Rapid Protein-Ligand Affinity Determination by Photoinduced Hyperpolarized NMR

Area of Science:

• Biochemistry
• Structural Biology
• Medicinal Chemistry

Background:

• Protein-ligand binding affinity determination is crucial for drug design.
• Current methods are often time-consuming and costly.
• Novel, rapid screening techniques are needed to accelerate drug discovery.

Purpose of the Study:

• To present a novel, rapid method for determining protein-ligand binding affinities.
• To establish a methodological framework for affinity measurements using photo-CIDNP NMR.
• To demonstrate the method's accuracy and applicability to drug discovery.

Main Methods:

• Utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR.
• Measuring binding affinities of peptides to a PDZ domain and fragment ligands to PIN1.
• Employing competition binding experiments for non-photo-CIDNP-polarizable ligands.
• Correlating photo-CIDNP NMR fragment screening with saturation transfer difference (STD) NMR.

Main Results:

• Determined protein-ligand binding affinities within 5-15 minutes using only 0.1 mg of protein.
• Validated the method's accuracy for peptide-PDZ domain and fragment-PIN1 interactions.
• Demonstrated successful application in competition binding assays.
• Showed a strong correlation between photo-CIDNP and STD NMR for fragment screening.

Conclusions:

• The photo-CIDNP NMR method provides a rapid and accurate approach for measuring protein-ligand affinities in the micro- to millimolar range.
• This technique significantly reduces experimental time and protein requirements compared to traditional methods.
• The method offers insights into binding epitopes in a single scan, enhancing early-stage drug discovery.