Tuesday, December 16, 2025 12/16/2025

Exploiting new approaches for selective inhibition of trypsins

Award Number: R01GM144393
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 01/01/2022
PERIOD OF PERFORMANCE END DATE: 11/30/2026

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Exploiting new approaches for selective inhibition of trypsins - PROJECT SUMMARY/ABSTRACT The human trypsin isoforms, trypsin 1, trypsin 2, and mesotrypsin, are proteases that have been implicated in disease processes in cancer and pancreatitis, and may offer viable therapeutic targets. Trypsins belong to a large family of trypsin-like enzymes with similar active site topology, and hence existing inhibitors lack selectivity. There is a need for selective trypsin inhibitors and isoform-selective trypsin inhibitors as pharmacological tools to better define the functions of these individual enzymes in disease, and to evaluate trypsin inhibition as a therapeutic strategy in preclinical models of disease. In this project, we will take a multipronged approach to develop new strategies for potent and selective inhibition of each of the human trypsin isoforms. (1) Our preliminary data reveal a previously unsuspected auto-inhibited conformation of mesotrypsin with a ligand-targetable allosteric site that may be exploited for inhibitory effect. We will use high- throughput virtual screening and structure-based hit-to-lead optimization to develop potent and selective allosteric inhibitors of mesotrypsin. We will also use structural and molecular dynamics analyses to evaluate whether similar strategies may hold potential for trypsins 1 and 2. (2) Our published studies have shown that Kunitz domains can be engineered to create more selective protein-based inhibitors of trypsin-like proteases by using a yeast surface display (YSD) platform for directed evolution. To enable further optimization of such inhibitors, we seek to generate comprehensive maps of the binding specificity landscapes that can, for any possible combination of mutations within an inhibitor, predict the consequences on inhibitor affinity and specificity toward a set of target proteases. We will accomplish this task by integrating YSD combinatorial library screening with next-generation sequencing (NGS), machine-learning (ML) approaches, and experimental calibration to enable quantitative prediction of the impact of multiple potentially interacting mutations of an inhibitor. These data will enable us to identify the most potent and selective Kunitz domains that can be achieved for targeting each of the human trypsins. (3) Our preliminary data demonstrate an enhancement of trypsin affinity by bivalent inhibitors capable of binding simultaneously to two molecules of mesotrypsin. Here, we will dissect the mechanisms responsible for these affinity enhancements and design strategies to exploit this information toward development of more potent and selective polyvalent trypsin inhibitors. In addition to developing three complementary strategies, each of which has high potential to produce the desired selective inhibitors of human trypsins, our project will provide broader insights that can aid future development of inhibitors for many other important trypsin-like proteases. Finally, the novel methodology developed here for mapping protein-protein interaction (PPI) affinity and specificity landscapes will be of broad utility for characterizing the sequence and structural constraints governing affinity and selectivity of functional protein interactions in many other diverse systems.


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 = $0 )
2026	2025	MAYO CLINIC JACKSONVILLE (A NONPROFIT CORPORATION)	4500 SAN PABLO RD S	JACKSONVILLE	FL	32224	DUVAL	USA	Biomedical Research and Research Training	000	4	10/27/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $293,116 )
2025	2025	MAYO CLINIC JACKSONVILLE (A NONPROFIT CORPORATION)	4500 SAN PABLO RD S	JACKSONVILLE	FL	32224	DUVAL	USA	Biomedical Research and Research Training	001	4	9/9/2025	NON-COMPETING CONTINUATION	$29,311
2025	2025	MAYO CLINIC JACKSONVILLE (A NONPROFIT CORPORATION)	4500 SAN PABLO RD S	JACKSONVILLE	FL	32224	DUVAL	USA	Biomedical Research and Research Training	000	4	11/14/2024	NON-COMPETING CONTINUATION	$263,805
														Subtotal = $293,116

Issue Date FY: 2024 ( Subtotal = $290,781 )
2024	2024	MAYO CLINIC JACKSONVILLE (A NONPROFIT CORPORATION)	4500 SAN PABLO RD S	JACKSONVILLE	FL	32224	DUVAL	USA	Biomedical Research and Research Training	001	3	6/12/2024	NON-COMPETING CONTINUATION	$29,078
2024	2024	MAYO CLINIC JACKSONVILLE (A NONPROFIT CORPORATION)	4500 SAN PABLO RD S	JACKSONVILLE	FL	32224	DUVAL	USA	Biomedical Research and Research Training	000	3	11/8/2023	NON-COMPETING CONTINUATION	$261,703
														Subtotal = $290,781

Issue Date FY: 2023 ( Subtotal = $293,784 )
2023	2023	MAYO CLINIC JACKSONVILLE (A NONPROFIT CORPORATION)	4500 SAN PABLO RD S	JACKSONVILLE	FL	32224	DUVAL	USA	Biomedical Research and Research Training	000	2	12/1/2022	NON-COMPETING CONTINUATION	$264,406
2023	2023	MAYO CLINIC JACKSONVILLE (A NONPROFIT CORPORATION)	4500 SAN PABLO RD S	JACKSONVILLE	FL	32224	DUVAL	USA	Biomedical Research and Research Training	001	2	2/14/2023	NON-COMPETING CONTINUATION	$29,378
														Subtotal = $293,784

Issue Date FY: 2022 ( Subtotal = $301,404 )
2022	2022	MAYO CLINIC JACKSONVILLE (A NONPROFIT CORPORATION)	4500 SAN PABLO RD S	JACKSONVILLE	FL	32224	DUVAL	USA	Biomedical Research and Research Training	000	1	12/17/2021	NEW	$301,404
														Subtotal = $301,404

Grand Total All Awards = $1,179,085

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Exploiting new approaches for selective inhibition of trypsins

Award Number: R01GM144393

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 01/01/2022

PERIOD OF PERFORMANCE END DATE: 11/30/2026

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