Biomolecular NMR: recent advances in liquids, solids and screening.

The past year has witnessed significant advances in NMR analysis of bio-macromolecules from a broad array of disciplines. First, great progress in the development of methods for measuring residual dipolar couplings in nematic media promises to increase both the size of systems to be studied and the accuracy with which structures can be determined. Second, the ability of solid-state NMR to provide structural information on biological systems is undergoing rapid expansion as a result of recent developments. The structural details that can be derived for biomolecules in the liquid and solid states can be used for the rational design of high-affinity ligands. Such studies are now complemented by NMR screening of synthetic and natural molecular libraries. Several NMR screening protocols have been designed that employ both rational and random elements.     

Exchange-transferred NOE spectroscopy and bound ligand structure determination

The exchange-transferred nuclear Overhauser effect of NMR spectroscopy provides information on small-molecule ligands in association with high-molecular-weight proteins or nucleic acids, or with biomolecular assemblies such as membranes. The method has proved particularly useful for the structural analysis of proton-rich, flexible ligands and for screening mixtures of ligands for binding activity. Recent analysis has established the accuracy of bound peptide structures determined from transferred nuclear Overhauser effect data and that intermolecular spin diffusion effects do not diminish the reliability of the structural result. New applications of the method involve systems of greater complexity, such as membrane-bound receptors and ribosomes. In addition, new experiments have been developed that exploit the transfer of other types of NMR signal (saturation, cross-correlation, dipolar coupling) to obtain structural information.     

Determining the optimal size of small molecule mixtures for high throughput NMR screening

High-throughput screening (HTS) using NMR spectroscopy has become a common component of the drug discovery effort and is widely used throughout the pharmaceutical industry. NMR provides additional information about the nature of small molecule-protein interactions compared to traditional HTS methods. In order to achieve comparable efficiency, small molecules are often screened as mixtures in NMR-based assays. Nevertheless, an analysis of the efficiency of mixtures and a corresponding determination of the optimum mixture size (OMS) that minimizes the amount of material and instrumentation time required for an NMR screen has been lacking. A model for calculating OMS based on the application of the hypergeometric distribution function to determine the probability of a hit for various mixture sizes and hit rates is presented. An alternative method for the deconvolution of large screening mixtures is also discussed. These methods have been applied in a high-throughput NMR screening assay using a small, directed library.     

Molecular biodiversity and recent analytical developments: A marriage of convenience

Nature has developed an outstanding bio- and molecular diversity as a result of billion years of evolution resulting in a tremendous number of secondary metabolites. Among them a small part has been so far explored for discovery of lead compounds. The lead discovery from natural sources is a technological challenge for the pharmaceutical industry. The bio- and molecular diversity in plants, animals and microorganisms, as sources for new leads, and the remarkable recent developments in NMR, mass spectrometry coupled with advanced separation techniques (LC and GC), high throughput screening, and structure-based virtual screening are discussed in this article.     

NMR screening in drug discovery

NMR methods in drug discovery have traditionally been used to obtain structural information for drug targets or target-ligand complexes. Recently, it has been shown that NMR may be used as an alternative approach for identification of ligands that bind to protein drug targets, shifting the emphasis of many NMR laboratories towards screening and design of potential drug molecules, rather than structural characterization.     

Cracking the molecular weight barrier: fragment screening of an aminotransferase using an NMR-based functional assay

NMR-based screening of protein targets has become a well established part of the drug discovery process especially with respect to fragments. However, as target size increases the two-dimensional spectra typically used for such screening become more crowded due to the increased number of signals, and the individual signals broaden due to the decreased rotational correlation time of the protein. Here we present an NMR-based functional assay for the branched-chain aminotransferase BCATc, a dimer with a total molecular weight of 88 kDa, which overcomes the limitations of the typical protein-based NMR screening method. BCATc is involved in glutamate production in the brain and is a therapeutic target for neuronal disorders involving a glutamatergic mechanism. Several fragments which inhibit BCATc were discovered using this assay and these may serve as novel cores for the development of potent BCATc inhibitors.     

Small angle X-ray scattering as a complementary tool for high-throughput structural studies

Structural crystallography and nuclear magnetic resonance (NMR) spectroscopy are the predominant techniques for understanding the biological world on a molecular level. Crystallography is constrained by the ability to form a crystal that diffracts well and NMR is constrained to smaller proteins. Although powerful techniques, they leave many soluble, purified structurally uncharacterized protein samples. Small angle X-ray scattering (SAXS) is a solution technique that provides data on the size and multiple conformations of a sample, and can be used to reconstruct a low-resolution molecular envelope of a macromolecule. In this study, SAXS has been used in a high-throughput manner on a subset of 28 proteins, where structural information is available from crystallographic and/or NMR techniques. These crystallographic and NMR structures were used to validate the accuracy of molecular envelopes reconstructed from SAXS data on a statistical level, to compare and highlight complementary structural information that SAXS provides, and to leverage biological information derived by crystallographers and spectroscopists from their structures. All the ab initio molecular envelopes calculated from the SAXS data agree well with the available structural information. SAXS is a powerful albeit low-resolution technique that can provide additional structural information in a high-throughput and complementary manner to improve the functional interpretation of high-resolution structures.     

Using NMR for ligand discovery and optimization

Several recent technology-driven advances in the area of NMR have rekindled an interest in the application of the technology to problems in drug discovery and development. A unique aspect of NMR is that it has applicability in broadly different areas of the drug discovery and optimization processes. NMR techniques for screening aimed at the discovery of novel ligands or low molecular weight structures for fragment-based build up procedures are being applied commonly in the industry. Application of NMR in structure-guided drug design and metabonomics are also becoming routine. We present an overview of some of the most recent NMR developments in these areas.     

The use of solid-state NMR and X-ray crystallography as complementary tools for studying molecular recognition

Solid-state NMR is rapidly becoming available as a routine technique for studying the structure of crystalline or noncrystalline solids. This technique has an advantage over crystallography in that single crystals are not necessary, but it has the disadvantage that the information obtained does not produce a direct picture of the molecule and its environment. On the other hand, solid-state NMR can be done on mixtures, and it gives information about phase distribution in a manner similar to that of X-ray powder pattern analysis.Crystallographic effects such as polymorphism, multiple molecules per asymmetric unit, disorder and salvation can frequently be detected using NMR. Sometimes molecular point group symmetry can also be deduced based on the number of independent nuclei that are detected. The NMR method is sensitive to changes in the electronic structure of a molecule as sensed by the nuclei, and the effects are measured as changes in the isotropic chemical shift of individual nuclei.In this paper, we will give examples of the combined use of X-ray crystallography and ¹³CP/MAS (cross polarization/magic angle spinning) NMR for studying hostguest materials and cocrystals. We have learned how to use NMR to tell us about keto/enol composition in the solid state, to detect the presence of trapped solvent molecules, to detect hydrogen-bond formation and to evaluate molecular conformation and unusual packing pattern effects. We will also present a brief background of the ¹³CP/MAS NMR technique and three case studies in which solid-state NMR and X-ray crystallography are used together to understand materials' structures and properties     

Fragment-based lead discovery: a chemical update

Fragment-based lead discovery constructs drug leads from small molecular fragments. In theory, this is a highly efficient method for drug discovery, and the technique has become enormously popular in the past few years. In this review, I describe how a variety of approaches in fragment-based lead discovery--including NMR, X-ray crystallography, mass spectrometry, functional screening, and in silico screening--have produced drug leads. Although the examples show that the technique can reliably generate potent molecules, there is still much work to be done to maintain the efficiency of molecules' binding affinities as fragments are linked, expanded, and otherwise improved.     

Probing protein dynamics by nuclear magnetic resonance

Proteins are dynamic molecules that often undergo conformational changes while performing their specific functions, such as target recognition, ligand binding and catalysis. NMR spectroscopy is uniquely suited to study protein dynamics, because site-specific information can be obtained for motions that span a broad range of time scales. The information obtained from NMR dynamics experiments has provided insights into specific structural changes or conformational energetics associated with molecular function. In the last decade, a number of new advancements in NMR methodologies have further extended our ability to characterize protein dynamics. Here, we present an overview of current NMR technology that is used to monitor the dynamic properties of proteins.     

Finding and Characterizing the Complexes of Drug Like Molecules with Quadruplex DNA: Combined Use of an Enhanced Hydroxyl Radical Cleavage Protocol and NMR

Structural information on the complexes of drug like molecules with quadruplex DNAs can aid the development of therapeutics and research tools that selectively target specific quadruplex DNAs. Screening can identify candidate molecules that require additional evaluation. An enhanced hydroxyl radical cleavage protocol is demonstrated that can efficiently provide structural information on the complexes of the candidate molecules with quadruplex DNA. NMR methods have been used to offer additional structural information about the complexes as well as validate the results of the hydroxyl radical approach. This multi-step protocol has been demonstrated on complexes of the chair type quadruplex formed by the thrombin binding aptamer, d(GGTTGGTGTGGTTGG). The hydroxyl radical results indicate that NSC 176319, Cain’s quinolinium that was found by screening, exhibits selective binding to the two TT loops. The NMR results are consistent with selective disruption of the hydrogen bonding between T4 and T13 as well as unstacking of these residues from the bottom quartet. Thus, the combination of screening, hydroxyl radical footprinting and NMR can find new molecules that selectively bind to quadruplex DNAs as well as provide structural information about their complexes.     

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.     

Computer-Aided Design of Fragment Mixtures for NMR-Based Screening

Fragment-based drug discovery is widely applied both in industrial and in academic screening programs. Several screening techniques rely on NMR to detect binding of a fragment to a target. NMR-based methods are among the most sensitive techniques and have the further advantage of yielding a low rate of false positives and negatives. However, NMR is intrinsically slower than other screening techniques; thus, to increase throughput in NMR-based screening, researchers often assay mixtures of fragments, rather than single fragments. Herein we present a fast and straightforward computer-aided method to design mixtures of fragments taken from a library that have minimized NMR signal overlap. This approach enables direct identification of one or several active fragments without the need for deconvolution. Our approach entails encoding of NMR spectra into a computer-readable format that we call a fingerprint, and minimizing the global signal overlap through a Monte Carlo algorithm. The scoring function used favors a homogenous distribution of the global signal overlap. The method does not require additional experimental work: the only data required are NMR spectra, which are generally recorded for each compound as a quality control measure before its insertion into the library.     

STD-NMR: application to transient interactions between biomolecules—a quantitative approach

Saturation transfer difference NMR (STD NMR) spectroscopy is one of the most powerful NMR techniques for detection and characterization of transient (fast) receptor–ligand interactions in solution. By observing the signals of a small molecule (ligand) with spectroscopic properties suitable for high-resolution studies, irrespective of receptor size, STD NMR enables quantitative structural and affinity information to be obtained about the molecular recognition process under study. Approximately one decade after its introduction, the technique has reached maturity, and is highly robust and useful. The objective of this article is to review the current status of this powerful technique, with particular emphasis on quantitative applications, within the framework of the (bio-)chemistry of molecular recognition.