Claudio Dalvit

WaterLOGSY as a method for primary NMR screening: Practical aspects and range of applicability

WaterLOGSY represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. Several relay pathways are used constructively in the experiment for transferring bulk water magnetization to the ligand. The method is particularly useful for the identification of novel scaffolds of micromolar affinity that can be then optimized using directed screening, combinatorial chemistry, medicinal chemistry and structure-based drug design. The practical aspects and range of applicability of the WaterLOGSY experiment are analyzed in detail here. Competition binding and titration WaterLOGSY permit, after proper correction, the evaluation of the dissociation binding constant. The high sensitivity of the technique in combination with the easy deconvolution of the mixtures for the identification of the active components, significantly reduces the amount of material and time needed for the NMR screening process.

Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water

A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described. The method exploits efficiently the large reservoir of H2O magnetization. The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which constitutes an important requirement for high throughput screening by NMR of large libraries of compounds. Application of the method to a compound mixture against the cyclin-dependent kinase 2 (cdk2) protein is presented.

Perspectives on NMR in drug discovery: a technique comes of age

In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

NMR methods in fragment screening: theory and a comparison with other biophysical techniques

Nuclear magnetic resonance, surface plasmon resonance and fluorescence spectroscopy (particularly fluorescence anisotropy and fluorescence lifetime) are techniques often applied to fragment screening. These methodologies are analyzed on the basis of their performance, strengths, limitations and pitfalls. NMR-based screening, despite its low intrinsic sensitivity, offers the largest dynamic range and is capable of capturing very weak interactions. Theoretical simulation demonstrates the power of some NMR experiments, in particular those with fluorine observation, in detecting protein interactions for fragments tested at concentrations orders of magnitude lower than their dissociation binding constants. This apparently counterintuitive finding enables the identification of hits that are insoluble at the concentrations required for detection by other biophysical techniques. However it is evident that several techniques should be applied and the data analyzed on the basis of their complementarities.

Inhibition of protein–protein interactions: The discovery of druglike β‐catenin inhibitors by combining virtual and biophysical screening

The interaction between β‐catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 Å2), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain “hot spots,” small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4‐binding surface on β‐catenin contains a well‐defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to β‐catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4‐competitive compounds with the tightest binder having a KD of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein–protein interactions. Proteins 2006.

Efficient multiple-solvent suppression for the study of the interactions of organic solvents with biomolecules

A modification of the excitation sculpting sequence [Hwang, T.-L. and Shaka, A.J. (1995) J. Magn. Reson., A112, 275–279] for achieving efficient multiple-solvents suppression in 1D and 2D 1H NMR experiments is presented. Implementation of this scheme in the ePHOGSY experiment allows rapid and sensitive detection of weak interactions between organic solvents or small molecules and biomolecules. Application of the techniques to the peptide Sandostatin® dissolved in H2O and DMSO is demonstrated.

Design and NMR-Based Screening of LEF, a Library of Chemical Fragments with Different Local Environment of Fluorine

A novel strategy for the design of a fluorinated fragment library that takes into account the local environment of fluorine is described. The procedure, based on a fluorine fingerprints descriptor, and the criteria used in the design, selection, and construction of the library are presented. The library, named LEF (Local Environment of Fluorine), combined with (19)F NMR ligand-based screening experiments represents an efficient and sensitive approach for the initial fragment identification within a fragment-based drug discovery project and for probing the presence of fluorophilic protein environments. Proper setup of the method, according to described theoretical simulations, allows the detection of very weak-affinity ligands and the detection of multiple ligands present within the same tested mixture, thus capturing all the potential fragments interacting with the receptor. These NMR hits are then used in the FAXS experiments for the fragment optimization process and for the follow-up screening aimed at identifying other chemical scaffolds relevant for the binding to the receptor.

Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures.

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.

Fluorine–Protein Interactions and 19F NMR Isotropic Chemical Shifts: An Empirical Correlation with Implications for Drug Design

An empirical correlation between the fluorine isotropic chemical shifts, measured by 19F NMR spectroscopy, and the type of fluorine–protein interactions observed in crystal structures is presented. The CF, CF2, and CF3 groups present in fluorinated ligands found in the Protein Data Bank were classified according to their 19F NMR chemical shifts and their close intermolecular contacts with the protein atoms. Shielded fluorine atoms, i.e., those with increased electron density, are observed primarily in close contact to hydrogen bond donors within the protein structure, suggesting the possibility of intermolecular hydrogen bond formation. Deshielded fluorines are predominantly found in close contact with hydrophobic side chains and with the carbon of carbonyl groups of the protein backbone. Correlation between the 19F NMR chemical shift and hydrogen bond distance, both derived experimentally and computed through quantum chemical methods, is also presented. The proposed “rule of shielding” provides some insight into and guidelines for the judicious selection of appropriate fluorinated moieties to be inserted into a molecule for making the most favorable interactions with the receptor.

Proton chemical shift anisotropy: Detection of cross-correlation with dipole-dipole interactions by double-quantum filtered two-dimensional NMR exchange spectroscopy

Abstract Cross-correlation between proton chemical shift anisotropy and proton-proton dipolar interactions is shown to be an important relaxation mechanism. Cross-correlation leads to a partial conversion of Zeeman order into longitudinal two-spin order. The build-up of longitudinal two-spin order can be observed selectively by means of a novel experiment that combines double-quantum filtration with two-dimensional nuclear Overhauser effect spectroscopy (DQF-NOESY).