Current Topics in Medicinal Chemistry - Volume 11, Issue 1, 2011

The intense activity in nuclear magnetic resonance (NMR) spectroscopy applied to biomedical and, in particular, pharmaceutical research represents, both, an opportunity and a challenge when reviewing the published work for an issue devoted to NMR in drug discovery. The wide success of NMR is partly due to the excellent sensitivity to chemical structure and molecular dynamics coupled with the non-invasive and non-destructive nature of the technique. These factors have contributed to the enormous advance that it has experienced since its beginnings, more than half-a-century ago. High resolution (HR) NMR spectroscopy allows the simultaneous detection and characterization of a large number of endogenous (metabolites) and exogenous (i.e., drugs) compounds in biological systems. Thus, NMR has a great potential in Medicinal Chemistry for monitoring the response of cells and living organisms to therapeutic agents. In this issue of Current Topics in Medicinal Chemistry, we have attempted to provide a fresh look at this field with focus on most recent work and a projection to the future. A total of seven papers are included. The first contribution by Moestue et al. describes the fundamentals of proton HR magic angle spinning (MAS) NMR spectroscopy of tissues and its application to the characterization of cancer metabolism and response to therapeutic intervention. In the second paper (Garcia et al.), the application of HR NMR to the investigation of drugs effects on cells and tissues and how this technique could be used to assist in the design and test of the bioactivity of new compounds are reviewed. The next two papers deal with the application on NMR to investigate interactions between small molecule ligands and macromolecular receptors for compound library screening and hit validation (Campos-Oliva), and in-cell analysis of structures, interactions, dynamics and stability of proteins (Ohno et al.). Most recent work describing in situ structural characterization of organic compounds attached to solid supports with HR MAS NMR is reviewed by Espinosa. The full potential of NMR spectroscopic techniques as valuable tools to assist in the translation of drug research results from laboratory to clinical practice is shown in the contribution by Ross et al. where the possibilities of in vivo spectroscopic magnetic resonance imaging (MRI) to assess brain metabolism in healthy and disease conditions are reviewed. The last contribution by Pacheco et al. reviews the basics of environmental contrast and the availability of MRI and magnetic resonance spectroscopy (MRS) contrast enhancing agents to visualize it. The potential applications of these agents in multimodal and molecular imaging approaches are discussed. The work reviewed here illustrates the high versatility of NMR. This would certainly contribute to maintain the NMR-based techniques alone and/or in combination with other analytical techniques, such as liquid chromatography and mass spectrometry, as essential tools in drug discovery and development. Moreover, the vibrant activity in NMR research would likely push the frontier of current technique limitations further (i.e., enhanced molecular detection with methods based on dynamic nuclear polarization) and provide new opportunities for exploring in situ the chemistry of living systems.

One of the central hallmarks of cancer is the rapid and infinite cellular proliferation. In order to cope with increased requirement for building blocks and energy, cancer cells develop abnormal metabolic properties. Detailed assessment of cancer cell metabolism can provide biological information for use in both drug discovery and development of personalized cancer therapy. Analysis of intact tissue using high resolution magic angle spinning (HR MAS) magnetic resonance spectroscopy (MRS) gives qualitative and quantitative metabolite measures with minimal sample preparation. Multivariate statistical methods are important tools for analysis of complex MR data and have in recent years been used for analysis of HR MAS data from intact tissue. HR MAS analysis of intact tissue allows combination of metabolomic data with genomic or proteomic data, and can therefore be used both for exploring the molecular biology of cancer and for clinical improvements in cancer diagnostics, prognostics and treatment planning. In this review, the basic concepts of HR MAS are presented, and its use in characterisation of cancer metabolism is discussed with specific focus on selected pathways such as choline metabolism and glycolysis. The use of HR MAS in analysis of amino acids and lipid metabolism in cancer is also reviewed. Finally, the expected role of HR MAS in metabolic characterisation in the near future is discussed.

In this article, the application of high resolution NMR spectroscopy to study the effect of therapeutic compounds on cells, tissues and organisms is reviewed. To illustrate how these NMR methods can provide useful information for a better understanding about the mechanism of action of drugs and their interactions with metabolic pathways, the emphasis is placed on most recent work about drug therapeutic intervention in biological models of diseases and in humans. Specifically, the application of NMR spectroscopy to investigate the effect of drugs on the treatment of neurological disorders, cancer, infectious diseases and diabetes is illustrated. In addition, NMR studies of drug-induced toxicity and multinuclear NMR for monitoring drug delivery and catabolism are described. Current progress in NMR instrumentation and methods will continue to improve the sensitivity and maintain this very versatile technique as powerful tool for research in the field of medicinal chemistry.

Over the past three decades nuclear magnetic resonance spectroscopy has been developed into a mature technique for the characterization of interactions of small molecule ligands with their corresponding protein and nucleic acid receptors. In fact, a significant number of industrial and academic laboratories employ NMR for screening small molecule compound collections for binding to defined macromolecular targets, thus potentially providing initial, low affinity hits for a fragment-based approach in the drug discovery process. NMR is also applied to interrogate hits obtained by high throughput screening using biochemical assays and by virtual screening methods, for their ability to physically interact with the target receptor. In favorable cases a variety of NMR-based methods can also provide essential information to validate the hit, rank the different hits according to affinity, and to structurally analyze the ligand-target complex, thus providing essential information for structure-based optimization and medicinal chemistry. In this review a comprehensive overview of the large variety of NMR methods to study interactions between small molecule ligands and macromolecular receptors is provided, summarizing the physico-chemical bases of the different receptor- and ligand-observed experiments. The application of these methods for compound library screening and hit validation, with special emphasis on their contribution to fragment-based drug discovery strategies, is illustrated by recent examples selected from the literature and work in my laboratory.

“In-cell nuclear magnetic resonance (NMR)” is a unique method for characterization of conformation, interaction and dynamics of proteins inside living cells at atomic level. Since the method was proposed by Dotch and co-workers in 2001(1), its application had been limited to bacterial cells and oocytes of Xenopus laevis(2). Recently, we reported the method for efficient delivery of 15N-labeled proteins into human HeLa cells by using cell-penetrating peptides, and measured high-resolution two-dimensional 1H-15N correlation spectra of proteins in the cells. The in-cell NMR spectroscopy in human cells is capable of analyzing structures, interactions, dynamics and stability of proteins inside cells. Of its possible applications, we propose that in-cell NMR spectroscopy can be utilized as an effective step in protein-targeted drug development process, by demonstrating that interaction of FKBP12 with immunosuppressants administered extracellularly was successfully observed in living cells. This observation suggests that drug delivery and capability of target proteins inside cells for interaction with drugs can be investigated by in-cell NMR spectroscopy. More recently, an alternative way for intracellular delivery of labeled proteins for in-cell NMR was reported on 293F cells by Shimada and co-workers. Here, we review recent technical development of in-cell NMR spectroscopy, and discuss potential usefulness for protein chemistry and drug screening process.

In situ structural characterization of organic compounds attached to solid supports can be achieved by high-resolution magic angle spinning NMR (HRMAS NMR), a technique that provides solution-like spectra for resin-bound molecules. This review outlines the principles of the technique, the influence of the solid support on data quality, and NMR experiments that are useful for obtaining valuable information. The review describes, with multiple examples mainly from the last 7 years, how HRMAS NMR has been applied to monitor solid-phase reactions, elucidate reaction products and quantify compound loading on a solid support. Other applications, such as conformational analysis of immobilized compounds and investigation of molecular interactions with compounds in solution, are also discussed.

We describe the details of the magnetic resonance spectroscopy and chemical shift imaging techniques for the human brain which have been developed over the last two decades. With these non-invasive tools, it is now readily possible to repeatedly assay up to 20 common brain metabolites. From the perspective of drug discovery, each of these metabolites could fulfill a number of useful functions: disease biomarker, surrogate marker of drug delivery, surrogate marker of drug efficacy and so on. To facilitate the possible utility of clinical magnetic resonance spectroscopy in future drug discovery, the major portion of the review is devoted to a detailed description of the well-validated neurochemical profiles of many common human brain disorders, for which MRS data now exists. Beyond proton, MRS, the commonest tool provided by the manufacturers of clinical MRI equipment, lays the world of heteronuclear NMR more familiar to chemists. Here too, with relatively little effort it has been possible to define neurochemical profiles of human brain disorders using 13C MRS. The future for drug discovery scientists is discussed. Finally, recognizing that a known feature of MR is the lack of sensitivity, we describe new efforts to harness hyperpolarization, with its 50,000 signal amplification, to conventional MRS.

Even though alterations in the microenvironmental properties of tissues underlie the development of the most prevalent and morbid pathologies, they are not directly observable in vivo by Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS) methods. This circumstance has lead to the development of a wide variety of exogenous paramagnetic and diamagnetic MRI and MRS probes able to inform non invasively on microenvironmental variables such as pH, pO2, ion concentration o even temperature. This review covers the fundamentals of environmental contrast and the current arsenal of endogenous and exogenous MRI and MRS contrast enhancing agents available to visualize it. We begin describing the physicochemical background necessary to understand paramagnetic and diamagnetic contrast enhancement with a special reference to novel magnetization transfer and 13C hiperpolarization strategies. We describe then the main macrocyclic structures used to support the environmentally sensitive paramagnetic sensors, including CEST and PARACEST pH sensitive probes, temperature probes and enzyme activity or gene expression activatable probes. Finally we address the most commonly used diamagnetic contrast agents including imidazolic derivatives to reveal extracellular pH and tissue pO2 values by MRS. The potential applications of these agents in mutimodal and molecular imaging approaches are discussed.