NMR spectroscopy tools for structure-aided drug design

Biomolecular NMR spectroscopy has expanded dramatically in recent years and is now a powerful tool for the study of structure, dynamics, and interactions of biomolecules. Previous limitations with respect to molecular size are no longer a primary barrier, and systems as large as 900 kDa were recently studied. NMR spectroscopy is already well-established as an efficient method for ligand screening. A number of recently developed techniques show promise as aids in structure-based drug design, for example, in the rapid determination of global protein folds, the structural characterization of ligand-protein complexes, and the derivation of thermodynamic parameters. An advantage of the method is that all these interactions can be studied in solution--time-consuming crystallization is not necessary. This Review focuses on recent developments in NMR spectroscopy and how they might be of value in removing some of the current "bottlenecks" in structure-based drug discovery.     

Rapid screening of binding constants by calibrated competitive 1H NMR spectroscopy

A calibrated competitive NMR method has been developed that is appropriate for the rapid screening of binding constants. This method involves the initial characterisation of a receptor-substrate binding event for which the (1)H NMR spectrum of a given receptor (calibrant) is modified by the substrate of interest at a range of concentrations. For all subsequent "unknown" receptors, K(a) values are then determined by using a competition assay (in the presence of the calibrant receptor) by measuring a single standard (1)H NMR spectrum. This enables a rapid assessment of the recognition properties of a library of potential receptors. Only the calibrant receptor needs to be NMR active, while the library of putative receptors, as well as the substrate, can be NMR silent. This method assumes the formation of complexes of 1:1 stoichiometry. To demonstrate this methodology, the binding of a number of crown ether type compounds with K+ ions has been studied. Comparison of the binding strengths obtained by using this approach with those in the literature shows excellent agreement. A range of new compounds that have recently been synthesised within our group has also been screened in order to illustrate how this approach can rapidly assess binding ability. This method has significance for chemists working in the fields of combinatorial receptor/substrate design and supramolecular chemistry as a means of rapid optimisation of binding strength.     

4,4'-dithiobisdipicolinic acid: a small and convenient lanthanide binding tag for protein NMR spectroscopy

Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structure restraints for studies of proteins by nuclear magnetic resonance spectroscopy. To elicit PCSs, the lanthanide must be attached site-specifically to the target protein. In addition, it needs to be attached rigidly to avoid averaging of the PCSs due to mobility with respect to the protein and it must not interfere with the function of the protein. Here, we present a dipicolinic acid reagent that spontaneously forms a disulfide bond with thiol groups of accessible cysteine residues. A minimal number of rotatable bonds between the cysteine side chain and the tag helps to minimise mobility. Combined with the small size of the tag and quantitative tagging yields, these features make it a highly attractive tool for generating structure restraints by paramagnetic lanthanides.     

Structural and binding study of modified siRNAs with the Argonaute 2 PAZ domain by NMR spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.     

An efficient use of the WATERGATE W5 sequence for observing a ligand binding with a protein receptor

An efficient pulse sequence for observing a ligand binding with a receptor has been developed by incorporating the WATERGATE W5 sequence. In the conventional water ligand observed via gradient spectroscopy (WaterLOGSY) techniques, the water resonance is selectively excited using, e.g. the double-pulsed field gradient spin-echo (DPFGSE) sequence at the initial portion of pulse sequence. In the current version, the modified WATERGATE W5 sequence is incorporated at the initial portion of the pulse sequence, and the resonance at the water frequency can be selectively reserved by the modified WATERGATE W5 sequence. The efficiency of ligand-observed NMR screening techniques has been demonstrated using the human serum albumin (HSA)-tryptophan complex.     

Application of NMR screening techniques for observing ligand binding with a protein receptor

Water ligand observed via gradient spectroscopy (WaterLOGSY), saturation transfer difference and NOE pumping NMR techniques were used to identify ligand binding with a receptor. Although these experiments were originally designed to observe ligands in complexes, their application is limited by the affinity of ligands towards target molecules. Here the improved WaterLOGSY pulse sequence was developed by incorporating the double pulsed field gradient spin-echo and gradient-tailored excitation WATERGATE sequences. The efficiency of these ligand-observed NMR screening techniques was investigated using the ribonuclease T1-inhibitor system.