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Defining the Translocation Mechanisms of SARS-CoV-2 nsp13 Helicase to Aid in Antiviral Development

Award Number: R01AI166050
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Project Summary SARS-CoV-2, the causative agent of COVID-19, has infected more than 103M people worldwide (February 2021) with more than 2.25M deaths, and represents a dire threat to the health and economic well-being of the entire world. Although vaccines seem to be effective against SARS-CoV-2, recent information regarding potential vaccine resistant strains highlights the importance of alternative strategies to combat this virus. The development of antiviral therapeutics on important mutation resistant viral proteins such as nsp13 is one such strategy. Improved knowledge of the molecular mechanisms utilized by nsp13 are necessary to rationally develop inhibitors. This project will address this deficiency utilizing an integrated multiscale modeling, protein crystallography, and biochemical approach to define how SARS-CoV-2 nsp13 helicase binds RNA and ATP substrates, transduces energy during ATP binding and hydrolysis, and changes conformation during ligand binding and catalysis. We propose the following: 1) Identification of molecular-level components of the RNA- binding and translocation mechanisms of nsp13. Preliminary all-atom molecular dynamics (aaMD) simulations of SARS-CoV-2 nsp13 have identified key protein-RNA interactions that will inform initial mutagenesis studies. Further simulation and protein crystallography will inform on the ATP-dependent protein-RNA interactions observed in the RNA cleft. Biochemical experiments will be performed to test the structure-function hypotheses generated by the structural-based approaches. 2) Identification of molecular-level features of the binding, hydrolysis and product release of ATP by nsp13. We have performed aaMD simulations of the SARS- CoV-2 nsp13 in all relevant substrate states. Soaked-in ATP and non-hydrolysable analogue protein crystallography will be performed to test these initial models. Subsequent quantum mechanical calculations will identify key components of the ATP hydrolysis reaction. Site-directed mutagenesis and well-established enzyme kinetics assays will be used to test effects predicted by these simulations. 3) Identification of allosteric networks in SARS-CoV-2 nsp13 that transduce energy from ATP binding and hydrolysis to perform RNA translocation. Utilizing network analyses of aaMD simulations, Motif V has been identified as a key allosteric contributor. Biochemical studies will be performed to verify that Motif V is necessary for nsp13 helicase function. Further work will be done to identify allosteric networks between additional components of the ATP pocket and RNA cleft identified in Aims 2 and 3. This work will produce unprecedented molecular-level insight into the translocation mechanism of SARS-CoV-2 nsp13 helicases. Key components of this mechanism represent new targets for antiviral development.


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: 2024 ( Subtotal = $439,024 )
2024	2024	OKLAHOMA STATE UNIVERSITY	401 WHITEHURST HALL	STILLWATER	OK	74078	PAYNE	USA	Allergy and Infectious Diseases Research	000	4	8/13/2024	NON-COMPETING CONTINUATION	$439,024
														Subtotal = $439,024

Issue Date FY: 2023 ( Subtotal = $439,004 )
2023	2023	OKLAHOMA STATE UNIVERSITY	401 WHITEHURST HALL	STILLWATER	OK	74078	PAYNE	USA	Allergy and Infectious Diseases Research	000	3	9/5/2023	NON-COMPETING CONTINUATION	$439,004
														Subtotal = $439,004

Issue Date FY: 2022 ( Subtotal = $434,531 )
2022	2022	OKLAHOMA STATE UNIVERSITY	401 WHITEHURST HALL	STILLWATER	OK	74078	PAYNE	USA	Allergy and Infectious Diseases Research	000	2	8/19/2022	NON-COMPETING CONTINUATION	$434,531
														Subtotal = $434,531

Issue Date FY: 2021 ( Subtotal = $479,232 )
2021	2021	OKLAHOMA STATE UNIVERSITY	401 WHITEHURST HALL	STILLWATER	OK	74078	PAYNE	USA	Allergy and Infectious Diseases Research	000	1	9/17/2021	NEW	$479,232
														Subtotal = $479,232

Grand Total All Awards = $1,791,791

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Defining the Translocation Mechanisms of SARS-CoV-2 nsp13 Helicase to Aid in Antiviral Development

Award Number: R01AI166050

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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