NMR-based methods and strategies for drug discovery

Nuclear Magnetic Resonance (NMR) spectroscopy has long been a favourite tool of chemists interested in host-guest systems because it permits access to a wealth of information about the molecular recognition reaction. NMR has evolved dramatically in the last 15 years and, in parallel with the development of NMR methods for the determination of protein structure, a variety of tools aimed at detecting protein ligand interactions have been proposed and are being now used both in industrial and academic laboratories as valuable tools for structure-based drug discovery. Very recent developments have considerably increased the fraction of therapeutic targets that can be tackled by NMR and significantly reduced the amount of sample required for analysis; in this tutorial review we outline the essential NMR-based techniques and describe some examples of their implementation as part of drug discovery programmes.     

Solution nuclear magnetic resonance spectroscopy techniques for probing intermolecular interactions

Nuclear magnetic resonance (NMR) spectroscopy in solution has evolved into a powerful technique for structure determination of proteins and nucleic acids. More recently, a number of NMR-based approaches have been developed to monitor and characterize intermolecular interactions. These approaches offer unique advantages over other techniques and find their utility in both structural biology and drug discovery. We will report on basic principles and recent examples of the application of such NMR methodologies to characterize protein-protein interactions and for ligand binding studies and drug discovery.     

NMR study of 1,7-diamino-4-oxyheptane-1,1,7,7-tetraphosphonic acid interaction with samarium(III) cation

1,7-Diamino-4-oxypentane-1,1,7,7-tetraphosphonic acid has been synthesized while searching for potential components for ¹⁵³Sm radiopharmaceuticals. Its interaction with samarium(III) cation has been studied by ¹³C and ³¹P NMR spectroscopy in comparison with 1,5-diamino-3-oxypentane-N,N'-tetra-(methylenephosphonic acid) which is the ligand component of the osteotropic drug Samarium ¹⁵³Sm oxabifore. NMR spectroscopy provides a simple, convenient, and informative method for studying interactions between the ligand and prospective radionuclide cation.     

Automated NMR resonance assignment of large proteins for protein-ligand interaction studies

The detection and structural characterization of protein-ligand interactions by solution NMR is central to functional biology research as well as to drug discovery. Here we present a robust and highly automated procedure for obtaining the resonance assignments necessary for studies of such interactions. The procedure relies on a combination of three automated projection spectroscopy (APSY) experiments, including the new 4D APSY-HNCACB, and the use of fractionally deuterated protein samples. This labeling pattern increases the experimental sensitivity on the one hand, but it leads to peak multiplets on the other hand. The latter complications are however overcome by the geometric APSY analysis of the projection spectra. The three APSY experiments thus provide high precision chemical shift correlations of the backbone and side chain methyl groups, allowing a reliable and robust assignment of the protein by suitable algorithms. The present approach doubles the molecular size limit of APSY-based assignments to 25 kDa, thus providing the basis for efficient characterization of protein-ligand interactions at atomic resolution by NMR, such as structure-based drug design. We show the application to two human proteins with molecular weights of 15 and 22 kDa, respectively, at concentrations of 0.4 mM and discuss the general applicability to studies of protein-protein and protein-nucleic acid complexes.     

NMR-based analysis of protein–ligand interactions

Physiological processes are mainly controlled by intermolecular recognition mechanisms involving protein–protein and protein–ligand (low molecular weight molecules) interactions. One of the most important tools for probing these interactions is high-field solution nuclear magnetic resonance (NMR) through protein-observed and ligand-observed experiments, where the protein receptor or the organic compounds are selectively detected. NMR binding experiments rely on comparison of NMR parameters of the free and bound states of the molecules. Ligand-observed methods are not limited by the protein molecular size and therefore have great applicability for analysing protein–ligand interactions. The use of these NMR techniques has considerably expanded in recent years, both in chemical biology and in drug discovery. We review here three major ligand-observed NMR methods that depend on the nuclear Overhauser effect—transferred nuclear Overhauser effect spectroscopy, saturation transfer difference spectroscopy and water–ligand interactions observed via gradient spectroscopy experiments—with the aim of reporting recent developments and applications for the characterization of protein–ligand complexes, including affinity measurements and structural determination.     

SEAL by NMR: Glyco‐Based Selenium‐Labeled Affinity Ligands Detected by NMR Spectroscopy

We report a method for the screening of interactions between proteins and selenium‐labeled carbohydrate ligands. SEAL by NMR is demonstrated with selenoglycosides binding to lectins where the selenium nucleus serves as an NMR‐active handle and reports on binding through ⁷⁷Se NMR spectroscopy. In terms of overall sensitivity, this nucleus is comparable to ¹³C NMR, while the NMR spectral width is ten times larger, yielding little overlap in ⁷⁷Se NMR spectroscopy, even for similar compounds. The studied ligands are singly selenated bioisosteres of methyl glycosides for which straightforward preparation methods are at hand and libraries can readily be generated. The strength of the approach lies in its simplicity, sensitivity to binding events, the tolerance to additives and the possibility of having several ligands in the assay. This study extends the increasing potential of selenium in structure biology and medicinal chemistry. We anticipate that SEAL by NMR will be a beneficial tool for the development of selenium‐based bioactive compounds, such as glycomimetic drug candidates.     

The process of structure-based drug design

The field of structure-based drug design is a rapidly growing area in which many successes have occurred in recent years. The explosion of genomic, proteomic, and structural information has provided hundreds of new targets and opportunities for future drug lead discovery. This review summarizes the process of structure-based drug design and includes, primarily, the choice of a target, the evaluation of a structure of that target, the pivotal questions to consider in choosing a method for drug lead discovery, and evaluation of the drug leads. Key principles in the field of structure-based drug design will be illustrated through a case study that explores drug design for AmpC beta-lactamase.     

Solid-state NMR studies of pharmaceutical solids in polymer matrices

Biodegradable drug-delivery systems can be formulated to release drug for hours to years and have been used for the controlled release of medications in animals and humans. An important consideration in developing a drug-delivery matrix is knowledge of the long-term stability of the form of the drug and matrix after formulation and any changes that might occur to the drug throughout the delivery process. Solid-state NMR spectroscopy is an effective technique for studying the state of both the drug and the matrix. Two systems that have been studied using solid-state NMR spectroscopy are presented. The first system studied involved bupivacaine, a local anesthetic compound, which was incorporated into microspheres composed of tristearin and encapsulated using a solid protein matrix. Solid-state ¹³C NMR spectroscopy was used to investigate the solid forms of bupivacaine in their bulk form or as incorporated into the tristearin/protein matrix. Bupivacaine free base and bupivacaine-HCl have very different solid-state NMR spectra, indicating that the molecules of these compounds pack in different crystal forms. In the tristearin matrix, the drug form could be determined at levels as low as 1:100 (w/w), and the form of bupivacaine was identified upon loading into the tristearin/protein matrix. In the second case, the possibility of using solid-state ¹³C NMR spectroscopy to characterize biomolecules lyophilized within polymer matrices is evaluated by studying uniformly ¹³C-labeled asparagine (Asn) in 1:250 (w/w) formulations with poly(vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA). This work shows the capability of solid-state NMR spectroscopy to study interactions between the amino acid and the polymer matrix for synthetic peptides and peptidomimetics containing selective ¹³C labeling at the Asn residue.     

High-throughput protein crystallography and drug discovery

Single crystal X-ray diffraction is the technique of choice for studying the interactions of small organic molecules with proteins by determining their three-dimensional structures; however the requirement for highly purified protein and lack of process automation have traditionally limited its use in this field. Despite these shortcomings, the use of crystal structures of therapeutically relevant drug targets in pharmaceutical research has increased significantly over the last decade. The application of structure-based drug design has resulted in several marketed drugs and is now an established discipline in most pharmaceutical companies. Furthermore, the recently published full genome sequences of Homo sapiens and a number of micro-organisms have provided a plethora of new potential drug targets that could be utilised in structure-based drug design programs. In order to take maximum advantage of this explosion of information, techniques have been developed to automate and speed up the various procedures required to obtain protein crystals of suitable quality, to collect and process the raw X-ray diffraction data into usable structural information, and to use three-dimensional protein structure as a basis for drug discovery and lead optimisation.This tutorial review covers the various technologies involved in the process pipeline for high-throughput protein crystallography as it is currently being applied to drug discovery. It is aimed at synthetic and computational chemists, as well as structural biologists, in both academia and industry, who are interested in structure-based drug design.     

Multinuclear Solid State NMR for the Study of Chemistry in Surface Science

This presentation will cover recent progress in the development and application of solid-state NMR techniques for characterizing surface chemistry. Several technological important systems, such as "ship-in-a-bottle" synthesis in zeolite, chemical immobilization on polymer surface and environmental pollutant-soil interactions, have been investigated by multinuclear solid- state NMR spectroscopy. An in situ sold-state NMR technique, GRASSHopper, has been introduced to study heterogeneous catalysis under flowing conditions.     

SOS-NMR: a saturation transfer NMR-based method for determining the structures of protein-ligand complexes

An NMR-based alternative to traditional X-ray crystallography and NMR methods for structure-based drug design is described that enables the structure determination of ligands complexed to virtually any biomolecular target regardless of size, composition, or oligomeric state. The method utilizes saturation transfer difference (STD) NMR spectroscopy performed on a ligand complexed to a series of target samples that have been deuterated everywhere except for specific amino acid types. In this way, the amino acid composition of the ligand-binding site can be defined, and, given the three-dimensional structure of the protein target, the three-dimensional structure of the protein-ligand complex can be determined. Unlike earlier NMR methods for solving the structures of protein-ligand complexes, no protein resonance assignments are necessary. Thus, the approach has broad potential applications--especially in cases where X-ray crystallography and traditional NMR methods have failed to produce structural data. The method is called SOS-NMR for structural information using Overhauser effects and selective labeling and is validated on two protein-ligand complexes: FKBP complexed to 2-(3'-pyridyl)-benzimidazole and MurA complexed to uridine diphosphate N-acetylglucosamine.     

Systems biology and systems chemistry: new directions for drug discovery

Improvements in drug design have historically been centered around structure-based optimization of molecule specificity for a targeted protein, in an effort to reduce unintentional binding to other proteins and off-target effects. Although the "one-to-one" drug design strategy has been successful in impairing function of targets associated with a number of diseases, recent reports of drug promiscuity, which are a potential source of adverse reactions in patients, make a case to refine the drug design strategy such that it includes an awareness of multiple interactions from both ligand and protein perspectives. Polypharmacology and chemical biology studies are amassing interaction data at rapid rates, and the integration of such data into an interpretable model requires zooming our perspective out from the single ligand-target level to the larger network-wide level. We review some of the recent developments in systems-level research for drug design and discovery, and discuss the directions that some drug design efforts are heading toward.     

Current trends in virtual high throughput screening using ligand-based and structure-based methods

High throughput in silico methods have offered the tantalizing potential to drastically accelerate the drug discovery process. Yet despite significant efforts expended by academia, national labs and industry over the years, many of these methods have not lived up to their initial promise of reducing the time and costs associated with the drug discovery enterprise, a process that can typically take over a decade and cost hundreds of millions of dollars from conception to final approval and marketing of a drug. Nevertheless structure-based modeling has become a mainstay of computational biology and medicinal chemistry, helping to leverage our knowledge of the biological target and the chemistry of protein-ligand interactions. While ligand-based methods utilize the chemistry of molecules that are known to bind to the biological target, structure-based drug design methods rely on knowledge of the three-dimensional structure of the target, as obtained through crystallographic, spectroscopic or bioinformatics techniques. Here we review recent developments in the methodology and applications of structure-based and ligand-based methods and target-based chemogenomics in Virtual High Throughput Screening (VHTS), highlighting some case studies of recent applications, as well as current research in further development of these methods. The limitations of these approaches will also be discussed, to give the reader an indication of what might be expected in years to come.     

Probing the Interactions of Porphyrins with Macromolecules Using NMR Spectroscopy Techniques

Porphyrinic compounds are widespread in nature and play key roles in biological processes such as oxygen transport in blood, enzymatic redox reactions or photosynthesis. In addition, both naturally derived as well as synthetic porphyrinic compounds are extensively explored for biomedical and technical applications such as photodynamic therapy (PDT) or photovoltaic systems, respectively. Their unique electronic structures and photophysical properties make this class of compounds so interesting for the multiple functions encountered. It is therefore not surprising that optical methods are typically the prevalent analytical tool applied in characterization and processes involving porphyrinic compounds. However, a wealth of complementary information can be obtained from NMR spectroscopic techniques. Based on the advantage of providing structural and dynamic information with atomic resolution simultaneously, NMR spectroscopy is a powerful method for studying molecular interactions between porphyrinic compounds and macromolecules. Such interactions are of special interest in medical applications of porphyrinic photosensitizers that are mostly combined with macromolecular carrier systems. The macromolecular surrounding typically stabilizes the encapsulated drug and may also modify its physical properties. Moreover, the interaction with macromolecular physiological components needs to be explored to understand and control mechanisms of action and therapeutic efficacy. This review focuses on such non-covalent interactions of porphyrinic drugs with synthetic polymers as well as with biomolecules such as phospholipids or proteins. A brief introduction into various NMR spectroscopic techniques is given including chemical shift perturbation methods, NOE enhancement spectroscopy, relaxation time measurements and diffusion-ordered spectroscopy. How these NMR tools are used to address porphyrin–macromolecule interactions with respect to their function in biomedical applications is the central point of the current review.     

Recent advances in protein NMR spectroscopy and their implications in protein therapeutics research

Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are the two main methods for protein three-dimensional structure determination at atomic resolution. According to the protein structures deposited in the Protein Data Bank, X-ray crystallography has become the dominant method for structure determination, particularly for large proteins and complexes. However, with the developments of isotope labeling, increase of magnetic field strength, common use of a cryogenic probe, and ingenious pulse sequence design, the applications of NMR spectroscopy have expanded in biological research, especially in characterizing protein dynamics, sparsely populated transient structures, weak protein interactions, and proteins in living cells at atomic resolution, which is difficult if not impossible by other biophysical methods. Although great advances have been made in protein NMR spectroscopy, its applications in protein therapeutics, which represents the fastest growing segment of the pharmaceutical industry, are still limited. Here we review the recent advances in the use of NMR spectroscopy in studies of large proteins or complexes, posttranslation modifications, weak interactions, and aggregation, and in-cell NMR spectroscopy. The potential applications of NMR spectroscopy in protein therapeutic assays are discussed.     

Conotoxins and structural biology: a prospective paradigm for drug discovery

Understanding the interactions between activating or antagonizing ligands and their cognate receptors at a molecular level offers promise for the development of pharmacological therapeutics for CNS disorders. The discovery of novel molecules that are capable of discriminating between the varied molecular subunits or isoforms of ion channels should provide a more detailed understanding of the pathophysiology of many CNS disorders. Abundant natural sources of pharmacologically active agents that demonstrate this refined selectivity and specificity are found in the animal toxins of venomous species including: snakes, spiders and the marine snail of the genus Conus. The uniquely fascinating combinatorial ability of the marine snail, genus Conus to modify the pharmacological properties of these neurotoxins or conopeptides within its venom is depicted throughout this review. The myriad of posttranslational modifications and disulfide bonded architectures that have been identified in the conopeptides, are described with an emphasis on the unique pharmacological properties and receptor target specificities that have been ascribed to each of these modifications. The ability of NMR spectroscopy to provide three-dimensional structural information within the interaction interface for both the ligand and target protein following complex formation and its application to conopeptide drug discovery are discussed. Similarly, the strength of merging NMR spectroscopy data with ab initio "restrained soft-docking" for rational pharmacophore design and the identification of lead compounds from in silico library screens will also be discussed. The initial phases of this stratagem are illustrated using two toxin antagonists and the recently determined structure of the KcsA potassium channel. These data exemplify the utility of this approach in elucidating important molecular interfaces of specific toxin-receptor/ion channel complexes, which can be further exploited in drug discovery initiatives.     

Spatial encoding strategies for ultrafast multidimensional nuclear magnetic resonance

Multidimensional spectroscopy plays a central role in contemporary magnetic resonance. A general feature of multidimensional NMR is its inherent multiscan nature, stemming from the methodology's reliance on a series of independent acquisitions to sample the spins' evolutions throughout the indirect time domains. Contrasting this traditional feature, an acquisition scheme has recently been reported that enables the collection of complete of multidimensional NMR data sets within one single scan. Provided that the signals to be observed are sufficiently strong, this new "ultrafast" protocol can thus shorten the acquisition times of multidimensional NMR experiments by several orders of magnitude. This new methodology operates by departing from temporal encoding principles used since the advent of Fourier-transform NMR, replacing them with a spatial encoding of the spin interactions. Spatial encoding operates in turn on the basis of novel radiofrequency irradiation and magnetic field gradient waveform manipulations, designed so as to impart on the sample a coherent spin magnetization pattern that reflects the internal interactions to be measured. Given the central role played by this new kind of spectroscopic-oriented manipulations in ultrafast NMR, we devote this review to surveying different variants that have hitherto been proposed for their implementation. These include both discrete and continuous versions, real- and constant-time implementations, as well as amplitude- and phase-modulated alternatives. The principles underlying these various spatial encoding approaches are treated, their operation is graphically illustrated as well as formally derived within suitable theoretical frameworks, and an in-depth comparison of their line shape characteristics is discussed.     

NMR: A good tool to ascertain σ-silane or σ-borane formulations?

NMR has played a major role in the characterization of dihydrogen complexes and a number of polyhydrides have been reformulated as dihydrogen complexes on the basis of NMR data. If dihydrogen complexes remain the most widely studied class of σ-complexes, silane compounds are also well recognized as an important family of σ-complexes and more recently a few σ-borane compounds have been isolated. One important problem is the discrimination between a σ-formulation and the corresponding hydrido(silyl) or hydrido(boryl) oxidative addition product. In this review we will discuss key literature data on silane and borane complexes to illustrate the benefit gained by using multinuclear NMR spectroscopy to better define the structures and bonding modes. Our goal is also to help the reader to appreciate the limits of the method and to provide valuable insights into the problem of secondary interactions.     

Structure-based drug design targeting biosynthesis of isoprenoids: a crystallographic state of the art of the involved enzymes

Biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), from three acetyl CoA moieties through mevalonate was studied extensively in the 1950s. For several decades, the mevalonate paradigm reigned supreme and a mevalonate origin was attributed to a growing number of natural products, in many cases erroneously. Besides this biosynthetic pathway, the existence of a second one leading to IPP and DMAPP through 1-deoxy-D-xylulose 5-phosphate and 2C-methyl-D-erythritol 4-phosphate was discovered more recently in plants and some eubacteria. This pathway is widely distributed in the bacterial kingdom including major human pathogens, such as Mycobacterium tuberculosis or Helicobacter pylori and is also essential in the malaria vector Plasmodium falciparum. During the last few years, the genes, enzymes, intermediates and mechanisms of the biosynthetic route have been elucidated by a combination of methods including comparative genomics, enzymology, advanced NMR technology and crystallography. The present crystallographic review of enzymes involved in isoprenoid biosynthesis will be useful for understanding the various catalytic mechanisms and could potentially help for structure-based drug design.     

Exploring experimental sources of multiple protein conformations in structure-based drug design

Receptor flexibility must be incorporated into structure-based drug design in order to portray a more accurate representation of a protein in solution. Our approach is to generate pharmacophore models based on multiple conformations of a protein and is very similar to solvent mapping of hot spots. Previously, we had success using computer-generated conformations of apo human immunodeficiency virus-1 protease (HIV-1p). Here, we examine the use of an NMR ensemble versus a collection of crystal structures, and we compare back to our previous study based on computer-generated conformations. To our knowledge, this is the first direct comparison of an NMR ensemble and a collection of crystal structures to incorporate protein flexibility in structure-based drug design. To provide an accurate comparison between the experimental sources, we used bound structures for our multiple protein structure (MPS) pharmacophore models. The models from an NMR ensemble and a collection of crystal structures were both able to discriminate known HIV-1p inhibitors from decoy molecules and displayed superior performance over models created from single conformations of the protein. Although the active-site conformations were already predefined by bound ligands, the use of MPS allows us to overcome the cross-docking problem and generate a model that does not simply reproduce the chemical characteristics of a specific ligand class. We show that there is more structural variation between 28 structures in an NMR ensemble than 90 crystal structures bound to a variety of ligands. MPS models from both sources performed well, but the model determined using the NMR ensemble appeared to be the most general yet accurate representation of the active site. This work encourages the use of NMR models in structure-based design.