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Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

Published on: February 23, 2016

Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization.

Yaewon Kim¹, Mengxiao Liu¹, Christian Hilty¹

¹Department of Chemistry, Texas A&M University , College Station, Texas 77843-3255, United States.

Analytical Chemistry

|October 11, 2016

Summary

This summary is machine-generated.

This study introduces a faster method for screening protein-ligand interactions using multiplexed nuclear magnetic resonance (NMR) and dissolution dynamic nuclear polarization (D-DNP). This technique significantly improves throughput for determining dissociation constants (K_D) in drug discovery.

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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

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Published on: September 13, 2014

Area of Science:

Biophysical Chemistry
Structural Biology
Drug Discovery

Background:

Nuclear magnetic resonance (NMR) spectroscopy is vital for screening protein-ligand interactions.
Dissolution dynamic nuclear polarization (D-DNP) enhances NMR sensitivity, enabling rapid dissociation constant (K_D) determination.
A key limitation of D-DNP is its lengthy polarization time, hindering high-throughput screening.

Purpose of the Study:

To develop a multiplexed NMR approach to overcome the limitations of D-DNP in protein-ligand interaction screening.
To enhance the throughput and accuracy of K_D measurements using hyperpolarized ligands.
To broaden the measurable K_D range in a single experiment.

Main Methods:

A novel multiplexed NMR experiment was designed, involving rapid mixing of a single hyperpolarized ligand with protein in two separate flow cells.
Simultaneous NMR detection was performed on both channels, enabling chemical shift-resolved spin relaxation measurements.
Reference compounds were utilized for accurate concentration quantification, and two protein-to-ligand concentration ratios were employed to expand the K_D measurement range.

Main Results:

The multiplexed D-DNP NMR method achieved simultaneous, chemical shift-resolved spin relaxation measurements.
Accurate K_D values for trypsin inhibitors benzamidine (12.6 ± 1.4 μM) and benzylamine (207 ± 22 μM) were determined.
The method demonstrated a broadened K_D measurement range (at least one order of magnitude) and improved throughput by a factor of two.

Conclusions:

The developed multiplexed NMR experiment significantly enhances the throughput of D-DNP for protein-ligand interaction screening.
This approach provides a more efficient and accurate method for determining dissociation constants (K_D), applicable to drug discovery and biophysical studies.
The technique is particularly beneficial for multipoint measurements traditionally requiring extensive titrations.