# Salmonella-specific therapeutics

> **NIH NIH R01** · OHIO STATE UNIVERSITY · 2021 · $635,257

## Abstract

Project Summary
Non-typhoidal salmonellosis is one of the most significant food-borne diseases in the U.S. and globally. We
recently used high-throughput genetic screening to identify the Salmonella fra locus, whose mutation causes
extreme attenuation of fitness in mice. We then determined that the fra locus encodes five genes involved with
the uptake and utilization of fructose-asparagine (F-Asn): fraR, fraB, fraD, fraA, and fraE. The fra locus is found
only in the non-typhoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of
Clostridium. Thus, targeting the products of this locus in Salmonella with novel antimicrobials is expected to
leave the normal microbiota largely intact. Our characterization of the mechanism of attenuation revealed that
mutations in fraB cause an accumulation of the FraB substrate – 6-phosphofructose-asparate (6-P-F-Asp) –
that is toxic to cells. We propose high-throughput screening (HTS) with three different assays to identify small
molecule inhibitors of FraB, a deglycase that converts 6-P-F-Asp to aspartate and glucose-6-P (Glc-6-P). One
assay utilizes purified FraB enzyme in a spectrophotometric assay, while another is a growth-based assay
utilizing a live-attenuated Salmonella and a ∆fra control. We tested the biochemical and cell-based assays at
the ICCB-Longwood facility at Harvard, and found them to be simple and robust with Z' ≥0.9 and ≥0.8,
respectively. We propose to identify FraB inhibitors using these two assays to screen up to 500,000
compounds at the ICCB-Longwood facility. In the third assay, we will use in silico structure-based virtual
screening of ~250,000 compounds from the NCI database. The hits from both of the ICCB-Longwood screens
and the computational screens will be tested again at our home institution. A second independent confirmation
will utilize a mass spectrometry-based assay to directly measure build-up of 6-P-F-Asp, the substrate of FraB,
in live cells. Hits will be characterized further with regard to their IC50, IC90, Ki, and specificity. Computational
chemistry will be employed to better understand the chemical profile of FraB inhibitors, and facilitate
quantitative structure-activity relationship (QSAR) studies. Moreover, to gain a structural basis for the potency
of hits, we will use X-ray crystallography to determine the atomic-resolution structure of FraB with and without
select inhibitors. Successful completion of these aims is expected to facilitate hit identification and
characterization, key pre-requisites for lead optimization and advancement to a much needed narrow-spectrum
therapeutic for non-typhoidal salmonellosis. Narrow-spectrum antibiotics will have two key advantages: (i) limit
the side effects caused by disruption of the normal microbiota, and (ii) avoid selecting for antimicrobial
resistance among the normal microbiota. We envision a future cocktail of species-specific drugs that could be
used to treat cases of human diarrhea witho...

## Key facts

- **NIH application ID:** 10215469
- **Project number:** 5R01AI140541-04
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** Brian M Ahmer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $635,257
- **Award type:** 5
- **Project period:** 2018-08-15 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10215469

## Citation

> US National Institutes of Health, RePORTER application 10215469, Salmonella-specific therapeutics (5R01AI140541-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10215469. Licensed CC0.

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