Determination of binding affinities using hyperpolarized NMR with simultaneous 4-channel detection

Area of Science:

• Nuclear Magnetic Resonance (NMR) Spectroscopy
• Hyperpolarized Chemistry
• Drug Discovery and Development

Background:

• Dissolution Dynamic Nuclear Polarization (D-DNP) enhances NMR sensitivity, aiding drug discovery by reducing ligand and protein concentration needs.
• Current D-DNP NMR screening methods suffer from low throughput due to the time required for hyperpolarization, delaying measurements.
• A fourfold increase in throughput is needed to make D-DNP NMR screening more broadly applicable.

Purpose of the Study:

• To develop a simultaneous 4-channel detection method for hyperpolarized 19F NMR to overcome throughput limitations in D-DNP NMR screening.
• To enable faster and more efficient assessment of ligand-target interactions in drug discovery.
• To determine dissociation constants (KDs) of ligands with high accuracy and a broad dynamic range.

Main Methods:

• Development of a multiplexed NMR spectrometer and probe for simultaneous 4-channel detection of hyperpolarized 19F NMR signals.
• Utilizing a hyperpolarized reporter ligand (TFBC) in conjunction with a concentration gradient across four flow cells to probe binding interactions.
• Employing T2 relaxation measurements of the reporter ligand in the presence of target protein (trypsin) and varying concentrations of competitor ligands (benzamidine, benzylamine).

Main Results:

• The 4-channel system successfully increased throughput fourfold, enabling simultaneous measurements.
• Concentration-dependent binding interactions between benzamidine and benzylamine with trypsin were observed.
• Accurate determination of dissociation constants (KDs) was achieved without individual experimental condition optimization for different affinities.
• Simulations indicated an extended screenable KD range of up to three orders of magnitude in a single experiment.

Conclusions:

• The developed 4-channel detection method significantly enhances throughput for D-DNP NMR screening.
• This method facilitates efficient and accurate determination of ligand-target binding affinities, including KDs.
• The approach holds promise for accelerating drug discovery by overcoming limitations of current D-DNP NMR screening techniques.