# Principles for Designing Stimulus-Responsive Enzymes

> **NIH NIH R01** · RUTGERS, THE STATE UNIV OF N.J. · 2021 · $438,029

## Abstract

Summary:
 The ability to design stimulus-responsiveness into any enzyme of choice would aid in our ability to
interrogate and intervene in biological processes with exquisitely high spatial and temporal precision. By
constructing stimulus-responsiveness into enzymes from the bottom up, insights into fundamental biophysical
principles underlying allosteric effects in natural systems will also be achieved (learning by building). The focus
of this proposal is the bottom-up development of stimulus-responsive prodrug-activating enzymes by
synergistically combining protein design through computational approaches (Khare) with protein engineering
through unnatural amino acid (UAA) mutagenesis (Deiters). Genetic incorporation of unnatural amino acids
allows introducing bio-orthogonal switches in a site-specific manner, and computational modeling enables
rational rewiring of the structure and conformational landscape of the protein active site and UAA
microenvironment with atomic resolution. By combining these state-of-the-art techniques with well-established
protocols for detailed kinetic, structural, and biophysical characterization, we hypothesize that a bottom up
framework for introducing stimulus-responsiveness in proteins will be deciphered. As our model systems, we
will use the carboxypeptidase G2/nitrogen mustard prodrug and cytosine deaminase/5-fluorouracil
enzyme/prodrug pairs, both of which have been extensively investigated in a chemotherapy application called
directed enzyme prodrug therapy (DEPT). Their utility in a therapeutic setting for DEPT will be enhanced by
rendering them conditionally activatable. In Aim 1, we will use azobenzene-containing photo-responsive UAAs
for rendering the designed enzymes photocontrollable. In Aim 2, we will develop methodology for designing
zymogenized versions of enzymes that can be activated by tissue-specific proteolytic enzymes, such as matrix
metalloproteases. In Aim 3, we will use a variety of structural and biophysical techniques to validate designed
enzymes and provide feedback for further design iterations and modeling methodology improvement. While
our focus is on the two enzymes mentioned, the methods we are developing, however, will be transferable to
the control of activity and delivery of a variety of other enzymes, making this a general approach to design new
stimulus-responsive enzymes and potentially also allowing unprecedented selectivity and optimal delivery of
chemotherapies.

## Key facts

- **NIH application ID:** 10207681
- **Project number:** 5R01GM132565-03
- **Recipient organization:** RUTGERS, THE STATE UNIV OF N.J.
- **Principal Investigator:** Sagar D Khare
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $438,029
- **Award type:** 5
- **Project period:** 2019-09-27 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10207681

## Citation

> US National Institutes of Health, RePORTER application 10207681, Principles for Designing Stimulus-Responsive Enzymes (5R01GM132565-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10207681. Licensed CC0.

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