# Advanced Nucleation Technologies for Membrane Protein Crystallization to Accelerate Structure-Based Drug Design for Substance Use Disorders

> **NIH NIH R44** · DENOVX, LLC · 2022 · $1,775,613

## Abstract

PROJECT SUMMARY
DeNovX creates innovative platform products that improve the crystallization of proteins and pharmaceuticals.
Of the ≈ 4700 human membrane proteins potentially involved in drug responses, ≈ 94% have yet to be
structurally characterized owing to difficulties in crystallization. The goal of Phase II is to adapt DeNovX’s
surface science agnostic approach to improving crystallization for use with membrane proteins to advance the
structure-based understanding of substance use disorders (SUDs) to benefit Public Health. DeNovX improves
crystallization using tunable substrates based on chemical interactions from bifunctional self-assembled
monolayers (SAMs); surface energy modifications from engineered nucleation features (ENFs); and a hybrid
strategy using chemically and energy modified ENFs (CENFs). A high confidence POC was achieved using the
membrane proteins quinol:fumarate reductase (QFR) and succinate:quinone oxidoreductase (SQR). Diffraction
quality QFR crystals were formed on a bifunctional SAM while no crystals formed on controls, and select ENFs
produced up to a 19-fold increase in QFR crystals vs. controls. The hypothesis is that bifunctional SAMs,
ENFs, or hybrid CENFs interacting with a membrane protein or its detergent envelope can facilitate
preorganization and crystal nucleation from supersaturated solutions. Specific Aim 1 - Conduct controlled,
replicate (n ≥ 6) studies of membrane protein crystallization outcomes using β-prototype bifunctional SAMs,
ENFs, and CENFs to identify those characteristics most favorably impacting crystal nucleation of the QFR and
SQR benchmark membrane proteins provided by Co-I Iverson under a subaward to Vanderbilt. These rigorous
and quantitative crystallization studies will be complemented with synchrotron X-ray diffraction to ensure
resolution that supports binding and conformational analyses through a subaward to Co-I Cohen at Stanford’s
Synchrotron Radiation Lightsource (SSRL). Specific Aim 2 - Incorporate surface characteristics most favorably
impacting membrane protein crystallization into bifunctional SAM, ENF, and CENFs on ≥ 12 𝛾-prototype
24/96/384 well HTS crystallization plates. Optimize for crystallization in detergent containing systems and
advance development of the top six nucleation surfaces showing reproducible (n ≥ 6) crystallization
improvements of ≥ 15% increase in hits, ≥ 20% reduction in onset times, or ≥ 25% increase in the quantity of
crystals generated vs. controls. Specific Aim 3 - Incorporating SUD relevant target guidance from NIH and
domain experts with a tractability assessment by the Co-Is, expand crystallization screening with optimized
nucleation surfaces to ≥ 8 membrane proteins of varying class that would most benefit from near atomic
resolution structural data. Generate crystals for ≥ 2 targets for study at SSRL. Specific Aim 4 - Demonstrate the
tangible benefits and deploy the surface science product suite for membrane protein crystallization screening
wi...

## Key facts

- **NIH application ID:** 10546186
- **Project number:** 2R44DA047146-02
- **Recipient organization:** DENOVX, LLC
- **Principal Investigator:** Andrew H. Bond
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,775,613
- **Award type:** 2
- **Project period:** 2022-09-30 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10546186

## Citation

> US National Institutes of Health, RePORTER application 10546186, Advanced Nucleation Technologies for Membrane Protein Crystallization to Accelerate Structure-Based Drug Design for Substance Use Disorders (2R44DA047146-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10546186. Licensed CC0.

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