NMR Screening Research Articles

Target Immobilization and NMR Screening of Fragments in Early Drug Discovery

Using localized NMR spectroscopy on immobilized targets provides us with a method to simultaneously assess binding of small molecules to two different samples. This Target Immobilized NMR Screening (TINS) has a number of advantages, not least is the requirement for minimal quantities of non-isotopically labeled protein and the applicability to insoluble or unstable targets. The technique is sensitive to binding with K(D) values in the range of 100 nM to 20 mM, while careful selection of the reference protein reduces the number of false positive hits. This combination ensures a maximal number of valid hits from which to select starting points for hit elaboration projects. Hits can be prioritized using biological assays when appropriate, as well as an array of biophysical techniques. So far a variety of soluble proteins, including kinases, GTPases, viral targets and proteases, as well as a membrane protein, have been successfully screened against our fragment library. Here we illustrate our experiences with a number of examples which emphasize the usefulness of the method in selecting and prioritizing fragment hits for elaboration towards leads.

Non-peptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide 1 is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3–41 μM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.

NMR-Based Multi Parametric Quality Control of Fruit Juices: SGF Profiling

With SGF Profiling™ we introduce an NMR-based screening method for the quality control of fruit juices. This method has been developed in a joint effort by Bruker BioSpin GmbH and SGF International e.V. The system is fully automated with respect to sample transfer, measurement, data analysis and reporting and is set up on an Avance 400 MHz flow-injection NMR spectrometer. For each fruit juice a multitude of parameters related to quality and authenticity are evaluated simultaneously from a single data set acquired within a few minutes. This multimarker/multi-aspect NMR screening approach features low cost-per-sample and is highly competitive with conventional and targeted fruit juice quality control methods.

A microscale protein NMR sample screening pipeline

As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30–200 μg in 8–35 μl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-009-9386-z) contains supplementary material, which is available to authorized users.

Automated system for high-throughput protein production using the dialysis cell-free method

High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14 h, including 8 h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).

Novel strategies to overcome expression problems encountered with toxic proteins: Application to the production of Lac repressor proteins for NMR studies

NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D 2O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.

Robust NMR Screening for Lead Compounds Using Tryptophan-Containing Proteins

NMR-based drug screening methods provide the most reliable characterization of binding propensities of ligands to their target proteins. Unique to NMR is its capability to detect weak microM-mM bindings. NMR assays are, however, one of the least effective methods in terms of the amount of protein required and the time needed for acquiring NMR experiments. We have recently described a time efficient 1D proton NMR assay for studying the effect of antagonists on protein-protein interactions. The method, named AIDA-NMR (for Antagonist Induced Dissociation Assay-NMR), can provide information on whether an antagonist of a protein-protein interaction is strong enough to dissociate the complex and, in addition, whether its interaction is through denaturation, precipitation, or release of a protein in its functional folded state. AIDA requires a large protein fragment (larger than ca. 30 kDa) to bind to a small reporter protein (smaller than ca.12 kDa). Here, we present an extension of this method, named SEI AIDA (SEI, for Selective Excitation-Inversion). The SEI AIDA uses tryptophan-bearing proteins, and by selectively exciting only the proton NMR signals of the (N)H(epsilon) indole side chains of tryptophans, the acquisition time of the AIDA experiment can be reduced by an order of magnitude relative to the corresponding 1D AIDA that uses hard pulses. Thus, at 600 MHz, the (N)H(epsilon) signal of a 35 microM protein complex can be acquired in only 2.5 min, making the SEI AIDA suitable for high-throughput screening pipelines in drug discovery.

Protein‐free ligand screening: simplification of chiral chromatographic development via novel adaptation of NMR screening methodologies

We demonstrate here a promising NMR method that provides evidence for chiral compound selector interaction as a first-pass screening method. A novel adaptation of commonly used protein-based screening technologies, this approach relies upon ligand-to-stationary phase interaction wherein the stationary phase is tethered to sepharose beads. At only minutes per experiment, this methodology significantly reduces the time required for chiral separation methodology development and complements currently available chromatographic purity technologies.

Design of compound libraries for fragment screening

Approaches to the design of libraries for fragment screening are illustrated with reference to a 20 k generic fragment screening library and a 1.2 k generic NMR screening library. Tools and methods for library design that have been developed within AstraZeneca are described, including Foyfi fingerprints and the Flush program for neighborhood characterization. It will be shown how Flush and the BigPicker, which selects maximally diverse sets of compounds, are used to apply the Core and Layer method for library design. Approaches to partitioning libraries into cocktails are also described.

A multi-step NMR screen for the identification and evaluation of chemical leads for drug discovery.

A multi-step NMR based screening assay is described for identifying and evaluating chemical leads for their ability to bind a target protein. The multi-step NMR assay provides structure-related information while being an integral part of a structure based drug discovery and design program. The fundamental principle of the multi-step NMR assay is to combine distinct 1D and 2D NMR techniques, in such a manner, that the inherent strengths and weakness associated with each technique is complementary to each other in the screen. By taking advantage of the combined strengths of 1D and 2D NMR experiments, it is possible to minimize protein requirements and experiment time and differentiate between non-specific and stoichiometric binders while being able to verify ligand binding, determine a semi-quantitative dissociation constant, identify the ligand binding site and rapidly determine a protein-ligand co-structure. Furthermore, the quality and physical behavior of the ligand is readily evaluated to determine its appropriateness as a chemical lead. The utility of the multi-step NMR assay is demonstrated with the use of PrgI from Salmonella typhimurium and human serum albumin (HSA) as target proteins.

Discovery of Ligands for a Novel Target, the Human Telomerase RNA, Based on Flexible-Target Virtual Screening and NMR

The human ribonucleoprotein telomerase is a validated anticancer drug target, and hTR-P2b is a part of the human telomerase RNA (hTR) essential for its activity. Interesting ligands that bind hTR-P2b were identified by iteratively using a tandem structure-based approach: docking of potential ligands from small databases to hTR-P2b via the program MORDOR, which permits flexibility in both ligand and target, with subsequent NMR screening of high-ranking compounds. A high percentage of the compounds tested experimentally were found via NMR to bind to the U-rich region of hTR-P2b; most have MW < 500 Da and are from different compound classes, and several possess a charge of 0 or +1. Of the 48 ligands identified, 24 exhibit a decided preference to bind hTR-P2b RNA rather than A-site rRNA and 10 do not bind A-site rRNA at all. Binding affinity was measured by monitoring RNA imino proton resonances for some of the compounds that showed hTR binding preference.

Organocuprate Conjugate Addition: Structural Features of Diastereomeric and Supramolecular π-Intermediates

In the reaction pathway of conjugate additions with organocuprate reagents, Cu(I) pi-complexes and Cu(III) sigma-complexes have been identified as central, NMR-detectable intermediate species. However, no experimental evidence for the structures of pi-intermediates with extensive chiral enones or the principal aggregation level and aggregate structure of pi-complexes in diethyl ether has been available so far. Furthermore, the structural characteristics of pi-complexes which are essential for their high reactivities and diastereoselectivities have not yet been rationalized experimentally. Therefore, the pi-intermediates of 4,4a,5,6,7,8-hexahydro-4a-methyl-naphthalen-2(3H)-one and Me2CuLi or Me2CuLi x LiX (X = I, CN) in diethyl ether are investigated in detail. For the first time, the formation of two intermediate cuprate enone pi-complexes on both sides of the double bond is observed. In addition, the conformation of the enone adopted in the major beta-face pi-complex rationalizes the exclusive syn addition observed in the synthetic product. For the investigation of the aggregation level and structure, a NMR screening of pi-complexes with Me2CuLi x LiX (X = I, CN) and three achiral enones is performed, which simplifies the spectra by the generation of enantiotopic pi-complexes. Thus, NMR diffusion experiments on cuprate intermediates and the detection of scalar couplings across copper without isotope labeling are possible for the first time. Extensive NMR studies, including those of cyclohexanone complexes, show that, in principle, salt-free dimethylcuprate is able to complex the carbonyl group. However, in the presence of salt, the carbonyl-complexing aggregates are composed of salt and cuprate moieties. These mixed aggregates cause the formation of large supramolecular pi-intermediate structures which control their reactivity. The pi-complexing cuprate units show a bent geometry as a general structural feature that is unaffected by the presence or kind of salt and the type of enone. Thus, the high diastereoselectivity and the reactivity of organocuprate 1,4-addition reactions are for the first time rationalized on the basis of structural characteristics of selected pi-intermediates.

Substrate and Catalyst Screening in Platinum-Catalyzed Asymmetric Alkylation of Bis(secondary) Phosphines. Synthesis of an Enantiomerically Pure C2-Symmetric Diphosphine

Platinum-catalyzed asymmetric alkylation of bis(secondary) phosphines was investigated. The modular design of the catalyst precursor Pt(diphos*)(R′)(Cl) and the substrates, a bis(secondary) phosphine HRP∼PHR and a benzyl halide, along with an efficient 31P NMR screening method, enabled rapid evaluation of the rate and diastereoselectivity of these reactions. These experiments identified a selective catalyst, Pt(DuPhos)(Ph)(Cl), and showed that the alkylation of PhHP(CH2)3PHPh (2) was faster and more selective than that of PhHP(CH2)2PHPh (1), MesHP(CH2)3PHMes (3), or 1,1′-(C5H4PHPh)2Fe (4). Alkylation of 1 with o-CF3C6H4CH2Br using the base NaOSiMe3 and the catalyst precursor Pt((R,R)-i-Pr-DuPhos)(Ph)(Cl), or the analogous Me-DuPhos complex, gave the diphosphine Ph(CH2o-CF3C6H4)P(CH2)2P(CH2o-CF3C6H4)Ph (7), which was prepared on a multigram scale and isolated as a borane adduct (6). The rac and meso diastereomers of 6 were separated by recrystallization, and enantiomerically pure 6 was isolated. Both (R,R)- and (S,S)-6, prepared separately in high ee using appropriate catalyst precursors, were characterized by X-ray crystallography, as was meso-7. Separate treatment of (S,S)-7 and meso-7 with Pt(COD)(Ph)(Cl) gave the complexes Pt((S,S)-7)(Ph)(Cl) ((S,S)-8) and Pt(meso-7)(Ph)(Cl) (meso-8). Both diastereomers of 8 were catalyst precursors for synthesis of 7 by alkylation of 1 with o-CF3C6H4CH2Br, but these reactions were unselective, because ligand 7 was rapidly displaced from its complex, 8.

Theoretical analysis of the competition ligand‐based NMR experiments and selected applications to fragment screening and binding constant measurements

AbstractCompetition ligand‐based NMR experiments have recently emerged as a powerful approach for fragment‐based screening in the drug discovery process. The robustness and reliability of these methodologies result in the identification of only specific binding ligands. In addition, the experiments, when properly performed, provide a quantitative measure of the affinity of the identified hits for the receptor. This is relevant for ranking the molecules according to their binding strength to the biomolecular target and for building a meaningful structure activity relationship (SAR) table. Several NMR experiments have been proposed for this purpose. The theory at the base of these different competition ligand‐based NMR experiments is analyzed in detail and several simulations with experimental conditions typically used in the NMR screening experiments are reported. Most of the presented theory applies also to the direct ligand‐based NMR screening experiments. In addition, the strategy for deriving the binding constant of the molecules is described, and selected applications to relevant drug discovery projects are presented. Finally, a novel sensitive NMR screening experiment that combines WaterLOGSY and transverse relaxation effects is proposed. © 2008 Wiley Periodicals, Inc. Concepts Magn Reson Part A 32A: 341–372, 2008.

NMR Screening for Lead Compounds Using Tryptophan-Mutated Proteins

NMR-based drug screening methods provide the most reliable characterization of binding propensities of ligands to their target proteins. They are, however, one of the least effective methods in terms of the amount of protein required and the time needed for acquiring an NMR experiment. We show here that the introduction of tryptophan to proteins permits rapid screening by monitoring a simple 1D proton NMR signal of the NH side chain ((N)H(epsilon)) of the tryptophan. The method could also provide quantitative characterization of the antagonist-protein and antagonist-protein-protein interactions in the form of KDs and fractions of the released proteins from their mutual binding. We illustrate the method with the lead compounds that block the Mdm2-p53 interaction and by studying inhibitors that bind to cyclin-dependent kinase 2 (CDK2).

An efficient use of the WATERGATE W5 sequence for observing a ligand binding with a protein receptor

An efficient pulse sequence for observing a ligand binding with a receptor has been developed by incorporating the WATERGATE W5 sequence. In the conventional water ligand observed via gradient spectroscopy (WaterLOGSY) techniques, the water resonance is selectively excited using, e.g. the double-pulsed field gradient spin-echo (DPFGSE) sequence at the initial portion of pulse sequence. In the current version, the modified WATERGATE W5 sequence is incorporated at the initial portion of the pulse sequence, and the resonance at the water frequency can be selectively reserved by the modified WATERGATE W5 sequence. The efficiency of ligand-observed NMR screening techniques has been demonstrated using the human serum albumin (HSA)-tryptophan complex.

Discovery of a Novel Warhead against β-Secretase through Fragment-Based Lead Generation

Fragment-based lead generation was applied to find novel small-molecule inhibitors of beta-secretase (BACE-1), a key target for the treatment of Alzheimer's disease. Fragment hits coming from a 1D NMR screen were characterized by BIAcore, and the most promising compounds were soaked into protein crystals to help the rational design of more potent hit analogues. Problems arising due to our inability to grow BACE-1 crystals at the biologically relevant pH at which the screen was run were overcome by using endothiapepsin as a surrogate aspartyl protease. Among others, we identified 6-substituted isocytosines as a novel warhead against BACE-1, and the accompanying paper in this journal describes how these were optimized to a lead series of nanomolar inhibitors.1.

Tuning Sensitivity in Paramagnetic NMR Detection of Ligand–DNA Interactions

Pulsed paramagnetics: The presence of an EDTA-functionalized nucleotide on a synthetic fragment of DNA permits the insertion of metal ions with various paramagnetic properties. Therefore, the paramagnetic contribution to the transverse relaxation rate of ligand protons, exploited to increase the sensitivity of NMR screening, can be tuned to get long-range effects and qualitative information on binding specificity.

Influence of Copper Salts, Solvents, and Ligands on the Structures of Precatalytic Phosphoramidite Copper Complexes for Conjugate Addition Reactions

For copper-catalyzed enantioselective conjugate addition reactions of organozinc reagents, the available knowledge about the mechanism and the structures involved is still insufficient to understand in detail the strong influences of solvent, salt, and ligand size, or to enable a rational control of this reaction. Screening with three phosphoramidite ligands and four copper(I) salts using NMR spectroscopy has revealed a binuclear copper complex with mixed trigonal/tetrahedral stereochemistry as the basic structural motif of the ground state of precatalysts with highly stereoselective ligands. Ligands with smaller amine moieties allow higher coordination numbers and higher aggregation levels, leading to reduced ee values. Since the ESI mass spectra of several precatalytic copper halide complexes show a striking correlation with the structures observed in solution, ESI-MS may be used as a fast tool to determine the maximum number of phosphoramidite ligands attached to copper.

Identification of novel fragment compounds targeted against the pY pocket of v‐Src SH2 by computational and NMR screening and thermodynamic evaluation

Discovery of small molecule inhibitors of protein-protein interactions is a major challenge to pharmaceutical development. Fragment-based approaches have begun to be widely adopted as an effective way of exploring chemical space on a protein surface with reduced library size. On completion of a fragment screen, the subsequent selection of appropriate "hit" molecules for development is a key decision point. Thermodynamic parameters can be used in this decision process. In this work, a fragment identification protocol based on a virtual fragment analysis and selection followed by 19F NMR screening was directed at the phosphotyrosine binding site of the Src SH2 domain. Three new ligands were identified. Isothermal titration calorimetry was used to provide thermodynamic parameters for the physiologically relevant ligand and the selected fragments. One of these fragments possesses a highly favorable enthalpic contribution to complex formation compared to other fragments and to the physiologically relevant ligand suggesting that it would make a good candidate for compound development.