Abstract

Dissolution dynamic nuclear polarization (D-DNP) is a powerful technique to improve NMR sensitivity by a factor of thousands. Combining D-DNP with NMR-based screening enables to mitigate solubility or availability problems of ligands and target proteins in drug discovery as it can lower the concentration requirements into the sub-micromolar range. One of the challenges that D-DNP assisted NMR screening methods face for broad application, however, is a reduced throughput due to additional procedures and time required to create hyperpolarization. These requirements result in a delay of several tens of minutes in-between each NMR measurement. To solve this problem, we have developed a simultaneous 4-channel detection method for hyperpolarized 19F NMR, which can increase throughput fourfold by utilizing a purpose-built multiplexed NMR spectrometer and probe. With this system, the concentration-dependent binding interactions were observed for benzamidine and benzylamine with the serine protease trypsin. A T2 relaxation measurement of a hyperpolarized reporter ligand (TFBC; CF3C6H4CNHNH2), which competes for the same binding site on trypsin with the other ligands, was used. The hyperpolarized TFBC was mixed with trypsin and the ligand of interest, and injected into four flow cells inside the NMR probe. Across the set of four channels, a concentration gradient was created. From the simultaneously acquired relaxation datasets, it was possible to determine the dissociation constant (KD) of benzamidine and benzylamine without the requirement for individually optimizing experimental conditions for different affinities. A simulation showed that this 4-channel detection method applied to D-DNP NMR extends the screenable KD range to up to three orders of magnitude in a single experiment.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.