NMR-based Screening Research Articles

Optimization of reverse NOE pumping experiments in the analysis of complex systems with densely crowded NMR spectra.

We present an optimization of Reverse NOE-pumping (RNP) in order to observe the 1H signals of ligands bound to proteins. Although various ligand-based NMR screening methods have been proposed, the most frequently used method has been Saturation-Transfer Difference (STD), owing to the relatively easy setup of experiments. Yet the critical point of STD is the selective irradiation of protein without irradiating ligand, and thus the STD technique is unable to observe 1H ligand signals, which resonate across the entire 1H spectral width. In the present study, the RNP experiment has been improved to develop an effective NMR-based screening technique. The optimized RNP spectra reveal less subtraction artifacts and phase distortion than the original RNP spectra, indicating its applicability to any type of ligand molecules.

Identification of new leads against ubiquitin specific protease-7 (USP7): a step towards the potential treatment of cancers.

Ubiquitin-specific protease-7 (USP7) is an important drug target as it regulates multiple proteins and genes (such as MDM2 and p53) with roles in cancer progression. Its inhibition can hinder the function of oncogenes, increase tumor suppression, and enhance immune response. The current study was designed to express USP7 in a prokaryotic system, followed by screening of small molecules against it using biophysical methods, primarily STD-NMR technique. Among them, 12 compounds showed interaction with USP7 as inferred from NMR-based screening. These compounds further caused destabilization of USP7 by reducing its melting temperature (T m) up to 6 °C in thermal shift assay. Molecular docking and simulation studies revealed that these compounds bind to the putative substrate binding pocket of USP7 and thus may block the entry of the substrate. Four compounds i.e., 4-hydroxy-diphenyl amine (2), phenyl-(2,3,4-trihydroxyphenyl) methanone (3), 4'-amino-2',5'-diethoxy benzanilide (5), and hydroquinone (12), showed anti-cancer activity against colorectal cancerous cells (HCT116) with IC50 values in the range of 31-143 μM. These compounds also down-regulated the mRNA expression of the MDM2 gene and up-regulated the mRNA expression of the p53 gene in HCT116 cells, as studied using qPCR analysis. This study thereby identifies several negative modulators of USP7 that can be studied further as potential anti-cancer agents.

Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl-1.

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein-protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein-protein interactions.

Microfluidics delivery of DARPP-32 into HeLa cells maintains viability for in-cell NMR spectroscopy

High-resolution structural studies of proteins and protein complexes in a native eukaryotic environment present a challenge to structural biology. In-cell NMR can characterize atomic resolution structures but requires high concentrations of labeled proteins in intact cells. Most exogenous delivery techniques are limited to specific cell types or are too destructive to preserve cellular physiology. The feasibility of microfluidics transfection or volume exchange for convective transfer, VECT, as a means to deliver labeled target proteins to HeLa cells for in-cell NMR experiments is demonstrated. VECT delivery does not require optimization or impede cell viability; cells are immediately available for long-term eukaryotic in-cell NMR experiments. In-cell NMR-based drug screening using VECT was demonstrated by collecting spectra of the sensor molecule DARPP32, in response to exogenous administration of Forskolin.

Boron NMR as a Method to Screen Natural Product Libraries for B-Containing Compounds.

Natural products are biologically relevant metabolites exploited for biomedicine and biotechnology. The frequent reisolation of known natural products questions whether existing discovery models are still capable of identifying novel compounds. As innovative NMR-based screening techniques can help overcome these challenges, we applied a phase cycling composite pulse sequence to 11B NMR experiments to enhance their sensitivity to screen libraries for novel boron-containing molecules. Aplasmomycin and autoinducer-2 were detected in crude and enhanced microbial fractions, via their boron signals, as proof of concept. Subsequently, a screen of 21 crude plant and 50 crude marine microbial extracts were chosen at random and analyzed with the optimized 11B experiment for feasibility as a high throughput discovery method. Eight of the plant samples and 13 of the microbial samples were identified as boron-containing, suggesting that there is a higher presence of boron metabolites available from natural sources than previously known due to a lack of appropriate discovery methods. As a result, we believe that this optimized 11B NMR experiment can serve as a robust method for quick and facile discovery of novel boron-containing metabolites from a variety of natural sources.

Bioactive recombinant human oncostatin M for NMR-based screening in drug discovery

Oncostatin M (OSM) is a pleiotropic, interleukin-6 family inflammatory cytokine that plays an important role in inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and cancer progression and metastasis. Recently, elevated OSM levels have been found in the serum of COVID-19 patients in intensive care units. Multiple anti-OSM therapeutics have been investigated, but to date no OSM small molecule inhibitors are clinically available. To pursue a high-throughput screening and structure-based drug discovery strategy to design a small molecule inhibitor of OSM, milligram quantities of highly pure, bioactive OSM are required. Here, we developed a reliable protocol to produce highly pure unlabeled and isotope enriched OSM from E. coli for biochemical and NMR studies. High yields (ca. 10 mg/L culture) were obtained in rich and minimal defined media cultures. Purified OSM was characterized by mass spectrometry and circular dichroism. The bioactivity was confirmed by induction of OSM/OSM receptor signaling through STAT3 phosphorylation in human breast cancer cells. Optimized buffer conditions yielded 1H, 15N HSQC NMR spectra with intense, well-dispersed peaks. Titration of 15N OSM with a small molecule inhibitor showed chemical shift perturbations for several key residues with a binding affinity of 12.2 ± 3.9 μM. These results demonstrate the value of bioactive recombinant human OSM for NMR-based small molecule screening.

A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site.

A fragment-based approach to discovery of Receptor for Advanced Glycation End products inhibitors.

The Receptor for Advanced Glycation End products (RAGE) is a pattern recognition receptor that signals for inflammation via the NF-κB pathway. RAGE has been pursued as a potential target to suppress symptoms of diabetes and is of interest in a number of other diseases associated with chronic inflammation, such as inflammatory bowel disease and bronchopulmonary dysplasia. Screening and optimization have previously produced small molecules that inhibit the activity of RAGE in cell-based assays, but efforts to develop a therapeutically viable direct-binding RAGE inhibitor have yet to be successful. Here, we show that a fragment-based approach can be applied to discover fundamentally new types of RAGE inhibitors that specifically target the ligand-binding surface. A series of systematic assays of structural stability, solubility, and crystallization were performed to select constructs of the RAGE ligand-binding domain and optimize conditions for NMR-based screening and co-crystallization of RAGE with hit fragments. An NMR-based screen of a highly curated ~14 000-member fragment library produced 21 fragment leads. Of these, three were selected for elaboration based on structure-activity relationships generated through cycles of structural analysis by X-ray crystallography, structure-guided design principles, and synthetic chemistry. These results, combined with crystal structures of the first linked fragment compounds, demonstrate the applicability of the fragment-based approach to the discovery of RAGE inhibitors.

NMR-based Fragment Screening in a Minimum Sample but Maximum Automation Mode.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

NMR-based screening for natural product subfraction to precisely identify ligand of target protein.

The inherent diversity of natural product extracts has not only made their value for biological activity attractive, but has also presented significant challenges for separation and detection techniques to enable rapid characterisation of the biologically active component present in the complicated mixture. Nuclear magnetic resonance (NMR) is routinely valued for its ability to shed light on molecular structure, when NMR is used as a promising tool in drug screening, it can detect and quantify molecular interactions, and at the same time provide detailed structural information with atomic level resolution. Here, we introduced an accurate and efficient strategy for discovering ligands from natural product extracts, by taking the advantage of NMR-based drug screening. The characteristic pre-purified subfraction libraries were brought into screening, and combinatorial ligand-observed NMR interaction detection experiments were performed, once hits were found from one subfraction, the repository of the subfraction would be subject to separation and preparation, and the structure of the hits would be easily identified. Screening procedure of Radix Polygoni Multiflori water extract against human serum albumin (HSA) was used as an example, to discuss and verify the detailed methodology. Furthermore, human fatty acid binding protein 4 (FABP4 ) was presented as an example target protein, to illustrate the utility of this method for discovering biologically active component. It is proved that suitable subfraction library and well-combined ligand-detected NMR experiments will make the screening streamline more accurate and efficient. NMR is a promising tool to integrate natural product extracts into modern drug screening.

HT-SuMD: making molecular dynamics simulations suitable for fragment-based screening. A comparative study with NMR

Fragment-based lead discovery (FBLD) is one of the most efficient methods to develop new drugs. We present here a new computational protocol called High-Throughput Supervised Molecular Dynamics (HT-SuMD), which makes it possible to automatically screen up to thousands of fragments, representing therefore a new valuable resource to prioritise fragments in FBLD campaigns. The protocol was applied to Bcl-XL, an oncological protein target involved in the regulation of apoptosis through protein–protein interactions. Initially, HT-SuMD performances were validated against a robust NMR-based screening, using the same set of 100 fragments. These independent results showed a remarkable agreement between the two methods. Then, a virtual screening on a larger library of additional 300 fragments was carried out and the best hits were validated by NMR. Remarkably, all the in silico selected fragments were confirmed as Bcl-XL binders. This represents, to date, the largest computational fragments screening entirely based on MD.

Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in tumour biology by promoting the stabilisation and activity of oncogenic ‘client’ proteins. Inhibition of Hsp90 by small-molecule drugs, acting via its ATP hydrolysis site, has shown promise as a molecularly targeted cancer therapy. Owing to the importance of Hop and other tetratricopeptide repeat (TPR)-containing cochaperones in regulating Hsp90 activity, the Hsp90-TPR domain interface is an alternative site for inhibitors, which could result in effects distinct from ATP site binders. The TPR binding site of Hsp90 cochaperones includes a shallow, positively charged groove that poses a significant challenge for druggability. Herein, we report the apo, solution-state structure of Hop TPR2A which enables this target for NMR-based screening approaches. We have designed prototype TPR ligands that mimic key native ‘carboxylate clamp’ interactions between Hsp90 and its TPR cochaperones and show that they block binding between Hop TPR2A and the Hsp90 C-terminal MEEVD peptide. We confirm direct TPR-binding of these ligands by mapping 1H–15N HSQC chemical shift perturbations to our new NMR structure. Our work provides a novel structure, a thorough assessment of druggability and robust screening approaches that may offer a potential route, albeit difficult, to address the chemically challenging nature of the Hop TPR2A target, with relevance to other TPR domain interactors.

Comprehensive and High-Throughput Exploration of Chemical Space Using Broadband 19 F NMR-Based Screening.

Fragment-based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease-relevant targets. Among the numerous screening techniques, fluorine-detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required. Here, we introduce a comprehensive method that enabled the efficient assembly of a high-quality and diverse library containing nearly 4000 fragments and screening for target-specific binders within days. At the core of the approach is a novel broadband relaxation-edited NMR experiment that covers the entire chemical shift range of drug-like 19 F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.

Incarnatapeptins A and B, Nonribosomal Peptides Discovered Using Genome Mining and 1H/15N HSQC-TOCSY.

Methods for the focused isolation of low-abundance natural products with specific chemical substructures could expand known bioactive chemical diversity for drug discovery. Here we report the combined use of genome mining and an 15N NMR-based screening method for the targeted isolation of the low-abundance piperazic-acid-containing peptides incarnatapeptins A (1) and B (3). Incarnatapeptin B (3) shows in vitro cytotoxicity to LNCaP prostate cancer cells.

Identification of Binding Epitopes of a Fluorinated Compound Bound to Proteins Using 1H and 19F NMR Spectroscopy.

1H/19F NMR-based screening methods were applied to a human serum albumin-fleroxacin complex. Fleroxacin contains three fluorine atoms in a molecule, which is suitable as a model fluorinated compound for NMR analysis with 1H and 19F detection. The 19F{1H} and 1H{1H} saturation transfer difference spectra were acquired and the 1H/19F spin-lattice relaxation rates were measured with and without any selective irradiation of protein resonance to identify the binding epitopes of fleroxacin. Because several 1H signals of fleroxacin resonated close to water, its precise signal intensities were unavailable. The 19F NMR-based screening methods successfully provide complementary information, indicating its importance in the analysis of fluorinated compounds.

NMR resonance assignments for the active and inactive conformations of the small G protein RalA

The Ral proteins (RalA and RalB) are small G proteins of the Ras family that have been implicated in exocytosis, endocytosis, transcriptional regulation and mitochondrial fission, as well as having a role in tumourigenesis. RalA and RalB are activated downstream of the master regulator, Ras, which causes the nucleotide exchange of GDP for GTP. Here we report the 1H, 15 N and 13C resonance assignments of RalA in its active form bound to the GTP analogue GMPPNP. We also report the backbone assignments of RalA in its inactive, GDP-bound form. The assignments give insight into the switch regions, which change conformation upon nucleotide exchange. These switch regions are invisible in the spectra of the active, GMPPNP bound form but the residues proximal to the switches can be monitored. RalA is also an important drug target due to its over activation in some cancers and these assignments will be extremely useful for NMR-based screening approaches.

Introducing the CSP Analyzer: A novel Machine Learning-based application for automated analysis of two-dimensional NMR spectra in NMR fragment-based screening

NMR-based screening, especially fragment-based drug discovery is a valuable approach in early-stage drug discovery. Monitoring fragment-binding in protein-detected 2D NMR experiments requires analysis of hundreds of spectra to detect chemical shift perturbations (CSPs) in the presence of ligands screened. Computational tools are available that simplify the tracking of CSPs in 2D NMR spectra. However, to the best of our knowledge, an efficient automated tool for the assessment and binning of multiple spectra for ligand binding has not yet been described. We present a novel and fast approach for analysis of multiple 2D HSQC spectra based on machine-learning-driven statistical discrimination. The CSP Analyzer features a C# frontend interfaced to a Python ML classifier. The software allows rapid evaluation of 2D screening data from large number of spectra, reducing user-introduced bias in the evaluation. The CSP Analyzer software package is available on GitHub https://github.com/rubbs14/CSP-Analyzer/releases/tag/v1.0 under the GPL license 3.0 and is free to use for academic and commercial uses.

Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach.

The insertase BamA is an essential protein of the bacterial outer membrane. Its 16-stranded transmembrane β-barrel contains a lateral gate as a key functional element. This gate is formed by the C-terminal half of the last β-strand. The BamA barrel was previously found to sample different conformations in aqueous solution, as well as different gate-open, gate-closed, and collapsed conformations in X-ray crystallography and cryo-electron microscopy structures. Here, we report the successful identification of conformation-selective nanobodies that stabilize BamA in specific conformations. While the initial candidate generation and selection protocol was based on established alpaca immunization and phage display selection procedures, the final selection of nanobodies was enhanced by a solution NMR-based screening step to shortlist the targets for crystallization. In this way, three crystal structures of BamA-nanobody complexes were efficiently obtained, showing two types of nanobodies that indeed stabilized BamA in two different conformations, i.e., with open and closed lateral gate, respectively. Then, by correlating the structural data with high resolution NMR spectra, we could for the first time assign the BamA conformational solution ensemble to defined structural states. The new nanobodies will be valuable tools towards understanding the client insertion mechanism of BamA and towards developing improved antibiotics.

Determination of binding affinities using hyperpolarized NMR with simultaneous 4-channel detection

Dissolution dynamic nuclear polarization (D-DNP) is a powerful technique to improve NMR sensitivity by a factor of thousands. Combining D-DNP with NMR-based screening enables to mitigate solubility or availability problems of ligands and target proteins in drug discovery as it can lower the concentration requirements into the sub-micromolar range. One of the challenges that D-DNP assisted NMR screening methods face for broad application, however, is a reduced throughput due to additional procedures and time required to create hyperpolarization. These requirements result in a delay of several tens of minutes in-between each NMR measurement. To solve this problem, we have developed a simultaneous 4-channel detection method for hyperpolarized 19F NMR, which can increase throughput fourfold by utilizing a purpose-built multiplexed NMR spectrometer and probe. With this system, the concentration-dependent binding interactions were observed for benzamidine and benzylamine with the serine protease trypsin. A T2 relaxation measurement of a hyperpolarized reporter ligand (TFBC; CF3C6H4CNHNH2), which competes for the same binding site on trypsin with the other ligands, was used. The hyperpolarized TFBC was mixed with trypsin and the ligand of interest, and injected into four flow cells inside the NMR probe. Across the set of four channels, a concentration gradient was created. From the simultaneously acquired relaxation datasets, it was possible to determine the dissociation constant (KD) of benzamidine and benzylamine without the requirement for individually optimizing experimental conditions for different affinities. A simulation showed that this 4-channel detection method applied to D-DNP NMR extends the screenable KD range to up to three orders of magnitude in a single experiment.

Identification of Thiourea-Based Inhibitors of the B-Cell Lymphoma 6 BTB Domain via NMR-Based Fragment Screening and Computer-Aided Drug Design.

Protein-protein interactions (PPI) between the transcriptional repressor B-cell lymphoma 6 (BCL6) BTB domain (BCL6BTB) and its corepressors have emerged as a promising target for anticancer therapeutics. However, identification of potent, drug-like inhibitors of BCL6BTB has remained challenging. Using NMR-based screening of a library of fragment-like small molecules, we have identified a thiourea compound (7CC5) that binds to BCL6BTB. From this hit, the application of computer-aided drug design (CADD), medicinal chemistry, NMR spectroscopy, and X-ray crystallography has yielded an inhibitor, 15f, that demonstrated over 100-fold improved potency for BCL6BTB. This gain in potency was achieved by a unique binding mode that mimics the binding mode of the corepressor SMRT in the aromatic and the HDCH sites. The structure-activity relationship based on these new inhibitors will have a significant impact on the rational design of novel BCL6 inhibitors, facilitating the identification of therapeutics for the treatment of BCL6-dependent tumors.