# Engineering protein-specific proteases: targeting signaling proteins

> **NIH NIH R01** · UNIV OF MARYLAND, COLLEGE PARK · 2022 · $521,252

## Abstract

Summary: Our objective is to develop general principles for engineering proteases that
can control signaling in cells. This requires seven steps: 1) Choice of signaling proteins
(Human RAS isoforms and oncogenic mutants); 2) Identification of dynamic regions
within the signaling proteins; 3) Identification of a target sequence within dynamic
regions; 4) Engineering/evolving high specificity against the target sequence; 5)
Engineering zymogen activation that is catalyzed by the target protein; 6) Testing
regulated RAS degradation in E. coli; 7) Testing control of signaling in human cell culture.
We will complete these steps using a multi-disciplinary approach that includes
computational and in vitro selection-based protein engineering, enzymology, multi-
dimensional NMR, X-ray crystallography, and cell-biology. The three Specific Aims are:
1) Evolve protein-specific proteases that target active RAS; 2) Engineer zymogen
activation catalyzed by active RAS; 3) Test promising proteases in cell model systems.
This project will develop general principles of recognition, folding, and catalysis based on
detailed understanding of structure and mechanism and use this knowledge to develop
tools to control signaling in a cell. The protease and its regulators will comprise an
enzymatic system that will sense the presence of active RAS and transduce a controlled
release of the active RAS-specific protease, thereby modulating RAS signaling. The
ability to cleave RAS in cell-based systems and thereby alter RAS signaling will
represent a significant milestone towards establishing the utility of protein-specific
proteases as exogenous signaling regulators. Although these proteases target active
RAS, the underlying design principles are fundamental and will be adaptable to many
target proteins.

## Key facts

- **NIH application ID:** 10431978
- **Project number:** 5R01GM141290-02
- **Recipient organization:** UNIV OF MARYLAND, COLLEGE PARK
- **Principal Investigator:** PHILIP N BRYAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $521,252
- **Award type:** 5
- **Project period:** 2021-07-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10431978

## Citation

> US National Institutes of Health, RePORTER application 10431978, Engineering protein-specific proteases: targeting signaling proteins (5R01GM141290-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10431978. Licensed CC0.

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