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High-throughput Screening and Characterization of Small-Molecule Inhibitors of N-Terminal Autoproteolysis of Coronavirus Main Protease Precursors

Award Number: R01AI201768
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: 09/03/2026
PERIOD OF PERFORMANCE END DATE: 08/31/2030

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High-throughput Screening and Characterization of Small-Molecule Inhibitors of N-Terminal Autoproteolysis of Coronavirus Main Protease Precursors - High-Throughput Screening and Characterization of Small-Molecule Inhibitors Targeting N-Terminal Autoproteolysis of Coronavirus Main Protease Precursors Project Summary Many viruses—including retroviruses and several pathogens of pandemic potential (PPPs) such as coronaviruses, flaviviruses, and picornaviruses—share a conserved replication strategy involving polyprotein precursors that contain a virus-encoded protease. These protease-containing precursors undergo regulated autoprocessing to release mature enzymes essential for viral replication. While mature viral proteases are proven antiviral drug targets, their precursor forms remain largely unexplored. Our team has demonstrated that the HIV- 1 protease precursor is enzymatically distinct from its mature counterpart and developed a cell-based high- throughput screening (HTS) platform to identify compounds that selectively interfere with precursor-mediated autoproteolysis. Using this approach, we recently identified several small-molecule hits that suppress both wild- type and multiple drug-resistant HIV variants with comparable potency, validating the precursor autoproteolysis as a promising pan-HIV antiviral target. To extend this strategy, we developed an analogous cell-based assay for N-terminal autoproteolysis of the coronavirus main protease (Mpro). Notably, as with HIV-1 protease, precursor forms of SARS-CoV-2 and MERS-CoV Mpro exhibit markedly reduced sensitivity (≥20-fold) to inhibitors designed for the mature enzyme. This platform also enables parallel screening for inhibitors targeting either precursor or mature Mpro forms. Pilot screens of 130 known protease inhibitors and 1,120 natural product extracts (NPEs: ~22,000 individual compounds) confirmed the assay's robustness and identified ~10 NPEs with reproducible, albeit modest, activities. Building on this foundation, the proposed project aims to identify small-molecule therapeutic candidates with pan-coronavirus activity that selectively interfere with the conserved N-terminal autoproteolysis of Mpro precursors. Specifically, we will: screen the SBP small-molecule library (~100,000 Enamine REAL compounds) using our cell-based functional assay to identify hit compounds that can inhibit precursor autoproteolysis (Aim 1); define active components from the positive NPEs through systematic subfractionation and characterization (Aim 2); establish and validate additional processing assays for other coronaviruses, including SARS-CoV, MERS-CoV, NL63, OC43, etc., to enable comprehensive hit characterization (Aim 3); evaluate confirmed hits in orthogonal biochemical, cellular, and virological assays to define mechanisms of action (MoAs) and distinguish broad-spectrum hits from subtype-specific inhibitors (Aim 4). In summary, this project advances a novel antiviral strategy that targets protease precursor autoproteolysis— a conserved process across multiple viral families—with the ultimate goal of identifying new therapeutic candidates for combination therapy and enhancing pandemic preparedness through the development of broad- spectrum antivirals.


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: 2026 ( Subtotal = $2,036,679 )
2026	2026	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	1	9/3/2026	NEW	$2,036,679
														Subtotal = $2,036,679

Grand Total All Awards = $2,036,679

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High-throughput Screening and Characterization of Small-Molecule Inhibitors of N-Terminal Autoproteolysis of Coronavirus Main Protease Precursors

Award Number: R01AI201768

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: 09/03/2026

PERIOD OF PERFORMANCE END DATE: 08/31/2030

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