Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency

Structure‐based drug development is often hampered by the lack of in vivo activity of promising compounds screened in vitro, due to low membrane permeability or poor intracellular binding selectivity. Herein, we show that ligand screening can be performed in living human cells by “intracellular protein‐observed” NMR spectroscopy, without requiring enzymatic activity measurements or other cellular assays. Quantitative binding information is obtained by fast, inexpensive ¹H NMR experiments, providing intracellular dose‐ and time‐dependent ligand binding curves, from which kinetic and thermodynamic parameters linked to cell permeability and binding affinity and selectivity are obtained. The approach was applied to carbonic anhydrase and, in principle, can be extended to any NMR‐observable intracellular target. The results obtained are directly related to the potency of candidate drugs, that is, the required dose. The application of this approach at an early stage of the drug design pipeline could greatly increase the low success rate of modern drug development.     

Solid-State NMR Spectroscopy: A Key Tool to Unravel the Supramolecular Structure of Drug Delivery Systems

In the past decades, nanosized drug delivery systems (DDS) have been extensively developed and studied as a promising way to improve the performance of a drug and reduce its undesirable side effects. DDSs are usually very complex supramolecular assemblies made of a core that contains the active substance(s) and ensures a controlled release, which is surrounded by a corona that stabilizes the particles and ensures the delivery to the targeted cells. To optimize the design of engineered DDSs, it is essential to gain a comprehensive understanding of these core–shell assemblies at the atomic level. In this review, we illustrate how solid-state nuclear magnetic resonance (ssNMR) spectroscopy has become an essential tool in DDS design.     

Functional Dynamics of Deuterated β2‐Adrenergic Receptor in Lipid Bilayers Revealed by NMR Spectroscopy

G‐protein‐coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β₂‐adrenergic receptor (β₂AR) in lipid bilayers of reconstituted high‐density lipoprotein (rHDL) increased by approximately 5‐fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β₂AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception.     

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.     

19F NMR Spectroscopy as a Highly Sensitive Method for the Direct Monitoring of Confined Crystallization within Nanoporous Materials

The introduction of fluorine into the structure of pharmaceuticals has been an effective strategy for tuning their pharmacodynamic properties, with more than 40 new drugs entering the market in the last 15 years. In this context, ¹⁹F NMR spectroscopy can be viewed as a useful method for investigating the host–guest chemistry of pharmaceuticals in nanosized drug‐delivery systems. Although the interest in confined crystallization, nanosized devices, and porous catalysts is gradually increasing, understanding of the complex phase behavior of organic molecules confined within nanochambers or nanoreactors is still lacking. Using ¹⁹F magic‐angle‐spinning NMR spectroscopy, we obtained detailed mechanistic insight into the crystallization of flufenamic acid (FFA) in a confined environment of mesoporous silica materials with different pore diameters (3.2–29 nm), providing direct experimental evidence for the formation of a molecular‐liquid‐like layer besides crystalline confined FFA form I.     

Two-Dimensional Solid-State NMR Spectroscopy: New Possibilities for the Investigation of the Structure and Dynamics of Solid Polymers [New Analytical Methods (38)]

NMR spectroscopy is an effective method not only for examining liquid samples but also for characterizing molecular sturcture, order and dynamics in amorphous and ordered solids. Recent developments in the area of solid-state NMR spectroscopy span from model-dependent studies of conventional one-dimensional spectra to the more definitive two-dimensional (2D) spectra which provide more specific information. For example, with 2D-NMR spectroscopy it is possible to determine the orientational distribution functions of molecular segments in drawn polymers and to distinguish different mechanisms of complex molecular motions. Following an introduction to basic NMR spectroscopy, an overview of the current state-of-the-art of 2D methods in solid-state NMR spectroscopy is presented and demonstrated with selected examples.     

Rational Design and Synthesis of HSF1-PROTACs for Anticancer Drug Development

PROTACs employ the proteosome-mediated proteolysis via E3 ligase and recruit the natural protein degradation machinery to selectively degrade the cancerous proteins. Herein, we have designed and synthesized heterobifunctional small molecules that consist of different linkers tethering KRIBB11, a HSF1 inhibitor, with pomalidomide, a commonly used E3 ligase ligand for anticancer drug development.     

183W NMR Spectroscopy Guides the Search for Tungsten Alkylidyne Catalysts for Alkyne Metathesis

Triarylsilanolates are privileged ancillary ligands for molybdenum alkylidyne catalysts for alkyne metathesis but lead to disappointing results and poor stability in the tungsten series. ¹H,¹⁸³W heteronuclear multiple bond correlation spectroscopy, exploiting a favorable ⁵J-coupling between the ¹⁸³W center and the peripheral protons on the alkylidyne cap, revealed that these ligands upregulate the Lewis acidity to an extent that the tungstenacyclobutadiene formed in the initial [2+2] cycloaddition step is over-stabilized and the catalytic turnover brought to a halt. Guided by the ¹⁸³W NMR shifts as a proxy for the Lewis acidity of the central atom and by an accompanying chemical shift tensor analysis of the alkylidyne unit, the ligand design was revisited and a more strongly π-donating all-alkoxide ligand prepared. The new expanded chelate complex has a tempered Lewis acidity and outperforms the classical Schrock catalyst, carrying monodentate tert-butoxy ligands, in terms of rate and functional-group compatibility.     

On the Tuning of High‐Resolution NMR Probes

Three optimum conditions for the tuning of NMR probes are compared: the conventional tuning optimum, which is based on radio‐frequency pulse efficiency, the spin noise tuning optimum based on the line shape of the spin noise signal, and the newly introduced frequency shift tuning optimum, which minimizes the frequency pushing effect on strong signals. The latter results if the radiation damping feedback field is not in perfect quadrature to the precessing magnetization. According to the conventional RLC (resistor–inductor–capacitor) resonant circuit model, the optima should be identical, but significant deviations are found experimentally at low temperatures, in particular on cryogenically cooled probes. The existence of different optima with respect to frequency pushing and spin noise line shape has important consequences on the nonlinearity of spin dynamics at high polarization levels and the implementation of experiments on cold probes.     

NMR Crystallography Enhanced by Quantum Chemical Calculations and Liquid State NMR Spectroscopy for the Investigation of Se-NHC Adducts**

N-heterocyclic carbene ligands (NHC) are widely utilized in catalysis and material science. They are characterized by their steric and electronic properties. Steric properties are usually quantified on the basis of their static structure, which can be determined by X-ray diffraction. The electronic properties are estimated in the liquid state; for example, via the ⁷⁷Se liquid state NMR of Se-NHC adducts. We demonstrate that ⁷⁷Se NMR crystallography can contribute to the characterization of the structural and electronic properties of NHC in solid and liquid states. Selected Se-NHC adducts are investigated via ⁷⁷Se solid state NMR and X-ray crystallography, supported by quantum chemical calculations. This investigation reveals a correlation between the molecular structure of adducts and NMR parameters, including not only isotropic chemical shifts but also the other chemical shift tensor components. Afterwards, the liquid state ⁷⁷Se NMR data is presented and interpreted in terms of the quantum chemistry modelling. The discrepancy between the structural and electronic properties, and in particular the π-accepting abilities of adducts in the solid and liquid states is discussed. Finally, the ¹³C isotropic chemical shift from the liquid state NMR and the ¹³C tensor components are also discussed, and compared with their ⁷⁷Se counterparts. ⁷⁷Se NMR crystallography can deliver valuable information about NHC ligands, and together with liquid state ⁷⁷Se NMR can provide an in-depth outlook on the properties of NHC ligands.     

Fast multidimensional localized parallel NMR spectroscopy for the analysis of samples

A parallel localized spectroscopy (PALSY) method is presented to speed up the acquisition of multidimensional NMR (nD) spectra. The sample is virtually divided into a discrete number of nonoverlapping slices that relax independently during consecutive scans of the experiment, affording a substantial reduction in the interscan relaxation delay and the total experiment time. PALSY was tested for the acquisition of three experiments 2D COSY, 2D DQF‐COSY and 2D TQF‐COSY in parallel, affording a time‐saving factor of 3–4. Some unique advantages are that the achievable resolution in any dimension is not compromised in any way: it uses conventional NMR data processing, it is not prone to generate spectral artifacts, and once calibrated, it can be used routinely with these and other combinations of NMR spectra. Copyright © 2010 John Wiley & Sons, Ltd.