RESUMEN
NMR spectroscopy is currently extensively used in binding assays for hit identification, but its use in dissociation constant determination is more limited when compared to other biophysical techniques, in particular for tight binders. Although NMR is quite suitable for measuring the binding strength of weak to medium affinity ligands with dissociation constant KD > 1 µM, it has some limitations in the determination of the binding strength of tight binders (KD < 1 µM). A theoretical analysis of the binding affinity determination of strong ligands using different types of NMR experiments is provided and practical guidelines are given for overcoming the limitations and for the proper set-up of the experiments. Some approaches require reagents with unique properties or highly specialized equipment, while others can be applied quite generally. We describe all approaches in detail, but give higher emphasis to the more general methods, like competition experiments, where we include actual experimental data and discuss the practical aspects.
RESUMEN
Ligand-based 19 F NMR screening is a highly effective and well-established hit-finding approach. The high sensitivity to protein binding makes it particularly suitable for fragment screening. Different criteria can be considered for generating fluorinated fragment libraries. One common strategy is to assemble a large, diverse, well-designed and characterized fragment library which is screened in mixtures, generated based on experimental 19 F NMR chemical shifts. Here, we introduce a complementary knowledge-based 19 F NMR screening approach, named 19 Focused screening, enabling the efficient screening of putative active molecules selected by computational hit finding methodologies, in mixtures assembled and on-the-fly deconvoluted based on predicted 19 F NMR chemical shifts. In this study, we developed a novel approach, named LEFshift, for 19 F NMR chemical shift prediction using rooted topological fluorine torsion fingerprints in combination with a random forest machine learning method. A demonstration of this approach to a real test case is reported.
Asunto(s)
Flúor , Imagen por Resonancia Magnética , Flúor/química , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Unión ProteicaRESUMEN
Fluorinated non-natural amino acids are useful tools for improving the bioavailability of peptides but can also serve as fluorinated probes in 19 F NMR-based enzymatic assays. We report herein that the use of the non-natural α-quaternarized (R)-α-trifluoromethylalanine ((R)-α-TfmAla) provides convenient and accurate monitoring of trypsin proteolytic activity and increases resistance towards pepsin degradation.
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Alanina/análogos & derivados , Péptido Hidrolasas/metabolismo , Péptidos/metabolismo , Alanina/química , Imagen por Resonancia Magnética con Fluor-19 , Estructura Molecular , Péptido Hidrolasas/química , Péptidos/química , EstereoisomerismoRESUMEN
The propensity of organic fluorine acting as a weak hydrogen bond acceptor (HBA) in intermolecular and intramolecular interactions has been the subject of many experimental and theoretical studies often reaching different conclusions. Over the last few years, new and stronger evidences have emerged for the direct involvement of fluorine in weak hydrogen bond (HB) formation. However, not all the fluorine atom types can act as weak HBA. In this work, the differential HBA propensity of various types of fluorine atoms was analyzed with a particular emphasis for the different types of alkyl fluorides. This was carried out by evaluating ab initio computed parameters, experimental 19 Fâ NMR chemical shifts and small molecule crystallographic structures (extracted from the CSD database). According to this analysis, shielded (with reference to the 19 Fâ NMR chemical shift) alkyl mono-fluorinated motifs display the highest HBA propensity in agreement with solution studies. Although much weaker than other well-characterized HB complexes, the fragile HBs formed by these fluorinated motifs have important implications for the chemical-physical and structural properties of the molecules, chemical reactions, and protein-ligand recognition.
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Fluoruros , Flúor , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , ProteínasRESUMEN
The substrate- or cofactor-based fluorine NMR screening, also known as n-FABS (n fluorine atoms for biochemical screening), represents a powerful method for performing a direct functional assay in the search of inhibitors or enhancers of an enzymatic reaction. Although it suffers from the intrinsic low sensitivity compared to other biophysical techniques usually applied in functional assays, it has some distinctive features that makes it appealing for tackling complex chemical and biological systems. Its strengths are represented by the easy set-up, robustness, flexibility, lack of signal interference and rich information content resulting in the identification of bona fide inhibitors and reliable determination of their inhibitory strength. The versatility of the n-FABS allows its application to either purified enzymes, cell lysates or intact living cells. The principles, along with theoretical, technical and practical aspects, of the methodology are discussed. Furthermore, several applications of the technique to pharmaceutical projects are presented.
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Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Enzimas/química , Flúor/química , Resonancia Magnética Nuclear Biomolecular/métodos , Amidohidrolasas/química , Catálisis , Células HEK293 , Halogenación , Humanos , Concentración 50 Inhibidora , Péptidos/química , Proteínas Proto-Oncogénicas c-akt/química , Tripsina/químicaRESUMEN
Ligand-based NMR screening represents a powerful method in fragment-based drug discovery for the identification of chemical matter interacting with the receptor of interest. The large dynamic range of these methods allows the detection of weakly binding ligands. However, the methodology has not been extensively used for quantifying the strength of these interactions. This knowledge is important for ranking fragments according to their binding strength and for prioritizing structure-based and medicinal chemistry activities. Rapid NMR methods for measuring the dissociation constant in direct and competition modes are presented here. The theory underpinning these methods are presented, along with their application to the measurement of the binding affinities of several ligands of the heat shock proteinâ 90.
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Proteínas HSP90 de Choque Térmico/química , Descubrimiento de Drogas , Ligandos , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
Ligand-based fluorine NMR screening has gained popularity in drug discovery projects during the past decade and has become a powerful methodology to produce high quality hits. Its high sensitivity to protein binding makes it particularly suitable for fragment screening, allowing detection and binding strength measurement of very weak affinity ligands. The screening can be performed in direct or competition format, and its versatility allows application to complex biological and chemical systems. As the potential of the methodology has now been recognized and successfully demonstrated in several relevant medicinal chemistry projects, it is now an appropriate time to report the learned lessons and point the way to the future. In this Perspective the principles of the methodology along with several applications to pharmaceutical projects are presented.
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Descubrimiento de Drogas/métodos , Flúor/química , Espectroscopía de Resonancia Magnética/métodos , Ligandos , Unión Proteica , Control de CalidadRESUMEN
Fluorine ligand-based NMR spectroscopy is now an established method for performing binding screening against a macromolecular target. Typically, the transverse relaxation rate of the fluorine signals is monitored in the absence and presence of the target. However, useful structural information can sometimes be obtained from the analysis of the fluorine isotropic chemical shift. This is particularly relevant for molecules that are racemates and/or display multiple conformers. The large difference in fluorine isotropic chemical shift between free and bound state deriving mainly from the breaking and/or making of intramolecular and/or intermolecular hydrogen bonds allows the detection of very weak affinity ligands. According to our experimental results, racemates should always be included in the generation of the fluorinated fragment libraries. The selection or the availability of only one of the enantiomers for the fluorinated screening library could result in missing relevant chemical scaffold motifs.
RESUMEN
Over the years a significant amount of effort has been put into the development of rapid and reliable methods to monitor the aggregation dynamics of the ß1-42 amyloid peptide in real time. We present an alternative approach based on a suitable reporter or spy molecule and three different NMR experiments: WaterLOGSY, 1 H selective T1 filter, and 19 F T2 filter, for monitoring the initial self-aggregation process kinetics of the ß1-42 amyloid peptide and identifying molecules that retard or accelerate the self-aggregation process. Although the proposed method is not a high-throughput assay, it avoids problems associated with interference events that are sometimes observed in fluorescence-based assays.
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Péptidos beta-Amiloides/química , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Agregado de Proteínas/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Cinética , LigandosRESUMEN
Chronic dysregulation of alternative complement pathway activation has been associated with diverse clinical disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinurea. Factor D is a trypsin-like serine protease with a narrow specificity for arginine in the P1 position, which catalyzes the first enzymatic reaction of the amplification loop of the alternative pathway. In this article, we describe two hit finding approaches leading to the discovery of new chemical matter for this pivotal protease of the complement system: in silico active site mapping for hot spot identification to guide rational structure-based design and NMR screening of focused and diverse fragment libraries. The wealth of information gathered by these complementary approaches enabled the identification of ligands binding to different subpockets of the latent Factor D conformation and was instrumental for understanding the binding requirements for the generation of the first known potent noncovalent reversible Factor D inhibitors.
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Inhibidores de Proteasas/farmacología , Dominio Catalítico , Factor D del Complemento/química , Diseño de Fármacos , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Inhibidores de Proteasas/químicaRESUMEN
Ligand-based 19 F 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 19 F transverse relaxation rate and isotropic chemical shift. However, there are few cases where the 19 F longitudinal relaxation rate should also be used. A theoretical and experimental analysis of the 19 F 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.
RESUMEN
It is known that strong hydrogen-bonding interactions play an important role in many chemical and biological systems. However, weak or very weak hydrogen bonds, which are often difficult to detect and characterize, may also be relevant in many recognition and reaction processes. Fluorine serving as a hydrogen-bond acceptor has been the subject of many controversial discussions and there are different opinions about it. It now appears that there is compelling experimental evidence for the involvement of fluorine in weak intramolecular or intermolecular hydrogen bonds. Using established NMR methods, we have previously characterized and measured the strengths of intermolecular hydrogen-bond complexes involving the fluorine moieties CH2 F, CHF2 , and CF3 , and have compared them with the well-known hydrogen-bond complex formed between acetophenone and the strong hydrogen-bond donor p-fluorophenol. We now report evidence for the formation of hydrogen bonds involving fluorine with significantly weaker donors, namely 5-fluoroindole and water. A simple NMR method is proposed for the simultaneous measurement of the strengths of hydrogen bonds between an acceptor and a donor or water. Important implications of these results for enzymatic/chemical reactions involving fluorine, for chemical and physical properties, and for ligand/protein (19) Fâ NMR screening are analyzed through experiments and theoretical simulations.
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Flúor/química , Proteínas/química , Enlace de Hidrógeno , Indoles/química , Ligandos , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Fenoles/química , AguaRESUMEN
Nuclear magnetic resonance (NMR)-based screening has been recognized as a powerful approach for the identification and characterization of molecules interacting with pharmaceutical targets. Indeed, several NMR methods have been developed and successfully applied to many drug discovery projects. Whereas most of these approaches have targeted isolated biomolecular receptors, very few cases are reported with the screening performed in intact cells and cell extracts. Here we report the first successful application of the fluorine NMR-based assay n-FABS (n-fluorine atoms for biochemical screening) in living mammalian cells expressing the membrane protein fatty acid amide hydrolase (FAAH). This method allows the identification of both weak and potent inhibitors and the measurement of their potency in a physiological environment.
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Amidohidrolasas/análisis , Resonancia Magnética Nuclear Biomolecular , Amidohidrolasas/metabolismo , Benzamidas/química , Benzamidas/metabolismo , Carbamatos/química , Carbamatos/metabolismo , Flúor/química , Células HEK293 , Humanos , Concentración 50 InhibidoraRESUMEN
ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand-target complexes in the gas phase and allows inference of ligand-target binding in solution. In this article we used this approach to investigate the interaction of ligands to the Heat Shock Protein 90 (Hsp90). This enzyme is a molecular chaperone involved in the folding and maturation of several proteins which has been subjected in the last years to intensive drug discovery efforts due to its key role in cancer. In particular, reference compounds, with a broad range of dissociation constants from 40pM to 100µM, were tested to assess the reliability of ESI-MS for the study of protein-ligand complexes. A good agreement was found between the values measured with a fluorescence polarization displacement assay and those determined by mass spectrometry. After this validation step we describe the setup of a medium throughput screening method, based on ESI-MS, suitable to explore interactions of therapeutic relevance biopolymers with chemical libraries. Our approach is based on an automated flow injection ESI-MS method (AFI-MS) and has been applied to screen the Nerviano Medical Sciences proprietary fragment library of about 2000 fragments against Hsp90. In order to discard false positive hits and to discriminate those of them interacting with the N-terminal ATP binding site, competition experiments were performed using a reference inhibitor. Gratifyingly, this group of hits matches with the ligands previously identified by NMR FAXS techniques and confirmed by X-ray co-crystallization experiments. These results support the use of AFI-MS for the screening of medium size libraries, including libraries of small molecules with low affinity typically used in fragment based drug discovery. AFI-MS is a valid alternative to other techniques with the additional opportunities to identify compounds interacting with unpredicted or allosteric sites, without the need of any binding probes.
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Descubrimiento de Drogas/métodos , Análisis de Inyección de Flujo , Proteínas HSP90 de Choque Térmico/metabolismo , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Espectrometría de Masa por Ionización de Electrospray , Automatización de Laboratorios , Sitios de Unión , Unión Competitiva , Cristalografía por Rayos X , Polarización de Fluorescencia , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/química , Ligandos , Espectroscopía de Resonancia Magnética , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
Hydrogen-bonding interactions play an important role in many chemical and biological systems. Fluorine acting as a hydrogen-bond acceptor in intermolecular and intramolecular interactions has been the subject of many controversial discussions and there are different opinions about it. Recently, we have proposed a correlation between the propensity of fluorine to be involved in hydrogen bonds and its (19)Fâ NMR chemical shift. We now provide additional experimental and computational evidence for this correlation. The strength of hydrogen-bond complexes involving the fluorine moieties CH2F, CHF2, and CF3 was measured and characterized in simple systems by using established and novel NMR methods and compared to the known hydrogen-bond complex formed between acetophenone and p-fluorophenol. Implications of these results for (19)Fâ NMR screening are analyzed in detail. Computed values of the molecular electrostatic potential at the different fluorine atoms and the analysis of the electron density topology at bond critical points correlate well with the NMR results.
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Simulación por Computador , Flúor/química , Diseño de Fármacos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , TermodinámicaRESUMEN
In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.
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Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Quinazolinas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Quinazolinas/síntesis química , Quinazolinas/farmacología , Relación Estructura-ActividadRESUMEN
Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral target. Here, we report the first successful application of (19)F-NMR fragment-based screening to identify chemically diverse compounds that bind to an RNA molecule such as TERRA. We have built a library of 355 fluorinated fragments, and checked their interaction with a long telomeric RNA as a target molecule. The screening resulted in the identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction with TERRA was confirmed by (19)F- and (1)H NMR as well as by CD melting experiments. We have also explored the selectivity of the ligands for RNA G-quadruplexes and found that some of the hits do not interact with other nucleic acids such as tRNA and duplex DNA and, most importantly, favor the propeller-like parallel conformation in telomeric DNA G-quadruplexes. This suggests a selective recognition of this particular quadruplex topology and that different ligands may recognize specific sites in propeller-like parallel G-quadruplexes. Such features make some of the resulting binders promising lead compounds for fragment based drug discovery.
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Antineoplásicos/química , G-Cuádruplex/efectos de los fármacos , ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Antineoplásicos/farmacología , Secuencia de Bases , Descubrimiento de Drogas , Halogenación , Humanos , Ligandos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Resonancia Magnética Nuclear Biomolecular , ARN/química , Secuencias Repetitivas de Ácidos Nucleicos/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Telómero/química , Telómero/metabolismoRESUMEN
In order to defend themselves against arthropod herbivores, maize plants produce 1,4-benzoxazin-3-ones (BXs), which are stored as weakly active glucosides in the vacuole. Upon tissue disruption, BXs come into contact with ß-glucosidases, resulting in the release of active aglycones and their breakdown products. While some aglycones can be reglucosylated by specialist herbivores, little is known about how they detoxify BX breakdown products. Here we report on the structure of an N-glucoside, 3-ß-d-glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc), purified from Spodoptera frugiperda faeces. In vitro assays showed that MBOA-N-Glc is formed enzymatically in the insect gut using the BX breakdown product 6-methoxy-2-benzoxazolinone (MBOA) as precursor. While Spodoptera littoralis and S. frugiperda caterpillars readily glucosylated MBOA, larvae of the European corn borer Ostrinia nubilalis were hardly able to process the molecule. Accordingly, Spodoptera caterpillar growth was unaffected by the presence of MBOA, while O. nubilalis growth was reduced. We conclude that glucosylation of MBOA is an important detoxification mechanism that helps insects tolerate maize BXs.
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Benzoxazoles/metabolismo , Glucósidos/metabolismo , Spodoptera/metabolismo , Zea mays/química , Animales , Benzoxazoles/química , Glucósidos/química , Inactivación Metabólica , Estructura Molecular , Spodoptera/químicaRESUMEN
The possibility of measuring the action of inhibitors of specific enzymatic reactions in intact cells, cell lysates or membrane preparations represents a major advance in the lead discovery process. Despite the relevance of assaying in physiological conditions, only a small number of biophysical techniques, often requiring complex set-up, are applicable to these sample types. Here, we demonstrate the first application of n-fluorine atoms for biochemical screening (n-FABS), a homogeneous and versatile assay based on (19) Fâ NMR spectroscopy, to the detection of high- and low-affinity inhibitors of a membrane enzyme in cell extracts and determination of their IC50 values. Our approach can allow the discovery of novel binding fragments against targets known to be difficult to purify or where membrane-association is required for activity. These results pave the way for future applications of the methodology to these relevant and complex biological systems.
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Membrana Celular/enzimología , Descubrimiento de Drogas/métodos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Membrana Celular/efectos de los fármacos , Flúor/análisis , Humanos , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
Fragment screening performed with (19) Fâ NMR spectroscopy is becoming increasingly popular in drug discovery projects. With this approach, libraries of fluorinated fragments are first screened using the direct-mode format of the assay. The choice of fluorinated motifs present in the library is fundamental in order to ensure a large coverage of chemical space and local environment of fluorine (LEF). Mono- and poly-fluorinated fragments to be included in the libraries for screening are selected from both in-house and commercial collections, and those that are ad hoc designed and synthesized. Additional fluorinated motifs to be included in the libraries derive from the fragmentation of compounds in development and launched on the market, and compounds contained in other databases (such as Integrity, PDB and ChEMBL). Complex mixtures of highly diverse fluorine motifs can be rapidly screened and deconvoluted in the same NMR tube with a novel on the fly combined procedure for the identification of the active molecule(s). Issues and problems encountered in the design, generation and screening of diverse fragment libraries of fluorinated compounds with (19) Fâ NMR spectroscopy are analyzed and technical solutions are provided to overcome them. The versatile screening methodology described here can be efficiently applied in laboratories with limited NMR setup and could potentially lead to the increasing role of (19) Fâ NMR in the hit identification and lead optimization phases of drug discovery projects.