Sensitivity improvement in 19F NMR-based screening experiments: theoretical considerations and experimental applications

NMR-based binding and functional screening performed with FAXS (fluorine chemical shift anisotropy and exchange for screening) and 3-FABS (three fluorine atoms for biochemical screening) represents a potential alternative approach to high-throughput screening for the identification of novel potential drug candidates. The major limitation of this method in its current status is its intrinsic low sensitivity that limits the number of tested compounds. One approach for overcoming this problem is the use of a cryogenically cooled (19)F probe that reduces the thermal noise in the receiver circuitry. Sensitivity improvement in the two screening techniques achieved with the novel cryogenic (19)F probe technology permits an increased throughput, detection of weaker binders and inhibitors (relevant in a fragment-based lead discovery program), detection of slow binders, and reduction in protein and substrate consumption. These aspects are analyzed with theoretical simulations and experimental quantitative performance evaluation. Application of 3-FABS combined with the cryogenic (19)F probe technology to rapid screening at very low enzyme concentrations and the current detection limits reached with this approach are also presented.     

A general NMR method for rapid, efficient, and reliable biochemical screening

High-throughput screening is usually the method of drug-lead discovery. It is now well accepted that, for a functional assay, quality is more important than quantity. The ligand-based or protein-based NMR screening methodologies for detecting compounds binding to the macromolecular target of interest are now well established. A novel and sensitive NMR method for rapid, efficient, and reliable biochemical screening is presented. The method named 3-FABS (three fluorine atoms for biochemical screening) requires the labeling of the substrate with a CF(3) moiety and utilizes (19)F NMR spectroscopy for the detection of the starting and enzymatically modified substrates. The method allows for high-quality screening of large compound or natural product extract collections and for measuring their IC(50) values. Applications of this technique to the screening of inhibitors of the Ser/Thr kinase AKT1 and the protease trypsin are presented. In addition, an interesting application of 3-FABS to functional genomics is also presented.     

19F NMR transverse and longitudinal relaxation filter experiments for screening: a theoretical and experimental analysis

Ligand‐based ¹⁹F NMR screening represents an efficient approach for performing binding assays. The high sensitivity of the methodology to receptor binding allows the detection of weak affinity ligands. The observable NMR parameters that are typically used are the ¹⁹F transverse relaxation rate and isotropic chemical shift. However, there are few cases where the ¹⁹F longitudinal relaxation rate should also be used. A theoretical and experimental analysis of the ¹⁹F NMR transverse and longitudinal relaxation rates at different magnetic fields is presented along with proposed methods for improving the sensitivity and dynamic range of these experiments applied to fragment‐based screening. Copyright © 2016 John Wiley & Sons, Ltd.     

Substrate phage as a tool to identify novel substrate sequences of proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.     

A fluorescence-based supramolecular tandem assay for monitoring lysine methyltransferase activity in homogeneous solution

The demand for practical and convenient enzyme assays for histone lysine methyltransferases (HKMTs) emerges along with the rapid development of this young class of enzymes. A supramolecular reporter pair composed of p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) has been used to monitor enzymatic trimethylation of lysine residues in peptide substrates. The assay affords a switch-ON fluorescence response and operates in a continuous, real-time, and label-free fashion. The underlying working principle relies on the higher affinity of the macrocycle towards the trimethylated product of the enzymatic reaction as compared to the substrate, which allows the assay to be carried out in the product-selective mode. The final product incorporates a trimethylammonium moiety, a known high-affinity binding motif for CX4. Two substrates corresponding to the H3 N-terminal tail, namely, S2 (RTKQTARKSTGGKAP) and S6 (QTARKSTGGS), were selected as model compounds for methylation with the Neurospora crassa Dim-5 enzyme and investigated by the newly developed supramolecular tandem HKMTs assay. Only the longer substrate S2 underwent methylation in solution. The potential of the assay for inhibitor screening was demonstrated by means of inhibition studies with 1,10-phenanthroline to afford an inhibition constant of (70±20)?μM.     

Technical and practical aspects of 19F NMR‐based screening: toward sensitive high‐throughput screening with rapid deconvolution

The technical and practical aspects of ¹⁹F NMR‐based screening against a macromolecular target are analyzed in detail. A novel method utilizing the relaxation of ¹⁹F homonuclear double quantum coherence is proposed for performing NMR‐based binding assays in a direct‐ or competition‐mode format. A combined strategy based on ¹⁹F NMR chemical shift prediction, 2D ¹⁹F NMR DOSY, and 2D ¹⁹F–¹H NMR long‐range COSY experiments is presented for the deconvolution of complex mixtures of fluorinated molecules generated by either addition of single compounds or by chemical synthesis. The approaches presented here allow the screening of complex mixtures, even in the case where the exact composition is not known, and the rapid identification of the binders contained in the mixtures. Copyright © 2012 John Wiley & Sons, Ltd.     

Structural dynamics of the cooperative binding of organic molecules in the human cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally administered drugs which exhibit an intriguing kinetic behavior typified by a sigmoidal dependence of the reaction velocity on the substrate concentration. There is evidence for the binding of two substrates in the active site of the enzyme, but the mechanism of this cooperative binding is unclear. Diazepam is such a drug that undergoes metabolism by CYP3A4 with sigmoidal dependence. Metabolism is initiated by hydrogen atom abstraction from the drug. To understand the factors that determine the cooperative binding and the juxtaposition of the C-H bond undergoing abstraction, we carried out molecular dynamics simulations for two enzymatic conformers and examined the differences between the substrate-free and the bound enzymes, with one and two diazepam molecules. Our results indicate that the effector substrate interacts both with the active substrate and with the enzyme, and that this interaction results in side chain reorientation with relatively minor long-range effects. In accord with experiment, we find that F304, in the interface between the active and effector binding sites, is a key residue in the mechanism of cooperative binding. The addition of the effector substrate stabilizes F304 and its environment, especially F213, and induces a favorable orientation of the active substrate, leading to a short distance between the targeted hydrogen for abstraction and the active species of the enzyme. In addition, in one conformer of the enzyme, residue R212 may strongly interact with F304 and counteract the effector's impact on the enzyme.     

A Fluorescence‐Based Supramolecular Tandem Assay for Monitoring Lysine Methyltransferase Activity in Homogeneous Solution

The demand for practical and convenient enzyme assays for histone lysine methyltransferases (HKMTs) emerges along with the rapid development of this young class of enzymes. A supramolecular reporter pair composed of p‐sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) has been used to monitor enzymatic trimethylation of lysine residues in peptide substrates. The assay affords a switch‐ON fluorescence response and operates in a continuous, real‐time, and label‐free fashion. The underlying working principle relies on the higher affinity of the macrocycle towards the trimethylated product of the enzymatic reaction as compared to the substrate, which allows the assay to be carried out in the product‐selective mode. The final product incorporates a trimethylammonium moiety, a known high‐affinity binding motif for CX4. Two substrates corresponding to the H3 N‐terminal tail, namely, S2 (RTKQTA RKSTG GKAP) and S6 (QTA RKSTG GS), were selected as model compounds for methylation with the Neurospora crassa Dim‐5 enzyme and investigated by the newly developed supramolecular tandem HKMTs assay. Only the longer substrate S2 underwent methylation in solution. The potential of the assay for inhibitor screening was demonstrated by means of inhibition studies with 1,10‐phenanthroline to afford an inhibition constant of (70±20) μM .     

Supramolecular tandem enzyme assays

We conceptualize a novel approach towards enzyme assays based on the reversible and competitive binding of a fluorescent dye and the substrate as well as product of an enzymatic reaction to a macrocyclic host. This method was termed "supramolecular tandem assay", and has been applied to inhibitor and activator screening, sensor array development, and enantiomeric excess determination of amino acids. The simple and rapid read-out by fluorescence allows their straightforward implementation into high-throughput screening.     

Application of chemical arrays in screening elastase inhibitors

Protein chip technology provides a new and useful tool for high-throughput screening of drugs because of its high performance and low sample consumption. In order to screen elastase inhibitors on a large scale, we designed a composite microarray integrating enzyme chip containing chemical arrays on glass slides to screen for enzymatic inhibitors. The composite microarray includes an active proteinase film, screened chemical arrays distributed on the film, and substrate microarrays to demonstrate change of color. The detection principle is that elastase hydrolyzes synthetic colorless substrates and turns them into yellow products. Because yellow is difficult to detect, bromochlorophenol blue (BPB) was added into substrate solutions to facilitate the detection process. After the enzyme had catalyzed reactions for 2 h, effects of samples on enzymatic activity could be determined by detecting color change of the spots. When chemical samples inhibited enzymatic activity, substrates were blue instead of yellow products. If the enzyme retained its activity, the yellow color of the products combined with blue of BPB to make the spots green. Chromogenic differences demonstrated whether chemicals inhibited enzymatic activity or not. In this assay, 11,680 compounds were screened, and two valuable chemical hits were identified, which demonstrates that this assay is effective, sensitive and applicable for high-throughput screening (HTS).     

Modeling of a control induced system for product formation in enzyme kinetics

Double substrate enzyme kinetics has a leading role for product quantification and optimization in different chemical and biochemical sectors. Mathematical approach for controlling these reactions in different stages by suitable parameters adds a new dimension in this interdisciplinary field of research. Applying control theoretic approach in the reversible backward stages of double substrate enzymatic model, time economization with regard to product formation is significant. In this article, we formulate a double substrate mathematical model of enzymatic dynamical reaction system with control measures with a view to observe the effect of changes of these measures with respect to the concentration of substrates, enzyme, complexes and finally product. Here, Pontryagin Minimum Principle is used for observing the effect of control measures in the system dynamics with the help of Hamiltonian. We compare the relevant numerical solutions for the substrates, enzyme, complexes and product concentration profile for a specified time interval with respect to control factors.     

Fluorine-NMR experiments for high-throughput screening: theoretical aspects,practical considerations,and range of applicability

Competition ligand-based NMR screening experiments have recently been introduced to overcome most of the problems associated with traditional ligand-based NMR screening. Molecules with marginal solubility and high affinity for a given target can be easily identified in a high-throughput manner by screening chemical mixtures against the target in the presence of a weak- to medium-affinity ligand of known binding constant. While the original competition-based approaches utilized (1)H detection, significant advantages are obtained using (19)F detection. The absence of spectral overlap permits the screening of large chemical mixtures and allows for automated analysis of the spectra, even in the presence of protonated buffers, solvents, and detergents. The large chemical shift anisotropy of fluorine and the significant exchange contribution allow for the selection of a weak-affinity spy molecule, thus resulting in a lower binding affinity threshold for the identified NMR hits. The method, labeled FAXS (fluorine chemical shift anisotropy and exchange for screening) is rapid and requires only a limited amount of protein and, therefore, compares favorably with the other established non-NMR techniques used in high-throughput screening. Herein the theoretical aspects of this powerful (19)F-based approach are presented and discussed in detail. The experimental conditions together with the detection limits and binding constant measurements are investigated using human serum albumin as the target.     

Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC–HR-MS

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures.     

Insight into Substrate Preference of Two Chimeric Esterases by Combining Experiment and Molecular Simulation

Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development. For this purpose, two chimeric esterases AAM7 and PAR, constructed via domain swapping between two ancient thermophilic esterases, were investigated on their molecular simulation(including homology modeling, substrates docking and substrate binding affinity validation) and enzymatic assay(specific activities and activation energies calculating). Our results indicate that the factors contributing to the substrate preference of many enzymes especially the broad-specificity enzymes like esterases are multiple and complicated, the substrate binding domains or binding pockets are important but not the only factor for substrate preference.     

Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.     

Chemical rescue of the distal histidine mutants of tryptophan 2,3-dioxygenase

Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen.     

Protein dynamics in cytochrome P450 molecular recognition and substrate specificity using 2D IR vibrational echo spectroscopy

Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. Protein dynamics have been implicated in cyt P450 substrate specificity. Here, 2D IR vibrational echo spectroscopy is employed to measure the dynamics of cyt P450(cam) from Pseudomonas putida on fast time scales using CO bound at the active site as a vibrational probe. The substrate-free enzyme and the enzyme bound to both its natural substrate, camphor, and a series of related substrates are investigated to explicate the role of dynamics in molecular recognition in cyt P450(cam) and to delineate how the motions may contribute to hydroxylation specificity. In substrate-free cyt P450(cam), three conformational states are populated, and the structural fluctuations within a conformational state are relatively slow. Substrate binding selectively stabilizes one conformational state, and the dynamics become faster. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of picosecond time scale contribute to the variation in activity of cyt P450(cam) toward different substrates.     

In silico strategies for the selection of chelating compounds with potential application in metal-promoted neurodegenerative diseases

The development of new strategies to find commercial molecules with promising biochemical features is a main target in the field of biomedicine chemistry. In this work we present an in silico-based protocol that allows identifying commercial compounds with suitable metal coordinating and pharmacokinetic properties to act as metal-ion chelators in metal-promoted neurodegenerative diseases (MpND). Selection of the chelating ligands is done by combining quantum chemical calculations with the search of commercial compounds on different databases via virtual screening. Starting from different designed molecular frameworks, which mainly constitute the binding site, the virtual screening on databases facilitates the identification of different commercial molecules that enclose such scaffolds and, by imposing a set of chemical and pharmacokinetic filters, obey some drug-like requirements mandatory to deal with MpND. The quantum mechanical calculations are useful to gauge the chelating properties of the selected candidate molecules by determining the structure of metal complexes and evaluating their stability constants. With the proposed strategy, commercial compounds containing N and S donor atoms in the binding sites and capable to cross the BBB have been identified and their chelating properties analyzed.     

Fluorine-19 NMR chemical shift probes molecular binding to lipid membranes

The binding of amphiphilic molecules to lipid bilayers is followed by 19F NMR using chemical shift and line shape differences between the solution and membrane-tethered states of -CF 3 and -CHF 2 groups. A chemical shift separation of 1.6 ppm combined with a high natural abundance and high sensitivity of 19F nuclei offers an advantage of using 19F NMR spectroscopy as an efficient tool for rapid time-resolved screening of pharmaceuticals for membrane binding. We illustrate the approach with molecules containing both fluorinated tails and an acrylate moiety, resolving the signals of molecules in solution from those bound to synthetic dimyristoylphosphatidylcholine bilayers both with and without magic angle sample spinning. The potential in vitro and in vivo biomedical applications are outlined. The presented method is applicable with the conventional NMR equipment, magnetic fields of several Tesla, stationary samples, and natural abundance isotopes.     

Evolution in microfluidic droplet

High-throughput screening (HTS) of enzymatic activity is important for directed evolution-based enzyme engineering. However, substrate and product diffusion can severely compromise these HTS assays. In this issue of Chemistry & Biology, Kintses and coworkers describe a microfluidic platform for the directed evolution of enzymes in droplets that allows for the screening of 10(7) mutants per round of evolution.