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Chemical tools to investigate chain-flipping in quorum signal synthases

Award Number: R21AI175749
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: 01/23/2023
PERIOD OF PERFORMANCE END DATE: 12/31/2025

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Chemical tools to investigate chain-flipping in quorum signal synthases - Abstract Gram-negative bacteria use acyl-homoserine lactone mediated quorum sensing to regulate key physiological activities that includes virulence, biofilm formation and toxin production. AHL synthases make AHL autoinducer signals by enzymatically coupling acyl-ACP and S-adenosyl-L-methionine metabolites to facilitate quorum sensing for the bacterium. Therefore, AHL synthase inhibitors hold significant promise as antimicrobials in treating multidrug resistant bacterial infections. Designing AHL synthase specific inhibitors, however, does remain a significant challenge. To develop QS selective inhibitors, we investigate unique functional aspects of AHL synthases that distinguish them from other enzymes utilizing either acyl-ACP or SAM substrates. One such unique aspect of the synthase is it’s ability to selectively bind and react with a specific acyl-ACP substrate thereby enforcing fidelity in AHL signal synthesis. In general, acyl-ACP binding to a partner enzyme is a minimal two-step process involving an initial electrostatic docking of ACP acidic residues on to a basic patch in the synthase (protein-protein binding) followed by the translocation of the acyl- chain cargo from the ACP to the enzyme’s active site pocket through a process referred to as “chain-flipping”. Decoupling the binding from the chain-flipping step was not possible until now due to lack of methods that could independently report on the chain flipping step in ACP utilizing enzyme reactions. To address this limitation, we placed a site-specific fluorescent label on the EsaI AHL synthase, conducted enzyme-ACP titrations in the pre-steady state kinetics regime using a stopped flow fluorometer and determined kon and koff for both fluorescent and non-fluorescent cargo chain flipping from ACP to the EsaI synthase. Building upon our preliminary data, we propose two aims in this application to validate a fluorescence based method for determining chain-flipping rate constants in AHL synthase enzymes. Successful completion of the proposed studies should a) enhance our understanding on the mechanistic basis of substrate recognition in AHL synthases b) aid in development of a fluorescence-based HTS assay for screening AHL synthase inhibitors and c) open doors for single molecule enzymological investigations on chain-flipping step in AHL synthesis and d) facilitate deployment of this tool to investigate chain-flipping kinetics over a broader range of medicinally important, carrier protein dependent enzymes.


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 = $126,541 )
2025	2025	BOISE STATE UNIVERSITY	1910 UNIVERSITY DR	BOISE	ID	83725	ADA	USA	Allergy and Infectious Diseases Research	000	3	2/12/2025	NON-COMPETING CONTINUATION	$126,541
														Subtotal = $126,541

Issue Date FY: 2024 ( Subtotal = $14,069 )
2024	2024	BOISE STATE UNIVERSITY	1910 UNIVERSITY DR	BOISE	ID	83725	ADA	USA	Allergy and Infectious Diseases Research	002	2	7/26/2024	NON-COMPETING CONTINUATION	$0
2024	2024	BOISE STATE UNIVERSITY	1910 UNIVERSITY DR	BOISE	ID	83725	ADA	USA	Allergy and Infectious Diseases Research	000	2	2/14/2024	NON-COMPETING CONTINUATION	$12,663
2024	2024	BOISE STATE UNIVERSITY	1910 UNIVERSITY DR	BOISE	ID	83725	ADA	USA	Allergy and Infectious Diseases Research	001	2	4/17/2024	NON-COMPETING CONTINUATION	$1,406
														Subtotal = $14,069

Issue Date FY: 2023 ( Subtotal = $217,496 )
2023	2023	BOISE STATE UNIVERSITY	1910 UNIVERSITY DR	BOISE	ID	83725	ADA	USA	Allergy and Infectious Diseases Research	000	1	1/23/2023	NEW	$217,496
														Subtotal = $217,496

Grand Total All Awards = $358,106

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Chemical tools to investigate chain-flipping in quorum signal synthases

Award Number: R21AI175749

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: 01/23/2023

PERIOD OF PERFORMANCE END DATE: 12/31/2025

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