Comprehensive and High-Throughput Exploration of Chemical Space Using Broadband 19F 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 ¹⁹F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.     

The 3F Library: Fluorinated Fsp3‐Rich Fragments for Expeditious 19F NMR Based Screening

Fragment‐based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide‐spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp³‐rich fragments is shape diverse and natural‐product‐like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two‐stage workflow of ¹⁹F NMR and subsequent ¹H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor‐made for ¹⁹F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.     

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.     

The 3F Library: Fluorinated Fsp3-Rich Fragments for Expeditious 19F NMR Based Screening

Fragment-based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide-spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp³-rich fragments is shape diverse and natural-product-like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two-stage workflow of ¹⁹F NMR and subsequent ¹H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor-made for ¹⁹F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.     

Target‐Based Whole‐Cell Screening by 1H NMR Spectroscopy

An NMR‐based approach marries the two traditional screening technologies (phenotypic and target‐based screening) to find compounds inhibiting a specific enzymatic reaction in bacterial cells. Building on a previous study in which it was demonstrated that hydrolytic decomposition of meropenem in living Escherichia coli cells carrying New Delhi metallo‐β‐lactamase subclass 1 (NDM‐1) can be monitored in real time by NMR spectroscopy, we designed a cell‐based NMR screening platform. A strong NDM‐1 inhibitor was identified with cellular IC₅₀ of 0.51 μM , which is over 300‐fold more potent than captopril, a known NDM‐1 inhibitor. This new screening approach has great potential to be applied to targets in other cell types, such as mammalian cells, and to targets that are only stable or functionally competent in the cellular environment.     

DNA‐Encoded Dynamic Combinatorial Chemical Libraries

Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA‐encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal‐to‐noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA‐encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high‐affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA‐templated chemical reactions.     

Synthesis and Evaluation of a 2,11‐Cembranoid‐Inspired Library

The 2,11‐cembranoid family of natural products has been used as inspiration for the synthesis of a structurally simplified, functionally diverse library of octahydroisobenzofuran‐based compounds designed to augment a typical medicinal chemistry library screen. Ring‐closing metathesis, lactonisation and SmI₂‐mediated methods were exemplified and applied to the installation of a third ring to mimic the nine‐membered ring of the 2,11‐cembranoids. The library was assessed for aqueous solubility and permeability, with a chemical‐space analysis performed for comparison to the family of cembranoid natural products and a sample set of a screening library. Preliminary investigations in cancer cells showed that the simpler scaffolds could recapitulate the reported anti‐migratory activity of the natural products.     

Live‐Cell‐Templated Dynamic Combinatorial Chemistry

Dynamic covalent chemistry combines in a single step the screening and synthesis of ligands for biomolecular recognition. In order to do that, a chemical entity is used as template within a dynamic combinatorial library of interconverting species, so that the stronger binders are amplified due to the efficient interaction with the target. Here we employed whole A549 living cells as template in a dynamic mixture of imines, for which amplification reflects the efficient and selective interaction with the corresponding extracellular matrix. The amplified polyamine showed strong interaction with the A549 extracellular matrix in on‐cell NMR experiments, while combination of NMR, SPR, and molecular dynamics simulations in model systems provided insights on the molecular recognition event. Notably, our work pioneers the use of whole living cells in dynamic combinatorial chemistry, which paves the way towards the discovery of new bioactive molecules in a more biorelevant environment.     

J‐Edited Pure Shift NMR for the Facile Measurement of nJHH for Specific Protons

We report a novel 1D J‐edited pure shift NMR experiment (J‐PSHIFT) that was constructed from a pseudo 2D experiment for the direct measurement of proton–proton scalar couplings. The experiment gives homonuclear broad‐band ¹H‐decoupled ¹H NMR spectra, which provide a single peak for chemically distinct protons, and only retain the homonuclear‐scalar‐coupled doublet pattern at the chemical‐shift positions of the protons in the coupled network of a specific proton. This permits the direct and unambiguous measurement of the magnitudes of the couplings. The incorporation of a 1D selective correlation spectroscopy (COSY)/ total correlation spectroscopy (TOCSY) block in lieu of the initial selective pulse, results in the exclusive detection of the correlated spectrum of a specific proton.     

A Strategy to Design Hyperpolarized 13C Magnetic Resonance Probes Using [1‐13C]α‐Amino Acid as a Scaffold Structure

Hyperpolarization is an emerging method that dramatically enhances NMR signal intensity. As a result of their increased sensitivity, hyperpolarized (HP) NMR molecular probes can be used to perform time‐resolved spectroscopy and imaging in vitro and in vivo. It is, however, challenging to design such probes de novo. Herein, the [1‐¹³C]α‐amino acid is reported as a scaffold structure to design HP ¹³C NMR molecular probes. The [1‐¹³C]α‐amino acid can be converted to various HP ¹³C chemical probes that show sufficient chemical shift change by altering the chemical state of the α nitrogen upon interaction with the target. Several previously reported HP probes could be explained by this design principle. To demonstrate the versatility of this approach, two α‐amino‐acid‐based HP ¹³C chemical probes, sensitive to pH and Ca²⁺ ion, were developed and used to detect targets.     

Development of a Terpenoid Alkaloid‐like Compound Library Based on the Humulene Skeleton

Many natural terpenoid alkaloid conjugates show biological activity because their structures contain both sp³‐rich terpenoid scaffolds and nitrogen‐containing alkaloid scaffolds. However, their biosynthesis utilizes a limited set of compounds as sources of the terpenoid moiety. The production of terpenoid alkaloids containing various types of terpenoid moiety may provide useful, chemically diverse compound libraries for drug discovery. Herein, we report the construction of a library of terpenoid alkaloid‐like compounds based on Lewis‐acid‐catalyzed transannulation of humulene diepoxide and subsequent sequential olefin metathesis. Cheminformatic analysis quantitatively showed that the synthesized terpenoid alkaloid‐like compound library has a high level of three‐dimensional‐shape diversity. Extensive pharmacological screening of the library has led to the identification of promising compounds for the development of antihypolipidemic drugs. Therefore, the synthesis of terpenoid alkaloid‐like compound libraries based on humulene is well suited to drug discovery. Synthesis of terpenoid alkaloid‐like compounds based on several natural terpenoids is an effective strategy for producing chemically diverse libraries.     

A comprehensive library‐based,automated screening procedure for 46 synthetic cannabinoids in serum employing liquid chromatography‐quadrupole ion trap mass spectrometry with high‐temperature electrospray ionization

Considering the vast variety of synthetic cannabinoids and herbal mixtures – commonly known as ‘Spice’ or ‘K2’ – on the market and the resulting increase of severe intoxications related to their consumption, there is a need in clinical and forensic toxicology for comprehensive up‐to‐date screening methods. The focus of this project aimed at developing and implementing an automated screening procedure for the detection of synthetic cannabinoids in serum using a liquid chromatography‐ion trap‐MS (LC‐MSⁿ) system and a spectra library‐based approach, currently including 46 synthetic cannabinoids and 8 isotope labelled analogues. In the process of method development, a high‐temperature ESI source (IonBooster^TM, Bruker Daltonik) and its effects on the ionization efficiency of the investigated synthetic cannabinoids were evaluated and compared to a conventional ESI source. Despite their structural diversity, all investigated synthetic cannabinoids benefitted from high‐temperature ionization by showing remarkably higher MS intensities compared to conventional ESI. The employed search algorithm matches retention time, MS and MS²/MS³ spectra. With the utilization of the ionBooster source, limits for the automated detection comparable to cut‐off values of routine MRM methods were achieved for the majority of analytes. Even compounds not identified when using a conventional ESI source were detected using the ionBooster‐source. LODs in serum range from 0.1 ng/ml to 0.5 ng/ml. The use of parent compounds as analytical targets offers the possibility of instantly adding new emerging compounds to the library and immediately applying the updated method to serum samples, allowing the rapid adaptation of the screening method to ongoing forensic or clinical requirements. The presented approach can also be applied to other specimens, such as oral fluid or hair, and herbal mixtures and was successfully applied to authentic serum samples. Quantitative MRM results of samples with analyte concentrations above the determined LOD were confirmed as positive findings by the presented method. Copyright © 2014 John Wiley & Sons, Ltd.     

Enantiodifferentiation through Frequency‐Selective Pure‐Shift 1H Nuclear Magnetic Resonance Spectroscopy

A frequency‐selective 1D ¹H nuclear magnetic resonance (NMR) experiment for the fast and sensitive determination of chemical‐shift differences between overlapped resonances is proposed. The resulting fully homodecoupled ¹H NMR resonances appear as resolved 1D singlets without their typical J(HH) coupling constant multiplet structures. The high signal dispersion that is achieved is then exploited in enantiodiscrimination studies by using chiral solvating agents.     

Theoretical and experimental 15N NMR study of enamine–imine tautomerism of 4‐trifluoromethyl[b]benzo‐1,4‐diazepine system

The tautomeric structure of 4‐trifluoromethyl[b]benzo‐1,4‐diazepine system in solution has been evaluated by means of the calculation of ¹⁵N NMR chemical shifts of individual tautomers in comparison with the averaged experimental shifts to show that the enamine–imine equilibrium is entirely shifted toward the imine form. The adequacy of the theoretical level used for the computation of ¹⁵N NMR chemical shifts in this case has been verified based on the benchmark calculations in the series of the push–pull and captodative enamines together with related azomethynes, which demonstrated a good to excellent agreement with experiment. Copyright © 2015 John Wiley & Sons, Ltd.     

Fast and accurate predictions of binding free energies using MM‐PBSA and MM‐GBSA

In the drug discovery process, accurate methods of computing the affinity of small molecules with a biological target are strongly needed. This is particularly true for molecular docking and virtual screening methods, which use approximated scoring functions and struggle in estimating binding energies in correlation with experimental values. Among the various methods, MM‐PBSA and MM‐GBSA are emerging as useful and effective approaches. Although these methods are typically applied to large collections of equilibrated structures of protein‐ligand complexes sampled during molecular dynamics in water, the possibility to reliably estimate ligand affinity using a single energy‐minimized structure and implicit solvation models has not been explored in sufficient detail. Herein, we thoroughly investigate this hypothesis by comparing different methods for the generation of protein‐ligand complexes and diverse methods for free energy prediction for their ability to correlate with experimental values. The methods were tested on a series of structurally diverse inhibitors of Plasmodium falciparum DHFR with known binding mode and measured affinities. The results showed that correlations between MM‐PBSA or MM‐GBSA binding free energies with experimental affinities were in most cases excellent. Importantly, we found that correlations obtained with the use of a single protein‐ligand minimized structure and with implicit solvation models were similar to those obtained after averaging over multiple MD snapshots with explicit water molecules, with consequent save of computing time without loss of accuracy. When applied to a virtual screening experiment, such an approach proved to discriminate between true binders and decoy molecules and yielded significantly better enrichment curves. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Design of Selenium‐Based Chiral Chemical Probes for Simultaneous Enantio‐ and Chemosensing of Chiral Carboxylic Acids with Remote Stereogenic Centers by NMR Spectroscopy

Selenium‐based enantiopure chiral chemical probes have been designed in a modular way starting from available amino alcohols. The probes developed were found to be efficient in chemoselective interaction with carboxylic functions of chiral substrates leading to diastereomeric amide formation and in sensing α‐, β‐, and remote (up to seven bonds away from the carboxylic group) chiral centers by using ⁷⁷Se NMR spectroscopy. As a result, it was possible to determine the enantiomeric ratio of structurally diverse individual chiral acids including polyfunctional compounds and drugs with high accuracy. An approach to analyzing the crude reaction mixtures has been successfully developed by using bifunctional selenium‐ and fluorine‐containing chiral probes. More importantly, it was revealed that, based on the ⁷⁷Se NMR data obtained, it is possible to obtain primary information about the location and nature of the substituents at the chiral center (chemo‐ and enantiosensing), which can simplify the structural elucidation of complex compounds. The derivatization procedure takes as little as 5 min and can be performed directly in an NMR tube followed by NMR measurements without any isolation and purification steps.     

Experimental and quantum chemical calculational studies on N‐[4‐(3‐Methyl‐3‐phenyl‐cyclobutyl)‐thiazol‐2‐yl]‐N′‐pyridin‐3ylmethylene‐hydrazine

The title molecule, N‐[4‐(3‐Methyl‐3‐phenyl‐cyclobutyl)‐thiazol‐2‐yl]‐N′‐pyridin‐3ylmethylene‐ hydrazine (C₂₀ H₂₀ N₄ S₁), was characterized by ¹H‐NMR, ¹³C‐NMR, IR, UV‐visible, and X‐ray determination. In addition to the molecular geometry from X‐ray experiment, the molecular geometry, vibrational frequencies and gauge including atomic orbital ¹H‐ and ¹³C‐NMR chemical shift values of the title compound in the ground state have been calculated using the Hartree‐Fock and density functional method (B3LYP) with 6‐31G(d, p) basis set. The calculated results show that optimized geometries can well reproduce the crystal structural parameters. By using time‐dependent density functional theory method, electronic absorption spectrum of the title compound has been predicted. © 2011 Wiley Periodicals, Inc.     

A 93Nb Solid‐State NMR and Density Functional Theory Study of Four‐ and Six‐Coordinate Niobate Systems

A variable B₀ field static (broadline) NMR study of a large suite of niobate materials has enabled the elucidation of high‐precision measurement of ⁹³Nb NMR interaction parameters such as the isotropic chemical shift (δ_iso), quadrupole coupling constant and asymmetry parameter (C_Q and η_Q), chemical shift span/anisotropy and skew/asymmetry (Ω/Δδ and κ/η_δ) and Euler angles (α, β, γ) describing the relative orientation of the quadrupolar and chemical shift tensorial frames. These measurements have been augmented with ab initio DFT calculations by using WIEN2k and NMR‐CASTEP codes, which corroborate these reported values. Unlike previous assertions made about the inability to detect CSA (chemical shift anisotropy) contributions from Nb^V in most oxo environments, this study emphasises that a thorough variable B₀ approach coupled with the VOCS (variable offset cumulative spectroscopy) technique for the acquisition of undistorted broad (?1/2?+1/2) central transition resonances facilitates the unambiguous observation of both quadrupolar and CSA contributions within these ⁹³Nb broadline data. These measurements reveal that the ⁹³Nb electric field gradient tensor is a particularly sensitive measure of the immediate and extended environments of the Nb^V positions, with C_Q values in the 0 to >80 MHz range being measured; similarly, the δ_iso (covering an approximately 250 ppm range) and Ω values (covering a 0 to approximately 800 ppm range) characteristic of these niobate systems are also sensitive to structural disposition. However, their systematic rationalisation in terms of the Nb? O bond angles and distances defining the immediate Nb^V oxo environment is complicated by longer‐range influences that usually involve other heavy elements comprising the structure. It has also been established in this study that the best computational method(s) of analysis for the ⁹³Nb NMR interaction parameters generated here are the all‐electron WIEN2k and the gauge included projector augmented wave (GIPAW) NMR‐CASTEP DFT approaches, which account for the short‐ and long‐range symmetries, periodicities and interaction‐potential characteristics for all elements (and particularly the heavy elements) in comparison with Gaussian 03 methods, which focus on terminated portions of the total structure.     

Tetrazole‐Based Probes for Integrated Phenotypic Screening,Affinity‐Based Proteome Profiling,and Sensitive Detection of a Cancer Biomarker

Target‐identification phenotypic screening has been a powerful approach in drug discovery; however, it is hindered by difficulties in identifying the underlying cellular targets. To address this challenge, we have combined phenotypic screening of a fully functionalized small‐molecule library with competitive affinity‐based proteome profiling to map and functionally characterize the targets of screening hits. Using this approach, we identified ANXA2, PDIA3/4, FLAD1, and NOS2 as primary cellular targets of two bioactive molecules that inhibit cancer cell proliferation. We further demonstrated that a panel of probes can label and/or image annexin A2 (a cancer biomarker) from different cancer cell lines, thus providing opportunities for potential cancer diagnosis and therapy.     

Substitution effects in the 15N NMR chemical shifts of heterocyclic azines evaluated at the GIAO‐DFT level

A systematic study of the accuracy factors for the computation of ¹⁵N NMR chemical shifts in comparison with available experiment in the series of 72 diverse heterocyclic azines substituted with a classical series of substituents (CH₃, F, Cl, Br, NH₂, OCH₃, SCH₃, COCH₃, CONH₂, COOH, and CN) providing marked electronic σ‐ and π‐electronic effects and strongly affecting ¹⁵N NMR chemical shifts is performed. The best computational scheme for heterocyclic azines at the DFT level was found to be KT3/pcS‐3//pc‐2 (IEF‐PCM). A vast amount of unknown ¹⁵N NMR chemical shifts was predicted using the best computational protocol for substituted heterocyclic azines, especially for trizine, tetrazine, and pentazine where experimental ¹⁵N NMR chemical shifts are almost totally unknown throughout the series. It was found that substitution effects in the classical series of substituents providing typical σ‐ and π‐electronic effects followed the expected trends, as derived from the correlations of experimental and calculated ¹⁵N NMR chemical shifts with Swain–Lupton's F and R constants.