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.     

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.     

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.     

Library Design Strategies To Accelerate Fragment-Based Drug Discovery

Fragment-based drug discovery (FBDD) has become an established approach for the generation of early lead candidates. However, despite its success and inherent advantages, hit-to-candidate progression for FBDD is not necessarily faster than that of traditional high-throughput screening. Thus, new technology-driven library design strategies have emerged as a means to facilitate more efficient fragment screening and/or subsequent fragment-to-hit chemistry. This minireview discusses such strategies, which cover the use of labeled fragments for NMR spectroscopy, X-ray crystallographic screening of specialized fragments, covalent linkage for mass spectrometry, dynamic combinatorial chemistry, and fragments optimized for easy elaboration.     

Expanding medicinal chemistry into 3D space: metallofragments as 3D scaffolds for fragment-based drug discovery

Fragment-based drug discovery (FBDD) is a powerful strategy for the identification of new bioactive molecules. FBDD relies on fragment libraries, generally of modest size, but of high chemical diversity. Although good chemical diversity in FBDD libraries has been achieved in many respects, achieving shape diversity – particularly fragments with three-dimensional (3D) structures – has remained challenging. A recent analysis revealed that >75% of all conventional, organic fragments are predominantly 1D or 2D in shape. However, 3D fragments are desired because molecular shape is one of the most important factors in molecular recognition by a biomolecule. To address this challenge, the use of inert metal complexes, so-called ‘metallofragments’ (mFs), to construct a 3D fragment library is introduced. A modest library of 71 compounds has been prepared with rich shape diversity as gauged by normalized principle moment of inertia (PMI) analysis. PMI analysis shows that these metallofragments occupy an area of fragment space that is unique and highly underrepresented when compared to conventional organic fragment libraries that are comprised of orders of magnitude more molecules. The potential value of this metallofragment library is demonstrated by screening against several different types of proteins, including an antiviral, an antibacterial, and an anticancer target. The suitability of the metallofragments for future hit-to-lead development was validated through the determination of IC₅₀ and thermal shift values for select fragments against several proteins. These findings demonstrate the utility of metallofragment libraries as a means of accessing underutilized 3D fragment space for FBDD against a variety of protein targets.

Fragment-based drug discovery (FBDD) using 3-dimensional metallofragments is a new strategy for the identification of bioactive molecules.     

The multiple roles of computational chemistry in fragment-based drug design

Fragment-based drug discovery (FBDD) represents a change in strategy from the screening of molecules with higher molecular weights and physical properties more akin to fully drug-like compounds, to the screening of smaller, less complex molecules. This is because it has been recognised that fragment hit molecules can be efficiently grown and optimised into leads, particularly after the binding mode to the target protein has been first determined by 3D structural elucidation, e.g. by NMR or X-ray crystallography. Several studies have shown that medicinal chemistry optimisation of an already drug-like hit or lead compound can result in a final compound with too high molecular weight and lipophilicity. The evolution of a lower molecular weight fragment hit therefore represents an attractive alternative approach to optimisation as it allows better control of compound properties. Computational chemistry can play an important role both prior to a fragment screen, in producing a target focussed fragment library, and post-screening in the evolution of a drug-like molecule from a fragment hit, both with and without the available fragment-target co-complex structure. We will review many of the current developments in the area and illustrate with some recent examples from successful FBDD discovery projects that we have conducted.     

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.     

Druggable Allosteric Sites in β-Propeller Lectins

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a β-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing ¹⁹F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure–activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol^?1 HA^?1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens.     

Specific Detection and Imaging of Enzyme Activity by Signal‐Amplifiable Self‐Assembling 19F MRI Probes

Specific turn‐on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal‐amplifiable self‐assembling ¹⁹F NMR/MRI probe for turn‐on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are “silent” when aggregated, but exhibit a disassembly driven turn‐on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these ¹⁹F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by ¹⁹F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by ¹⁹F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn‐on ¹⁹F probes may serve as a screening platform for the activity of MMPs.     

Monitoring Glycan–Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH–π Interactions

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan–protein interactions that involve N‐acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc‐containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal ¹H and ¹⁹F atoms that allow both ¹H and ¹⁹F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc‐ (or GalNAc‐) moieties over a range of binding affinities. The CHF₂CONH‐ moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic‐sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF₃CONH‐ analogue differs more significantly.     

Fragment screening of cyclin G-associated kinase by weak affinity chromatography

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K (D))?=?mM?-?μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46?%) with K (D)?相似文献   

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.     

Partially Saturated Bicyclic Heteroaromatics as an sp3‐Enriched Fragment Collection

Fragment‐based lead generation has proven to be an effective means of identifying high‐quality lead compounds for drug discovery programs. However, the fragment screening sets often used are principally comprised of sp²‐rich aromatic compounds, which limits the structural (and hence biological) diversity of the library. Herein, we describe strategies for the synthesis of a series of partially saturated bicyclic heteroaromatic scaffolds with enhanced sp³ character. Subsequent derivatization led to a fragment collection featuring regio‐ and stereo‐controlled introduction of substituents on the saturated ring system, often with formation of new stereocenters.     

Fluorinated Carbohydrates as Lectin Ligands: Dissecting Glycan–Cyanovirin Interactions by Using 19F NMR Spectroscopy

NMR spectroscopy and isothermal titration calorimetry (ITC) are powerful methods to investigate ligand–protein interactions. Here, we present a versatile and sensitive fluorine NMR spectroscopic approach that exploits the ¹⁹F nucleus of ¹⁹F‐labeled carbohydrates as a sensor to study glycan binding to lectins. Our approach is illustrated with the 11 kDa Cyanovirin‐N, a mannose binding anti‐HIV lectin. Two fluoro‐deoxy sugar derivatives, methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside and methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside were utilized. Binding was studied by ¹⁹F NMR spectroscopy of the ligand and ¹H–¹⁵N HSQC NMR spectroscopy of the protein. The NMR data agree well with those obtained from the equivalent reciprocal and direct ITC titrations. Our study shows that the strategic design of fluorinated ligands and fluorine NMR spectroscopy for ligand screening holds great promise for easy and fast identification of glycan binding, as well as for their use in reporting structural and/or electronic perturbations that ensue upon interaction with a cognate lectin.     

Parahydrogen‐Induced Polarization Transfer to 19F in Perfluorocarbons for 19F NMR Spectroscopy and MRI

Fluorinated substances are important in chemistry, industry, and the life sciences. In a new approach, parahydrogen‐induced polarization (PHIP) is applied to enhance ¹⁹F MR signals of (perfluoro‐n‐hexyl)ethene and (perfluoro‐n‐hexyl)ethane. Unexpectedly, the end‐standing CF₃ group exhibits the highest amount of polarization despite the negligible coupling to the added protons. To clarify this non‐intuitive distribution of polarization, signal enhancements in deuterated chloroform and acetone were compared and ¹⁹F–¹⁹F NOESY spectra, as well as ¹⁹F T₁ values were measured by NMR spectroscopy. By using the well separated and enhanced signal of the CF₃ group, first ¹⁹F MR images of hyperpolarized linear semifluorinated alkenes were recorded.     

19F‐NMR Reveals the Role of Mobile Loops in Product and Inhibitor Binding by the São Paulo Metallo‐β‐Lactamase

Resistance to β‐lactam antibiotics mediated by metallo‐β‐lactamases (MBLs) is a growing problem. We describe the use of protein‐observe ¹⁹F‐NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM‐1) from β‐lactam‐resistant Pseudomonas aeruginosa . Cysteinyl variants on the α3 and L3 regions, which flank the di‐Zn^II active site, were selectively ¹⁹F‐labeled using 3‐bromo‐1,1,1‐trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β‐lactam products to SPM‐1. These results have implications for the mechanisms and inhibition of MBLs by β‐lactams and non‐β‐lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers.     

Identification,synthesis and evaluation of CSF1R inhibitors using fragment based drug design

Colony-stimulating factor 1 receptor is a type III receptor protein tyrosine kinase belonging to PDGFR family. CSF1R signaling is essential for differentiation, proliferation and survival of macrophages. Aberrant expression of CSF1R appears to be an attractive target in several cancer types. Higher expression of CSF1R ligands correlates to tumor progression. CSF1R inhibitors have been shown to suppress cancers. We have attempted an in silico fragment derived drug discovery approach by screening ˜25,000 in-house compounds as potential CSF1R inhibitors. Using FBDD approach we have identified six diverse fragments that exhibit affinity towards hinge region of CSF1R. Some of the fragments 5-nitroindole and 7-azaindole and their derivatives were synthesized for further evaluation. The in silico and in vitro enzyme activity studies reveal moderate inhibition of CSF1R kinase activity by 5-nitroindole and good inhibition by 7-azaindole fragments. Bio and chemiinformatics studies have shown that 7-azaindole compounds have better membrane permeability and enzyme inhibition properties. Molecular docking studies show that the amino acid residues 664–666 in the hinge region of the cytosolic domain of CSF1R to be the preferred region of binding for nitroindole and azaindole derivatives. Further optimization and biological analysis would identify these fragments as potential and promising leads as CSF1R inhibitors.     

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC₅₀ value of 54 nm , which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization.