Hyperpolarized Water to Study Protein–Ligand Interactions

Abstract

ABSTRACT:The affinity between a chosen target protein and small molecules is a key aspect of drug discovery. Screening by popular NMR methods such as Water-LOGSY suffers from low sensitivity and from false positives caused by aggregated or denatured proteins. This work demonstrates that the sensitivity of Water-LOGSY can be greatly boosted by injecting hyperpolarized water into solutions of proteins and ligands. Ligand binding can be detected in a few seconds, whereas about 30 min is usually required without hyperpolarization. Hyperpolarized water also enhances proton signals of proteins at concentrations below 20 μM so that one can verify in a few seconds whether the proteins remain intact or have been denatured D rug discovery aspires to identify new lead compounds that bind with high affinity to target proteins or other biomolecules. Robust assays are required in order to determine binding affinities of extensive libraries of potential ligands.

Filterable Agents for Hyperpolarization of Water, Metabolites, and Proteins

“…Figure b shows the longitudinal relaxation of hyperpolarized HDO after FLAP has been removed by filtration. Compared to samples with dissolved nitroxide radicals without FLAP in our last experiments, the relaxation time could be extended by a factor of 3.7 from T 1 ( 1 H)=9.8±1.1 s to 36.6±0.3 s, whereas polarization could be enhanced by a factor of 1.5 from P ( 1 H)=4 % to P ( 1 H)=6 % …”

“…Hyperpolarizing H 2 O or HDO represents a particular challenge, because even low concentrations of free radicals cause the protons to relax significantly after dissolution, during transfer and in the NMR or MRI systems. Hyperpolarized H 2 O or HDO can be used in DNP‐enhanced Water‐LOGSY (Water‐Ligand Observed via Gradient SpectroscopY) experiments to detect ligand binding to macromolecules, or as a contrast agent for angiography . Unfortunately, such applications are hampered by the short T 1 ( 1 H) of HDO arising from the presence of free radicals.…”

“…Unfortunately, such applications are hampered by the short T 1 ( 1 H) of HDO arising from the presence of free radicals. Following methods used in a previous DNP‐enhanced Water‐LOGSY study, we determined a disappointingly short T 1 ( 1 H)=9.8 s in HDO after dissolution at 500 MHz, whereas T 1 ( 1 H)=37 s in degassed and pure HDO …”

Optimizing dissolution dynamic nuclear polarization

“…High contrast-to-noise images can be obtained in a fraction of seconds [31]. In our laboratory, 1 H d-DNP was combined with long-lived proton states to enhanced both the sensitivity and contrast in drug screening experiments [32], and hyperpolarized water was used for protein-ligand interaction studies in combination with the so called WaterLOGSY technique [33].…”

Untersuchung von intrinsisch unstrukturierten Proteinen mithilfe des Austausches mit hyperpolarisiertem Wasser

“…Die Bestrahlung der paramagnetischen Zentren mit Mikrowellen von ca. 188.2 GHz führte zu Hyperpolarisierung der Protonen von P ( 1 H)=89 % im gefrorenen Glas . Die 200 μL des hyperpolarisierten Feststoffes werden mit 5 mL (25‐fache Verdünnung) überhitztem D 2 O (180 °C, 10.5 bar) rasch geschmolzen und anschließend binnen ca.…”