, this Core will
provide extracellular drug concentration determinations for the in vitro hollow fiber and mouse studies. Taken
together, the Mechanistic Assay Core will provide novel mechanistic insights to support rational optimization of
combination dosage regimens in Project #1, and to prospectively validate the synergistic bacterial killing an...

## Key facts

- **NIH application ID:** 10763468
- **Project number:** 1P01AI179409-01
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Yinzhi Lang
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $555,021
- **Award type:** 1
- **Project period:** 2024-08-08 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10763468

## Citation

> US National Institutes of Health, RePORTER application 10763468, Core 2 Mechanistic Assay Core (1P01AI179409-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10763468. Licensed CC0.

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