# Converting human butyrylcholinesterase into a metalloenzyme for catalytic hydrolysis of organophosphates

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2024 · $192,331

## Abstract

PROJECT SUMMARY/ABSTRACT
Exposure to toxic organophosphorus (OP) compounds in the form of insecticides or chemical warfare nerve
agents (CWNAs) remains a persistent concern for both civilian and military populations. One countermeasure
to OP exposure that has been shown to be effective in animal models is the use of intravenously administered
butyrylcholinesterase (BChE), which scavenges free OP molecules in the bloodstream. A significant limitation
of BChE, however, is that it simply binds OP compounds stoichiometrically, resulting in the inactivation of both
molecules. The resulting requirement for large doses of BChE is exacerbated by difficulties associated with its
manufacture, thereby complicating its use as an OP defense measure. An important advance would therefore
be the modification of the human BChE enzyme to enable catalytic degradation of OP compounds, thus
providing a therapeutic enzyme with minimal potential immunogenicity. Several lines of evidence support the
feasibility of this approach: microbial enzymes that exhibit high OP hydrolysis activity are known, and studies
by our lab and others have shown that BChE can be made modestly catalytic for OP hydrolysis through the
introduction of mutations or by adding certain oxime compounds. With this in mind, our ultimate goal is to
develop catalytic human BChE enzymes that can functionally mimic microbial or oxime-coupled OP
degradation reactions, but which also exhibit long serum half-lives, negligible side effects, and minimal
immunogenicity. A critical and novel aspect of our work is the rational introduction of a zinc-binding site within
the substrate gorge of the BChE enzyme, which is designed to mimic the catalytic center of the highly efficient
bacterial organophosphate hydrolase (OPH) and other engineered hydrolytic zinc enzymes. Three basic
design strategies are described for initial evaluation, while additional and subsequent optimized mutants will be
developed through multiscale computer-guided design. BChE mutants will be expressed in HEK293E cells,
which we have successfully used to produce properly glycosylated BChE tetramers in quantities sufficient for
characterization. Evaluation of the metal binding site will be assessed through spectroscopic and analytical
techniques, while hydrolytic reactivity towards a range of OP model compounds will be measured through
colorimetric assays. Due to their enhanced safety profiles, these engineered enzymes would provide
enhancements to conventional clinical interventions for acute OP exposures, especially when combined with
post-exposure oxime treatments. In future work, these studies will be used to optimize BChE metalloenzymes
that are effective against highly toxic and difficult-to-assay targets, for which evolutionary approaches are
intractable, and ultimately develop effective formulations for storage and delivery for post-exposure indications.
The goal of the introductory work proposed here is the initial development and valid...

## Key facts

- **NIH application ID:** 10928701
- **Project number:** 5R21NS126163-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Hannah S Shafaat
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $192,331
- **Award type:** 5
- **Project period:** 2023-09-15 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10928701

## Citation

> US National Institutes of Health, RePORTER application 10928701, Converting human butyrylcholinesterase into a metalloenzyme for catalytic hydrolysis of organophosphates (5R21NS126163-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10928701. Licensed CC0.

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