# De novo design of generalizable allosteric modulators and peptide ligands for G protein coupled receptors

> **NIH NIH R21** · INSTITUTE FOR PROTEIN INNOVATION, INC. · 2021 · $179,776

## Abstract

Project Summary
Roughly one third of all FDA approved drugs target G protein coupled receptors (GPCRs), and this protein
family holds myriad therapeutic opportunities yet to be discovered. GPCRs signal through allosteric changes in
protein structure, and there is a large unmet need for effective, highly generalizable tools for conformational
control of these receptors. ​This project has two Specific Aims, both of which employ computational protein
design with Rosetta to build proteins ​de novo that bind and modulate GPCR conformation. Upon ligand binding
and activation, GPCRs associate with and signal through G proteins. In the first Aim, we design proteins ​de
novo which mimic the interaction domain of the endogenous Gα protein subunit. Because the interaction
between receptor and Gα is highly conserved, the protein tools we propose to develop can be applied broadly.
Synthetic genes encoding ​de novo Gα mimetics have been expressed, and protein was purified from
Escherichia coli.​ Binding to several different detergent-solubilized GPCRs was demonstrated ​via pull-down and
Western blotting. Functional Gα mimetic proteins will be structurally characterized and used as templates for
re-engineering a small suite of mimetics for all Gα subtypes. Additionally, we propose to leverage the Gα
mimetics to enhance the expression and purification of GPCRs. To demonstrate this system, we will attempt to
produce three different GPCRs for the first time: super conserved receptor expressed in brain 1-3. In the
second aim, we will design disulfide constrained peptides to serve as agonists and antagonists for all
structure-enabled family B GPCRs. This receptor family recognizes endogenous peptide ligands, and they are
difficult to drug with small molecules. Structures that detail ligand recognition are available for 60% of family B
receptors; we will use these as the basis for ​de novo design of high-affinity antagonist ligands based on
disulfide-constrained peptides. We will then screen hundreds of thousands of rationally designed ​de novo
peptides using yeast display, fluorescence activated cell sorting, and deep sequencing. Our unique design
approach enables us to custom tailor the peptide topology so that we can readily convert our antagonists into
high affinity agonists by extending the peptide N-terminus. The efficacy of designed ligands identified ​via the
high-throughput screen will be assessed individually using a luciferase reporter assay for GPCR signaling. The
research proposed here represents the first time that computational ​de novo protein design will be applied to
control GPCR conformation. This work has the potential to create valuable tools to empower the GPCR
research community and accelerate discovery of lead therapeutics for many diseases.

## Key facts

- **NIH application ID:** 10160902
- **Project number:** 5R21EB028342-02
- **Recipient organization:** INSTITUTE FOR PROTEIN INNOVATION, INC.
- **Principal Investigator:** Christopher David Bahl
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $179,776
- **Award type:** 5
- **Project period:** 2020-05-07 → 2022-01-27

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10160902

## Citation

> US National Institutes of Health, RePORTER application 10160902, De novo design of generalizable allosteric modulators and peptide ligands for G protein coupled receptors (5R21EB028342-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10160902. Licensed CC0.

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