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The performance of molecular docking can be improved by comparing the shape similarity of the flexibly sampled poses against the target proteins' inverted binding cavities. The effectiveness of these pseudo-ligands or negative image-based models in docking rescoring is boosted further by performing enrichment-driven optimization. Here, we introduce a novel shape-focused pharmacophore modeling algorithm O-LAP that generates a new class of cavity-filling models by clumping together overlapping atomic content via pairwise distance graph clustering. Top-ranked poses of flexibly docked active ligands were used as the modeling input and multiple alternative clustering settings were benchmark-tested thoroughly with five demanding drug targets using random training/test divisions. In docking rescoring, the O-LAP modeling typically improved massively on the default docking enrichment; furthermore, the results indicate that the clustered models work well in rigid docking. The C+ +/Qt5-based algorithm O-LAP is released under the GNU General Public License v3.0 via GitHub ( https://github.com/jvlehtonen/overlap-toolkit ). SCIENTIFIC CONTRIBUTION: This study introduces O-LAP, a C++/Qt5-based graph clustering software for generating new type of shape-focused pharmacophore models. In the O-LAP modeling, the target protein cavity is filled with flexibly docked active ligands, the overlapping ligand atoms are clustered, and the shape/electrostatic potential of the resulting model is compared against the flexibly sampled molecular docking poses. The O-LAP modeling is shown to ensure high enrichment in both docking rescoring and rigid docking based on comprehensive benchmark-testing.
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Molecular docking is a key method used in virtual screening (VS) campaigns to identify small-molecule ligands for drug discovery targets. While docking provides a tangible way to understand and predict the protein-ligand complex formation, the docking algorithms are often unable to separate active ligands from inactive molecules in practical VS usage. Here, a novel docking and shape-focused pharmacophore VS protocol is demonstrated for facilitating effective hit discovery using retinoic acid receptor-related orphan receptor gamma t (RORγt) as a case study. RORγt is a prospective target for treating inflammatory diseases such as psoriasis and multiple sclerosis. First, a commercial molecular database was flexibly docked. Second, the alternative docking poses were rescored against the shape/electrostatic potential of negative image-based (NIB) models that mirror the target's binding cavity. The compositions of the NIB models were optimized via iterative trimming and benchmarking using a greedy search-driven algorithm or brute force NIB optimization. Third, a pharmacophore point-based filtering was performed to focus the hit identification on the known RORγt activity hotspots. Fourth, free energy binding affinity evaluation was performed on the remaining molecules. Finally, twenty-eight compounds were selected for in vitro testing and eight compounds were determined to be low µM range RORγt inhibitors, thereby showing that the introduced VS protocol generated an effective hit rate of ~29%.
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Descubrimiento de Drogas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Simulación del Acoplamiento Molecular , Factores de Transcripción , Receptores de Ácido Retinoico , Tretinoina , LigandosRESUMEN
Despite the pivotal role of molecular docking in modern drug discovery, the default docking scoring functions often fail to recognize active ligands in virtual screening campaigns. Negative image-based rescoring improves docking enrichment by comparing the shape/electrostatic potential (ESP) of the flexible docking poses against the target protein's inverted cavity volume. By optimizing these negative image-based (NIB) models using a greedy search, the docking rescoring yield can be improved massively and consistently. Here, a fundamental modification is implemented to this shape-focused pharmacophore modelling approach-actual ligand 3D coordinates are incorporated into the NIB models for the optimization. This hybrid approach, labelled as ligand-enhanced brute-force negative image-based optimization (LBR-NiB), takes the best from both worlds, i.e., the all-roundedness of the NIB models and the difficult to emulate atomic arrangements of actual protein-bound small-molecule ligands. Thorough benchmarking, focused on proinflammatory targets, shows that the LBR-NiB routinely improves the docking enrichment over prior iterations of the R-NiB methodology. This boost can be massive, if the added ligand information provides truly essential binding information that was lacking or completely missing from the cavity-based NIB model. On a practical level, the results indicate that the LBR-NiB typically works well when the added ligand 3D data originates from a high-quality source, such as X-ray crystallography, and, yet, the NIB model compositions can also sometimes be improved by fusing into them, for example, with flexibly docked solvent molecules. In short, the study demonstrates that the protein-bound ligands can be used to improve the shape/ESP features of the negative images for effective docking rescoring use in virtual screening.
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Descubrimiento de Drogas , Sitios de Unión , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Electricidad EstáticaRESUMEN
COVID-19 is more virulent and challenging to human life. In India, the Ministry of AYUSH recommended some strategies through Siddha, homeopathy, and other methods to effectively manage COVID-19 (Guidelines for AYUSH Clinical Studies in COVID-19, 2020). Kabasura Kudineer and homeopathy medicines are in use for the prevention and treatment of COVID-19 infection; however, the mechanism of action is less explored. This study aims to understand the antagonist activity of natural compounds found in Kabasura Kudineer and homeopathy medicines against the SARS-CoV-2 using computational methods. Potential compounds were screened against NSP-12, NSP-13, NSP-14, NSP-15, main protease, and spike proteins. Structure-based virtual screening results shows that, out of 14,682 Kabasura Kudineer compounds, the 250395, 129677029, 44259583, 44259584, and 88583189 compounds and, out of 3,112 homeopathy compounds, the 3802778, 320361, 5315832, 14590080, and 74029795 compounds have good scoring function against the SARS-CoV-2 structural and nonstructural proteins. As a result of docking, homeopathy compounds have a docking score ranging from -5.636 to 13.631 kcal/mol, while Kabasura Kudineer compounds have a docking score varying from -8.290 to -13.759 kcal/mol. It has been found that the selected compounds bind well to the active site of SARS-CoV-2 proteins and form hydrogen bonds. The molecular dynamics simulation study shows that the selected compounds have maintained stable conformation in the simulation period and interact with the target. This study supports the antagonist activity of natural compounds from Kabasura Kudineer and homeopathy against SARS-CoV-2's structural and nonstructural proteins.
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Rab geranylgeranyltransferase (GGTase-II, RGGT) catalyses the post-translational modification of eukaryotic Rab GTPases, proteins implicated in several pathologies, including cancer, diabetes, neurodegenerative, and infectious diseases. Thus, RGGT inhibitors are believed to be a potential platform for the development of drugs and tools for studying processes related to the abnormal activity of Rab GTPases. Here, a series of new α-phosphonocarboxylates have been prepared in the first attempt of rational design of covalent inhibitors of RGGT derived from non-covalent inhibitors. These compounds were equipped with electrophilic groups capable of binding cysteines, which are present in the catalytic cavity of RGGT. A few of these analogues have shown micromolar activity against RGGT, which correlated with their ability to inhibit the proliferation of the HeLa cancer cell line. The proposed mechanism of this inhibitory activity was rationalised by molecular docking and mass spectrometric measurements, supported by stability and reactivity studies.
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Transferasas Alquil y Aril , Transferasas Alquil y Aril/metabolismo , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Molecular docking is a key in silico method used routinely in modern drug discovery projects. Although docking provides high-quality ligand binding predictions, it regularly fails to separate the active compounds from the inactive ones. In negative image-based rescoring (R-NiB), the shape/electrostatic potential (ESP) of docking poses is compared to the negative image of the protein's ligand binding cavity. While R-NiB often improves the docking yield considerably, the cavity-based models do not reach their full potential without expert editing. Accordingly, a greedy search-driven methodology, brute force negative image-based optimization (BR-NiB), is presented for optimizing the models via iterative editing and benchmarking. Thorough and unbiased training, testing and stringent validation with a multitude of drug targets, and alternative docking software show that BR-NiB ensures excellent docking efficacy. BR-NiB can be considered as a new type of shape-focused pharmacophore modeling, where the optimized models contain only the most vital cavity information needed for effectively filtering docked actives from the inactive or decoy compounds. Finally, the BR-NiB code for performing the automated optimization is provided free-of-charge under MIT license via GitHub (https://github.com/jvlehtonen/brutenib) for boosting the success rates of docking-based virtual screening campaigns.
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Programas Informáticos , Sitios de Unión , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Electricidad EstáticaRESUMEN
Projects in chemo- and bioinformatics often consist of scattered data in various types and are difficult to access in a meaningful way for efficient data analysis. Data is usually too diverse to be even manipulated effectively. Sdfconf is data manipulation and analysis software to address this problem in a logical and robust manner. Other software commonly used for such tasks are either not designed with molecular and/or conformational data in mind or provide only a narrow set of tasks to be accomplished. Furthermore, many tools are only available within commercial software packages. Sdfconf is a flexible, robust, and free-of-charge tool for linking data from various sources for meaningful and efficient manipulation and analysis of molecule data sets. Sdfconf packages molecular structures and metadata into a complete ensemble, from which one can access both the whole data set and individual molecules and/or conformations. In this software note, we offer some practical examples of the utilization of sdfconf.
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Biología Computacional , Manejo de Datos , Análisis de Datos , Programas InformáticosRESUMEN
Receptor tyrosine kinases play an important role in many cellular processes, and their dysregulation leads to diseases, most importantly cancer. One such receptor tyrosine kinase is c-Kit, a type-III receptor tyrosine kinase, which is involved in various intracellular signaling pathways. The role of different mutant isoforms of c-Kit has been established in several types of cancers. Accordingly, promising c-Kit inhibition results have been reported for the treatment of different cancers (e.g., gastrointestinal stromal tumors, melanoma, acute myeloid leukemia, and other tumors). Therefore, lots of effort has been put to target c-Kit for the treatment of cancer. Here, we provide a comprehensive compilation to provide an insight into c-Kit inhibitor discovery. This compilation provides key information regarding the structure, signaling pathways related to c-Kit, and, more importantly, pharmacophores, binding modes, and SAR analysis for almost all small-molecule heterocycles reported for their c-Kit inhibitory activity. This work could be used as a guide in understanding the basic requirements for targeting c-Kit, and how the selectivity and efficacy of the molecules have been achieved till today.
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Neoplasias/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Humanos , Neoplasias/fisiopatología , Transducción de SeñalRESUMEN
Steroid hormones play an essential role in a wide variety of actions in the body, such as in metabolism, inflammation, initiating and maintaining sexual differentiation and reproduction, immune functions, and stress response. Androgen, aromatase, and sulfatase pathway enzymes and nuclear receptors are responsible for steroid biosynthesis and sensing steroid hormones. Changes in steroid homeostasis are associated with many endocrine diseases. Thus, the discovery and development of novel drug candidates require a detailed understanding of the small molecule structure-activity relationship with enzymes and receptors participating in steroid hormone synthesis, signaling, and metabolism. Here, we show that simple coumarin derivatives can be employed to build cost-efficiently a set of molecules that derive essential features that enable easy discovery of selective and high-affinity molecules to target proteins. In addition, these compounds are also potent tool molecules to study the metabolism of any small molecule.
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Proteínas Portadoras/metabolismo , Cumarinas/farmacología , Esteroides/metabolismo , Animales , Humanos , Unión Proteica/fisiología , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Of the three enzymes in the human cytochrome P450 family 1, CYP1A2 is an important enzyme mediating metabolism of xenobiotics including drugs in the liver, while CYP1A1 and CYP1B1 are expressed in extrahepatic tissues. Currently used CYP substrates, such as 7-ethoxycoumarin and 7-ethoxyresorufin, are oxidized by all individual CYP1 forms. The main aim of this study was to find profluorescent coumarin substrates that are more selective for the individual CYP1 forms. Eleven 3-phenylcoumarin derivatives were synthetized, their enzyme kinetic parameters were determined, and their interactions in the active sites of CYP1 enzymes were analyzed by docking and molecular dynamic simulations. All coumarin derivatives and 7-ethoxyresorufin and 7-pentoxyresorufin were oxidized by at least one CYP1 enzyme. 3-(3-Methoxyphenyl)-6-methoxycoumarin (19) was 7-O-demethylated by similar high efficiency [21-30 ML/(min·mol CYP)] by all CYP1 forms and displayed similar binding in the enzyme active sites. 3-(3-Fluoro-4-acetoxyphenyl)coumarin (14) was selectively 7-O-demethylated by CYP1A1, but with low efficiency [0.16 ML/(min mol)]. This was explained by better orientation and stronger H-bond interactions in the active site of CYP1A1 than that of CYP1A2 and CYP1B1. 3-(4-Acetoxyphenyl)-6-chlorocoumarin (20) was 7-O-demethylated most efficiently by CYP1B1 [53 ML/(min·mol CYP)], followed by CYP1A1 [16 ML/(min·mol CYP)] and CYP1A2 [0.6 ML/(min·mol CYP)]. Variations in stabilities of complexes between 20 and the individual CYP enzymes explained these differences. Compounds 14, 19, and 20 are candidates to replace traditional substrates in measuring activity of human CYP1 enzymes.
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The novel SARS-CoV-2 uses ACE2 (Angiotensin-Converting Enzyme 2) receptor as an entry point. Insights on S protein receptor-binding domain (RBD) interaction with ACE2 receptor and drug repurposing has accelerated drug discovery for the novel SARS-CoV-2 infection. Finding small molecule binding sites in S protein and ACE2 interface is crucial in search of effective drugs to prevent viral entry. In this study, we employed molecular dynamics simulations in mixed solvents together with virtual screening to identify small molecules that could be potential inhibitors of S protein -ACE2 interaction. Observation of organic probe molecule localization during the simulations revealed multiple sites at the S protein surface related to small molecule, antibody, and ACE2 binding. In addition, a novel conformation of the S protein was discovered that could be stabilized by small molecules to inhibit attachment to ACE2. The most promising binding site on RBD-ACE2 interface was targeted with virtual screening and top-ranked compounds (DB08248, DB02651, DB03714, and DB14826) are suggested for experimental testing. The protocol described here offers an extremely fast method for characterizing key proteins of a novel pathogen and for the identification of compounds that could inhibit or accelerate spreading of the disease.
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COVID-19/virología , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/farmacología , Sitios de Unión , COVID-19/metabolismo , Biología Computacional , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Interacciones Microbiota-Huesped/efectos de los fármacos , Interacciones Microbiota-Huesped/fisiología , Humanos , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/efectos de los fármacos , Solventes , Interfaz Usuario-Computador , Tratamiento Farmacológico de COVID-19RESUMEN
Rational drug discovery relies heavily on molecular docking-based virtual screening, which samples flexibly the ligand binding poses against the target protein's structure. The upside of flexible docking is that the geometries of the generated docking poses are adjusted to match the residue alignment inside the target protein's ligand-binding pocket. The downside is that the flexible docking requires plenty of computing resources and, regardless, acquiring a decent level of enrichment typically demands further rescoring or post-processing. Negative image-based screening is a rigid docking technique that is ultrafast and computationally light but also effective as proven by vast benchmarking and screening experiments. In the NIB screening, the target protein cavity's shape/electrostatics is aligned and compared against ab initio-generated ligand 3D conformers. In this chapter, the NIB methodology is explained at the practical level and both its weaknesses and strengths are discussed candidly.
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Descubrimiento de Drogas/métodos , Simulación del Acoplamiento Molecular/métodos , Proteínas/química , Algoritmos , Sitios de Unión , Cristalografía por Rayos X , Ciclooxigenasa 2/química , Bases de Datos de Proteínas , Ligandos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Curva ROC , Bibliotecas de Moléculas Pequeñas/química , Programas Informáticos , Electricidad Estática , Interfaz Usuario-ComputadorRESUMEN
Molecular docking produces often lackluster results in real-life virtual screening assays that aim to discover novel drug candidates or hit compounds. The problem lies in the inability of the default docking scoring to properly estimate the Gibbs free energy of binding, which impairs the recognition of the best binding poses and the separation of active ligands from inactive compounds. Negative image-based rescoring (R-NiB) provides both effective and efficient way for re-ranking the outputted flexible docking poses to improve the virtual screening yield. Importantly, R-NiB has been shown to work with multiple genuine drug targets and six popular docking algorithms using demanding benchmark test sets. The effectiveness of the R-NiB methodology relies on the shape/electrostatics similarity between the target protein's ligand-binding cavity and the docked ligand poses. In this chapter, the R-NiB method is described with practical usability in mind.
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Descubrimiento de Drogas/métodos , Simulación del Acoplamiento Molecular/métodos , Proteínas/química , Algoritmos , Área Bajo la Curva , Sitios de Unión , Cristalografía por Rayos X , Ciclooxigenasa 2/química , Bases de Datos de Proteínas , Ligandos , Conformación Molecular , Neuraminidasa/química , Unión Proteica , Programas Informáticos , Electricidad EstáticaRESUMEN
CYP2A13 enzyme is expressed in human extrahepatic tissues, while CYP2A6 is a hepatic enzyme. Reactions catalysed by CYP2A13 activate tobacco-specific nitrosamines and some other toxic xenobiotics in lungs.To compare oxidation characteristics and substrate-enzyme active site interactions in CYP2A13 vs CYP2A6, we evaluated CYP2A13 mediated oxidation characteristics of 23 coumarin derivatives and modelled their interactions at the enzyme active site.CYP2A13 did not oxidise six coumarin derivatives to corresponding fluorescent 7-hydroxycoumarins. The Km-values of the other coumarins varied 0.85-97 µM, Vmax-values of the oxidation reaction varied 0.25-60 min-1, and intrinsic clearance varied 26-6190 kL/min*mol CYP2A13). Km of 6-chloro-3-(3-hydroxyphenyl)-coumarin was 0.85 (0.55-1.15 95% confidence limit) µM and Vmax 0.25 (0.23-0.26) min-1, whereas Km of 6-hydroxy-3-(3-hydroxyphenyl)-coumarin was 10.9 (9.9-11.8) µM and Vmax 60 (58-63) min-1. Docking analyses demonstrated that 6-chloro or 6-methoxy and 3-(3-hydroxyphenyl) or 3-(4-trifluoromethylphenyl) substituents of coumarin increased affinity to CYP2A13, whereas 3-triazole or 3-(3-acetate phenyl) or 3-(4-acetate phenyl) substituents decreased it.The active site of CYP2A13 accepts more diversified types of coumarin substrates than the hepatic CYP2A6 enzyme. New sensitive and convenient profluorescent CYP2A13 substrates were identified, such as 6-chloro-3-(3-hydroxyphenyl)-coumarin having high affinity and 6-hydroxy-3-(3-hydroxyphenyl)-coumarin with high intrinsic clearance.
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Hidrocarburo de Aril Hidroxilasas , Hidrocarburo de Aril Hidroxilasas/metabolismo , Cumarinas , Citocromo P-450 CYP2A6 , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Cinética , Simulación del Acoplamiento MolecularRESUMEN
The COVID-19 pandemic, caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a severe global health crisis now. SARS-CoV-2 utilizes its Spike protein receptor-binding domain (S-protein) to invade human cell through binding to Angiotensin-Converting Enzyme 2 receptor (ACE2). S-protein is the key target for many therapeutics and vaccines. Potential S-protein-ACE2 fusion inhibitor is expected to block the virus entry into the host cell. In many countries, traditional practices, based on natural products (NPs) have been in use to slow down COVID-19 infection. In this study, a protocol was applied that combines mixed solvent molecular dynamics simulations (MixMD) with high-throughput virtual screening (HTVS) to search NPs to block SARS-CoV-2 entry into the human cell. MixMD simulations were employed to discover the most promising stable binding conformations of drug-like probes in the S-protein-ACE2 interface. Detected stable sites were used for HTVs of 612093 NPs to identify molecules that could interfere with the S-protein-ACE2 interaction. In total, 19 NPs were selected with rescoring model. These top-ranked NP-S-protein complexes were subjected to classical MD simulations for 300 ns (3 replicates of 100 ns) to estimate the stability and affinity of binding. Three compounds, ZINC000002128789, ZINC000002159944 and SN00059335, showed better stability in all MD runs, of which ZINC000002128789 was predicted to have the highest binding affinity, suggesting that it could be effective modulator in RBD-ACE2 interface to prevent SARS-CoV-2 infection. Our results support that NPs may provide tools to fight COVID-19.
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Streptococcus pneumoniae causes invasive infections such as otitis media, pneumonia and meningitis. It produces the pneumolysin (Ply) toxin, which forms a pore onto the host cell membrane and has multiple functions in the pathogenesis of S. pneumoniae. The Ply C-terminal domain 4 mediates binding to membrane cholesterol and induces the formation of pores composed of up to 40 Ply monomers. Ply has a key role in the establishment of nasal colonization, pneumococcal transmission from host to host and pathogenicity. Altogether, 27 hydrolysable tannins were tested for Ply inhibition in a hemolysis assay and a tannin-protein precipitation assay. Pentagalloylglucose (PGG) and gemin A showed nanomolar inhibitory activity. Ply oligomerization on the erythrocyte surface was inhibited with PGG. PGG also inhibited Ply cytotoxicity to A549 human lung epithelial cells. Molecular modelling of Ply interaction with PGG suggests that it binds to the pocket formed by domains 2, 3 and 4. In this study, we reveal the structural features of hydrolysable tannins that are required for interaction with Ply. Monomeric hydrolysable tannins containing three to four flexible galloyl groups have the highest inhibitory power to Ply cytotoxicity and are followed by oligomers. Of the oligomers, macrocyclic and C-glycosidic structures were weaker in their inhibition than the glucopyranose-based oligomers. Accordingly, PGG-type monomers and oligomers might have therapeutic value in the targeting of S. pneumoniae infections.
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in vivo methods, such as spectrophotometric, fluorometric, mass spectrometric,and radioactivity-based techniques. In fluorescence-based assays, the reaction produces a fluorescentproduct from a nonfluorescent substrate or vice versa. Fluorescence-based enzyme assays areusually highly sensitive and specific, allowing measurements on small specimens of tissues withlow enzyme activities. Fluorescence assays are also amenable to miniaturization of the reactionmixtures and can thus be done in high throughput. 7-Hydroxycoumarin and its derivatives arewidely used as fluorophores due to their desirable photophysical properties. They possess a large -conjugated system with electron-rich and charge transfer properties. This conjugated structure leadsto applications of 7-hydroxycoumarins as fluorescent sensors for biological activities. We describe inthis review historical highlights and current use of coumarins and their derivatives in evaluatingactivities of the major types of xenobiotic-metabolizing enzyme systems. Traditionally, coumarinsubstrates have been used to measure oxidative activities of cytochrome P450 (CYP) enzymes. For thispurpose, profluorescent coumarins are very sensitive, but generally lack selectivity for individual CYPforms. With the aid of molecular modeling, we have recently described several new coumarin-basedsubstrates for measuring activities of CYP and conjugating enzymes with improved selectivity.
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Cumarinas/química , Cumarinas/metabolismo , Xenobióticos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Fluorescencia , Humanos , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Umbeliferonas/metabolismo , Xenobióticos/químicaRESUMEN
Enzymes in the cytochrome P450 family 1 (CYP1) catalyze metabolic activation of procarcinogens and deactivation of certain anticancer drugs. Inhibition of these enzymes is a potential approach for cancer chemoprevention and treatment of CYP1-mediated drug resistance. We characterized inhibition of human CYP1A1, CYP1A2, and CYP1B1 enzymes by the novel inhibitor N-(3,5-dichlorophenyl)cyclopropanecarboxamide (DCPCC) and α-naphthoflavone (ANF). Depending on substrate, IC50 values of DCPCC for CYP1A1 or CYP1B1 were 10-95 times higher than for CYP1A2. IC50 of DCPCC for CYP1A2 was 100-fold lower than for enzymes in CYP2 and CYP3 families. DCPCC IC50 values were 10-680 times higher than the ones of ANF. DCPCC was a mixed-type inhibitor of CYP1A2. ANF was a competitive tight-binding inhibitor of CYP1A1, CYP1A2, and CYP1B1. CYP1A1 oxidized DCPCC more rapidly than CYP1A2 or CYP1B1 to the same metabolite. Molecular dynamics simulations and binding free energy calculations explained the differences of binding of DCPCC and ANF to the active sites of all three CYP1 enzymes. We conclude that DCPCC is a more selective inhibitor for CYP1A2 than ANF. DCPCC is a candidate structure to modulate CYP1A2-mediated metabolism of procarcinogens and anticancer drugs.
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Amidas/química , Benzoflavonas/química , Ciclopropanos/química , Inhibidores Enzimáticos del Citocromo P-450/química , Familia 1 del Citocromo P450/antagonistas & inhibidores , Amidas/metabolismo , Benzoflavonas/metabolismo , Sitios de Unión , Dominio Catalítico , Cumarinas/química , Cumarinas/metabolismo , Ciclopropanos/metabolismo , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/antagonistas & inhibidores , Citocromo P-450 CYP1B1/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Familia 1 del Citocromo P450/metabolismo , Humanos , Hígado/enzimología , Simulación de Dinámica Molecular , Oxidación-ReducciónRESUMEN
Sulfonation is an important high affinity elimination pathway for phenolic compounds.In this study sulfonation of 7-hydroxycoumarin and 13 its derivatives were evaluated in liver cytosols of human and six animal species. 7-hydroxycoumarin and its derivatives are strongly fluorescent, and their sulfate conjugates are nonfluorescent at excitation 405 nm and emission 460 nm. A convenient fluorescence based kinetic assay of sulfonation was established.The sulfonation rate of most of the 7-hydroxycoumarin derivatives was low in liver cytosol of human and pig, whereas it was high with most compounds in dog and intermediate in rat, mouse, rabbit, and sheep. Sulfonation of the 7-hydroxycoumarin derivatives followed Michaelis-Menten kinetics with Km values of 0.1-12 µM, Vmax of 0.005-1.7 µmol/(min * g protein) and intrinsic clearance (Vmax/Km) of 0.004-1.9 L/(min * g cytosolic protein).Fluorescence based measurement of sulfonation of 7-hydroxycoumarin derivatives provides a sensitive and convenient high-throughput assay to determine sulfonation rate in different species and tissues and can be applied to evaluate sulfonation kinetics and inhibition.
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Citosol/metabolismo , Umbeliferonas/metabolismo , Animales , Perros , Humanos , Ratones , Conejos , Ratas , Ovinos , PorcinosRESUMEN
Cyanidin-3-glucoside (C3G) is a natural pigment, found in many colorful fruits and vegetables. It has many health benefits, including anti-inflammation, cancer prevention, and anti-diabetes. Although C3G is assumed to be an antioxidant, it has been reported to affect cell-matrix adhesions. However, the underlying molecular mechanism is unknown. Here, we show that the expression of talin1, a key regulator of integrins and cell adhesions, negatively correlated with the survival rate of colon cancer patients and that depletion of talin1 inhibited 3D spheroid growth in colon cancer cells. Interestingly, C3G bound to talin and promoted the interaction of talin with ß1A-integrin. Molecular docking analysis shows that C3G binds to the interface of the talin-ß-integrin complex, acting as an allosteric regulator and altering the interaction between talin and integrin. Moreover, C3G promoted colon cancer cell attachment to fibronectin. While C3G had no significant effect on colon cancer cell proliferation, it significantly inhibited 3D spheroid growth in fibrin gel assays. Since C3G has no or very low toxicity, it could be potentially used for colon cancer prevention or therapy.