Elisabetta Chiarparin

Selective cross-polarization in solution state NMR

In two influential paper, Ernst and coworkers argued that cross-polarization, originally proposed by Hartmann and Hahn, can be very useful in isotropic liqs. to transfer coherence between scalar-coupled nuclei such as protons and carbon-13 (Maudsley, A. A., Muller, L., and Ernst, R. R., 1977; J. Magn. Reson., 28, 463; Muller, L., and Ernst, R. R., 1979, Molec. Phys., 38, 963). Unfortunately, the efficiency of cross-polarization in liqs. tends to be strongly attenuated if the radiofrequency fields are not perfectly homogeneous. In this paper, it is demonstrated by expts. and simulations that imperfections in RF fields have little effect on the efficiency of magnetization transfer, provided that the RF amplitudes are comparable with the magnitudes of the heteronuclear scalar coupling consts. A comparison between selective cross-polarization and selective INEPT shows clearly that cross-polarization is more efficient. Selective cross-polarization does not require any careful calibration and is insensitive to exptl. instabilities. [on SciFinder (R)]

Efficient determination of angles subtended by C(alpha)-H(alpha) and N-H(N) vectors in proteins via dipole-dipole cross-correlation

The angle ΘCαHα,NHN subtended by the internuclear vectors 13Cα-Hα and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13Cα and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle ΘCαHα,NHN. Assuming planarity of the peptide bond, the dihedral angle Ψ (between Cα and C′) can be determined from the knowledge of ΘCαHα,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.

Exploring weak ligand-protein interactions by long-lived NMR states: improved contrast in fragment-based drug screening.

Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long-lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N-terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants KD. This property makes the LLS method particularly attractive for the initial steps of fragment-based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.

Simultaneous determination of y and F angles in proteins from measurements of cross-correlated relaxation effects

AbstractA method is presented to determine both φ and ψ backbone angles in proteins simultaneously. This is achieved by measuring the effect on two-spin coherences of cross-correlation between 15 N-1HN and 13

Normalized one-dimensional NOE measurements in isotopically labeled macromolecules using two-way cross-polarization

{}^{{\text{13}}}{\text{C}}^\alpha{\text{ - }}{}^{\text{1}}{\text{H}}^\alpha

Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

vectors. The cross-correlation rates are obtained by comparing two complementary three-dimensional experiments.

Hydrogen bonds in RNA base pairs investigated by cross-correlated relaxation of multiple-quantum coherence in NMR.

The concepts of drug efficiency (D(eff) ) and Drug Efficiency Index (DEI) have been recently introduced as useful parameters to optimize the absorption, distribution, metabolism, elimination/excretion, and toxicity properties and in vivo efficacy potential of molecules during lead optimization and at pre-clinical stages. The available free drug concentration relative to dose depends on the compounds bioavailability, clearance, and the nonspecific binding to proteins and phospholipids. In this paper, we have demonstrated, using the data of over 115 known drug molecules, that the nonspecific binding can be determined in vitro very efficiently using biomimetic high-performance liquid chromatography measurements. DEI can therefore be estimated from in vitro measurements. The data show that high in vitro DEI values can be associated with lower efficacious dose. A strategy is described of how to use the DEI parameter during early lead optimization. An example is given to highlight the advantages of optimizing on DEI value rather than on potency alone. In order to facilitate the in silico compound design, correlation between in vitro DEI and in silico ligand efficiency parameters such as ligand lipophilicity efficiency has been revealed, suggesting the potential use of these efficiency-related parameters across lead optimization.

Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-Linked Inhibitor of Apoptosis Protein (XIAP).

A novel one-dimensional NOE experiment is presented where a selected proton is excited by two-way heteronuclear cross- polarization between protons and nitrogen-15 or carbon-13. The utility of the method is demonstrated for a sample of 15N labeled human ubiquitin. Inter- and intra-residue NOEs are clearly observed in a very time-effective manner. The signal intensities can be easily normalized.