Paramagnetic relaxation enhancement for protein-observed ¹⁹F NMR as an enabling approach for efficient fragment screening

Abstract

Protein-observed 19F (PrOF) NMR is an emerging tool for ligand discovery. To optimize the efficiency of PrOF NMR experiments, paramagnetic relaxation enhancement through the addition of chelated Ni(II) was used to shorten longitudinal relaxation time without causing significant line broadening. Thus enhancing relaxation time leads to shorter experiments without perturbing the binding of low- or high-affinity ligands. This method allows for time-efficient screening of potential ligands for a wide variety of proteins in the growing field of fragment-based ligand discovery.

Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis

“…In addition to understanding biologically relevant and weak protein-protein interactions, 19 F lineshape analysis complements other methods for drug screening, discovery, and development (1,2,(15)(16)(17)(18)(19)(20), particularly because many initial hits are weak and often accompanied by transient kinetics. In the service of drug and fragment screening, 19 F lineshape analysis enables facile acquisition of quantitative data from simple spectra of labeled proteins or ligands.…”

“…Additional advantages include the high sensitivity of 19 F (83% that of 1 H), its large chemical shift range, the 100% abundance of 19 F, the near nonexistence of background, the absence of water suppression, the minimal pulse program, and the simplicity of spectra (14). These advantages have led to the increased use of protein-and ligand-observed 19 F NMR for the screening of compound and fragment libraries for drug discovery (1,2,(15)(16)(17)(18)(19)(20). The method used here, 19 F NMR lineshape analysis, provides both affinities (K D ) and rate constants (k on and k off ).…”

Hydrolysis, polarity, and conformational impact of C-terminal partially fluorinated ethyl esters in peptide models

“…Fluorine-containing groups can be incorporated into biopolymers by various approaches, including those that utilize biosynthesis [ 5 – 7 ], enzymatic conversion [ 8 ], chemical synthesis [ 9 – 10 ], and ligation reactions [ 11 ]. Depending on the research target, 19 F NMR measurements can be used to study ligand–protein [ 12 ] and protein–protein interactions [ 13 ]; membrane proteins [ 14 – 16 ] and membrane-associated peptides [ 17 – 18 ]; equilibria among conformations of RNA [ 19 ], DNA [ 20 ], and peptide nucleic acids (PNA) [ 21 ]; and many others. Particularly recent is the development of peptide-based contrast agents for 19 F imaging [ 22 ].…”

Protein-Engineered Nanoscale Micelles for Dynamic 19F Magnetic Resonance and Therapeutic Drug Delivery

“…45 19 F-incorporated peptides and proteins have also been explored, but to date have only been studied using NMR and phantom MRI. 42,51–55 Solid-phase synthesis of peptides has been used to incorporate fluorinated amino acids; 51 however, this technique can be limited by low yield with increasing peptide chain length. 56 Structural biologists and protein chemists have also biosynthetically incorporated 19 F nuclei into proteins, either site-specifically 52 or residue-specifically, 42,53,57 for use as NMR probes to understand structure-function relationships involved in ligand binding 53 and protein folding.…”

“…19 F MRI and MRS have been used for a variety of biomedical applications including tumor imaging − and measuring tumor cell proliferation, cell tracking, ,, assessing physiological oxygen tension and inflammation, detecting venous thrombosis, and monitoring enzyme activity. − Recently developed 19 F agents take the form of perfluorocarbon nanoemulsions, ,,, perfluoropolyethers, ,,, polymer- , and peptide-based nanoprobes, small molecular weight ligands and substrates, , lipid nanoemulsions, liposomes, metal organic frameworks, nanogels, nanocrystals, and dendrimers ,− Solid-phase synthesis of peptides has been used to incorporate fluorinated amino acids; however, this technique can be limited by low yield with increasing peptide chain length . Structural biologists and protein chemists have also biosynthetically incorporated 19 F nuclei into proteins, either site-specifically or residue-specifically, ,, for use as NMR probes to understand structure–function relationships involved in ligand binding and protein folding …”