High-field solution NMR spectroscopy as a tool for assessing protein interactions with small molecule ligands

Abstract

The ability of a small molecule to bind and modify the activity of a protein target at a specific site greatly impacts the success of drugs in the pharmaceutical industry. One of the most important tools for evaluating these interactions has been high-field solution nuclear magnetic resonance (NMR) because of its unique ability to examine even weak protein-drug interactions at high resolution. NMR can be used to evaluate the structural, thermodynamic and kinetic aspects of a binding reaction. The basis of NMR screening experiments is that binding causes a perturbation in the physical properties of both molecules. Unique properties of small and macromolecules allow selective detection of either the protein target or ligand, even in a mixture of compounds. This review outlines current methodologies for assessing protein-ligand interactions from the perspectives of the protein target and ligand and delineates the fundamental principles for understanding NMR approaches in drug research. Advances in instrumentation, pulse sequences, isotopic labeling strategies, and the development of competition experiments support the study of higher molecular weight protein targets, facilitate higher-throughput and expand the range of binding affinities that can be evaluated, enhancing the utility of NMR for identifying and characterizing potential therapeutics to druggable protein targets.

Figure 1. Exchange regimens observed with NMR

A) Two resonances appear because the observed nucleus exchanges slower than the NMR scale. B) One resonance is observed because the observed nucleus exchanges faster than the NMR time scale. The peak represents a weighted averaged of the two exchanging environments.

Figure 2. Multidimensional NMR

A) ¹H NMR spectrum of para-Nitrophenyl phosphate in 50mM HEPES, pH=7.5. The two resonances at approximately 7 and 8 ppm correspond to the two aromatic protons. The water peak resonates at 4.703 ppm and the remaining peaks below 4 ppm correspond to HEPES. B) ¹H NMR spectrum of phosphatase of regenerating liver-1 (PRL-1), a protein target implicated in cancer metastasis. The peaks above 6.0 ppm are the protein’s backbone amides and side chains. The water peak resonates at 4.703 ppm and the remaining signals represent the protein’s aliphatic protons. The signals are too numerous and overlapped to make any specific assignments. C) ¹H-¹⁵N HSQC of PRL-1. Each peak represents a single NH group in the protein, including backbone and side-chain amides. Using three-dimensional correlation experiments, each peak can be assigned to a given amino acid in the protein sequence to yield structural information that can guide structure-based inhibitor design to the protein target.

Figure 3. Protein backbone

Each amino acid is connected via a peptide bond between the carbonyl carbon of the first amino acid and the amide of the next amino acid, which is highlighted by the dashed square box. The ¹H-¹⁵N HSQC experiment detects protons directly coupled to nitrogen (designated by the circle), and the resulting spectrum contains one peak for every amino acid in the protein. The HNCO experiment (see Isotopic Labeling Techniques under Advances in NMR) correlates the carbonyl carbon of amino acid (i) to the amide proton of the proceeding amino acid (i-1).

Figure 4. Δδ vs [ligand]

Changes in chemical shift are monitored over a series of ligand concentrations and plotted to determine the dissociation constant.

Figure 5. SAR by NMR

Binding of a first ligand is determined by detecting changes in the 1H-15N HSQC of the protein target upon titration of a mixture of ligands. After a binding ligand is identified and optimized, a mixture is screened for a second low affinity binder to an adjacent site on the protein target. Once optimized, the ligands are synthetically linked together to create one high-affinity lead drug compound.

Figure 6. Nuclear relaxation

A) Longitudinal Relaxation. After an rf-pulse of energy is applied to the nuclei in solution, the atoms must relax back to their equilibrium position in line with the z-axis by releasing energy in the form of heat to its surrounding. B) Transverse Relaxation. After an rf-pulse of energy is applied to the nuclei in solution, the signals corresponding to the different nuclei fan out away from the x-axis at different rates due to chemical shift dispersion and diffusion and the transfer of energy from one spin to another spin, which proceeds until the net magnetization becomes zero. For small molecules the rate of transverse relaxation often equals the rate of longitudinal relaxation because there lacks sufficient interaction between the atoms to permit the transfer of excitation energy. For larger molecules, especially proteins, the rate of transverse relaxation is generally faster than longitudinal relaxation because the transfer of energy between spins occurs much more quickly and efficiently than the transfer of energy to the surroundings.

Figure 7. Diffusion Ordered SpectroscopY (DOSY)

A one-dimensional NMR experiment is extrapolated into a pseudo second dimension that encodes the diffusion coefficient of the molecule. In the above figure, the diffusion coefficient is used to distinguish free sucrose from sucrose bound to phospholipid vesicles. Reproduced with permission from J. Am. Chem. Soc. 1992, 114, 3139–3134. © 1992 American Chemical Society.

Figure 8. Difference spectroscopy with relaxation filtering

A) Relaxation-edited ¹H NMR spectrum of a mixture of nine compounds in the absence of protein. B) Relaxation-edited ¹H NMR spectrum of a mixture of nine compounds in the presence of protein after correcting for residual protein signals by subtracting a spectrum of protein alone. C) Difference spectrum obtained by subtracting B from A, which reveals only signal for the bound ligand. D) Reference spectrum of the binding ligand alone. E) Difference spectrum obtained by screening a mixture of non-binding ligands. This served as a negative control. Reproduced with permission from J. Am. Chem. Soc. 1997, 194, 12257–12261. © 1992 American Chemical Society.