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TrmD-targeting actinobacterial natural products as next generation antibiotics

Award Number: R01AI164490
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 06/25/2021
PERIOD OF PERFORMANCE END DATE: 05/31/2026

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TrmD-targeting actinobacterial natural products as next generation antibiotics - Project Summary. Discovering new antibiotics for Gram-negative bacteria is uniquely challenging, due to their double-membrane structure that acts as a permeability barrier to drugs and as an anchor for efflux pumps. Efforts that target one membrane protein or one efflux pump at a time are ineffective, due to rapid rise of resistance mutations. We will target the TrmD-catalyzed m1G37 methylation of tRNA to inhibit biosynthesis of multiple classes of membrane proteins, with the potential to accelerate bactericidal action. TrmD is a bacteria-specific S- adenosyl-methionine (AdoMet)-dependent methyl transferase that controls accuracy of the protein-synthesis reading frame. Loss of TrmD increases +1 frameshifting and causes cell death. We have shown that genes for multiple membrane proteins and efflux pumps in E. coli and in other Gram-negative bacteria contain TrmD- dependent codons near the start of the reading frame. We hypothesize that targeting TrmD will reduce protein synthesis of all of these genes, thus offering a novel solution to an unmet need. While AstraZeneca (AZ), GSK, and academic labs have attempted to target TrmD by screening small molecular compound libraries, isolated hits lack the cell-permeability needed to exhibit an antibacterial effect. Here, we propose to screen a large collection of microbial extracts and fractions for cell-permeable and TrmD-targeting natural products (NPs) that are potent and selective over the human counterpart Trm5. We will use a cell-based assay, consisting of a 1:1 mix of an E. coli (Ec) TrmDmCh strain (dependent on trmD for survival and expressing mCh (mCherry) as a fluorescence marker) and an Ec Trm5YFP strain (dependent on trm5 for survival and expressing YFP), in a high- throughput screening (HTS) campaign to isolate NPs that selectively inhibit the TrmDmCh strain. We perform this assay in Ec tolC+ cells, which maintain the entire Gram-negative efflux machinery including the major efflux pump encoded by tolC, to screen for NPs that are cell-permeable and resistant to efflux. A pilot screen with this tolC+ cell-based assay has identified an attractive hit, demonstrating the HTS-readiness of the assay. In Aim 1, we will use this tolC+ cell-based assay to screen 74,770 actinobacterial extracts and fractions available at The Scripps Research Institute (TSRI). We will assess hits in secondary assays, remove false positives, evaluate their activity at the whole-cell level, and test them for permeability and efflux in a panel of Gram-negative bacteria. In Aim 2, we will de-replicate the top 20 hits to isolate the active NPs, determine their structures, and use a combination of genome sequencing and mining to identify their biosynthetic gene clusters (BGCs) for developing biotechnology platforms to scale up their production. In Aim 3, we will test active NPs for conferring TrmD- deficient phenotypes in whole-cell assays, determine their potency, selectivity, mechanism of action, and assess their risk of resistance. These NPs represent novel leads in a new paradigm of antibiotic discovery that addresses the multi-drug resistance problem of Gram-negative bacteria.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $1,179,849 )
2025	2025	THOMAS JEFFERSON UNIVERSITY	1020 WALNUT ST STE 1	PHILADELPHIA	PA	19107	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	5	7/23/2025	NON-COMPETING CONTINUATION	$1,179,849
														Subtotal = $1,179,849

Issue Date FY: 2024 ( Subtotal = $483,241 )
2024	2024	THOMAS JEFFERSON UNIVERSITY	1020 WALNUT ST STE 1	PHILADELPHIA	PA	19107	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	001	4	7/10/2024	NON-COMPETING CONTINUATION	$483,241
2024	2023	THOMAS JEFFERSON UNIVERSITY	1020 WALNUT ST	PHILADELPHIA	PA	19107	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	3	12/6/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $483,241

Issue Date FY: 2023 ( Subtotal = $847,145 )
2023	2023	THOMAS JEFFERSON UNIVERSITY	1020 WALNUT ST STE 1	PHILADELPHIA	PA	19107	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	3	6/2/2023	NON-COMPETING CONTINUATION	$847,145
														Subtotal = $847,145

Issue Date FY: 2022 ( Subtotal = $831,545 )
2022	2022	THOMAS JEFFERSON UNIVERSITY	1020 WALNUT ST STE 1	PHILADELPHIA	PA	19107	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	001	2	5/20/2022	NON-COMPETING CONTINUATION	$831,545
2022	2021	THOMAS JEFFERSON UNIVERSITY	1020 WALNUT ST STE 1	PHILADELPHIA	PA	19107	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	1	3/24/2022	NEW	$0
														Subtotal = $831,545

Issue Date FY: 2021 ( Subtotal = $845,545 )
2021	2021	THOMAS JEFFERSON UNIVERSITY	1020 WALNUT ST STE 1	PHILADELPHIA	PA	19107	PHILADELPHIA	USA	Allergy and Infectious Diseases Research	000	1	6/25/2021	NEW	$845,545
														Subtotal = $845,545

Grand Total All Awards = $4,187,325

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TrmD-targeting actinobacterial natural products as next generation antibiotics

Award Number: R01AI164490

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 06/25/2021

PERIOD OF PERFORMANCE END DATE: 05/31/2026

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