# Characterization of MsbA inhibitors as potential antibiotic leads to treat carbapenem-resistant Enterobacteriaceae (CRE)

> **NIH NIH R21** · PROKARYOTICS, INC. · 2021 · $140,000

## Abstract

In 2013, the CDC designated carbapenem-resistant Enterobacteriaceae (CRE) an Urgent Threat, and in 2017,
the WHO designated it a Priority 1 “critical superbug”. As few therapies remain to treat CRE, the risk of “pan-
resistant” CRE untreatable by any currently available antibiotic also exists. Entirely new agents with novel
mechanisms of action (MOA) not cross-resistant to SOC agents languish. Development of such a ‘first-in-class’
agent also offers the potential for much-needed orally-administered CRE therapeutic, thus providing a new
step-down therapy to reduce hospital stay and growing healthcare costs. Our proposal aims to develop an
entirely novel therapeutic to treat life-threatening bacterial infections due to multidrug resistant (MDR)
Enterobacteriaceae, including CRE. Using an innovative overexpression-based co-culture screen in
Escherichia coli (Ec), we identified four structurally distinct series of small molecule inhibitors targeting MsbA,
an essential and broadly conserved Gram-negative (GN) ABC transporter responsible for lipopolysaccharide
(LPS) biogenesis and construction of the Gram-negative outer membrane (OM). Building upon a solid
foundation of preliminary data, our Aims are:
Aim (A) 1. Expanded MOA studies. Aim 1 studies will expand upon our preliminary MOA and microbiological
characterization of the MsbA inhibitor (MsbAi) hits to include other GN bacteria, particularly Klebsiella
pneumoniae (Kp). Specifically, we will demonstrate Kp MsbA target inhibition in (1) an in vitro biochemical assay
and (2) a whole-cell context by determining MICs of our MsbAis against a Kp ΔtolC strain and subsequently
selecting for MsbAisR mutants in this Kp background.
Aim 2. Demonstrate chemical tractability of MsbAis. The goal of Aim 2 is to perform Hit to Lead (H2L)
medicinal chemistry structure activity relationship (SAR) studies for each MsbAi and demonstrate >1 series is
chemically tractable and that quantifiable pre-lead optimization criteria can be established, including i) Ec and
Kp MsbA in vitro potency (IC50 < 0.25 µg/ml), ii) Ec and Kp WT activity (<16 µg/ml), and iii) > 90% HepG2 cell
viability at 50X MIC against EcΔtolC. MsbAis achieving these milestones serve as a justifiable starting point for
a future Lead Optimization leveraging the resulting SAR gained in Aim 2 and SBDD information from Aim 3.
Aim 3. Obtain Ec and Kp MsbAi-MsbA co-crystal structures in collaboration with SSGCID. The goal of
Aim 3 activities is to initiate a collaboration between Prokaryotics, Genentech, and the Seattle Structural
Genomics Center for Infectious Disease (SSGCID) led by Dr. Peter Myler to optimize conditions suitable for
obtaining high resolution x-ray crystal structures of Kp MsbA in the apo form as well as Ec and/or Kp MsbA
proteins in complex with MsbAis.

## Key facts

- **NIH application ID:** 10242174
- **Project number:** 5R21AI146541-02
- **Recipient organization:** PROKARYOTICS, INC.
- **Principal Investigator:** Terry Roemer
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $140,000
- **Award type:** 5
- **Project period:** 2020-08-19 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10242174

## Citation

> US National Institutes of Health, RePORTER application 10242174, Characterization of MsbA inhibitors as potential antibiotic leads to treat carbapenem-resistant Enterobacteriaceae (CRE) (5R21AI146541-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10242174. Licensed CC0.

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