RESUMEN
Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.
Asunto(s)
Ácidos Aristolóquicos , Colesterol , Ácido Flufenámico , Receptores Acoplados a Proteínas G , Gusto , Humanos , Ácidos Aristolóquicos/metabolismo , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/farmacología , Sitios de Unión/efectos de los fármacos , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacología , Microscopía por Crioelectrón , Ácido Flufenámico/química , Ácido Flufenámico/metabolismo , Ácido Flufenámico/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructura , Gusto/efectos de los fármacos , Gusto/fisiología , Transducina/química , Transducina/metabolismoRESUMEN
The YAP-TEAD transcriptional complex is responsible for the expression of genes that regulate cancer cell growth and proliferation. Dysregulation of the Hippo pathway due to overexpression of TEAD has been reported in a wide range of cancers. Inhibition of TEAD represses the expression of associated genes, demonstrating the value of this transcription factor for the development of novel anti-cancer therapies. We report herein the design, synthesis and biological evaluation of LM98, a flufenamic acid analogue. LM98 shows strong affinity to TEAD, inhibits its autopalmitoylation and reduces the YAP-TEAD transcriptional activity. Binding of LM98 to TEAD was supported by 19 F-NMR studies while co-crystallization experiments confirmed that LM98 is anchored within the palmitic acid pocket of TEAD. LM98 reduces the expression of CTGF and Cyr61, inhibits MDA-MB-231 breast cancer cell migration and arrests cell cycling in the S phase during cell division.
Asunto(s)
Antineoplásicos/farmacología , Ácido Flufenámico/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción de Dominio TEA/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Ácido Flufenámico/química , Humanos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción de Dominio TEA/metabolismo , Células Tumorales CultivadasRESUMEN
Transcriptional enhanced associate domain (TEAD) is a family of transcription factors that plays a significant role during embryonic developmental processes, and its dysregulation is responsible for tumour progression. TEAD is considered as druggable targets in various diseases, namely cancer, cardiovascular diseases and neurodegenerative disorders. Previous structural studies revealed the importance of the central hydrophobic pocket of TEAD as a potential target for small-molecule inhibitors and demonstrated flufenamic acid (FLU) (a COX-2 enzyme inhibitor) to bind and inhibit TEAD2 functions. However, to date, no drug candidates that bind specifically to TEAD2 with high selectivity and efficacy have been developed or proposed. Within this framework, we present here a case study where we have identified potential TEAD2 inhibitor candidates by integrating multiple computational approaches. Among the candidates, the top two ranked compounds ZINC95969481 (LG1) which is a fused pyrazole derivative and ZINC05203789 (LG2), a fluorene derivative resulted in much favourable binding energy scores than the reference ligand, FLU. The drug likeliness of the best compounds was also evaluated in silico to ensure the bioavailability of these compounds particularly LG1 as compared to FLU thus providing a strong rationale for their development as leads against TEAD. Molecular dynamics simulations results highlighted the role of key residues contributing to favourable interactions in TEAD2-LG1 complex with much favourable interaction and binding free energy values with respect to the reference compound. Altogether, this study provides a starting platform to be more exploited by future experimental research towards the development of inhibitors against TEAD, a persuasive strategy for therapeutic intervention in cancer treatment.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Descubrimiento de Drogas/métodos , Ácido Flufenámico/metabolismo , Neoplasias/metabolismo , Preparaciones Farmacéuticas/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Sitios de Unión , Cristalización , Proteínas de Unión al ADN/química , Ácido Flufenámico/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Ácido Niflúmico/química , Ácido Niflúmico/metabolismo , Preparaciones Farmacéuticas/química , Unión Proteica , Factores de Transcripción de Dominio TEA , Factores de Transcripción/químicaRESUMEN
Non-classical crystallisation (NCC) pathways are widely accepted, however there is conflicting evidence regarding the intermediate stages of crystallisation, how they manifest and further develop into crystals. Evidence from direct observations is especially lacking for small organic molecules, as distinguishing these low-electron dense entities from their similar liquid-phase surroundings presents signal-to-noise ratio and contrast challenges. Here, Liquid Phase Electron Microscopy (LPEM) captures the intermediate pre-crystalline stages of a small organic molecule, flufenamic acid (FFA), a common pharmaceutical. High temporospatial imaging of FFA in its native environment, an organic solvent, suggests that in this system a Pre-Nucleation Cluster (PNC) pathway is followed by features exhibiting two-step nucleation. This work adds to the growing body of evidence that suggests nucleation pathways are likely an amalgamation of multiple existing non-classical theories and highlights the need for the direct evidence presented by in situ techniques such as LPEM.
Asunto(s)
Cristalización , Ácido Flufenámico/química , Microscopía Electrónica/métodosRESUMEN
A prominent medical application of nanotechnology is represented in drug delivery. In this work, carboxymethyl cellulose (CMC) and poly(vinyl alcohol) (PVA) were used for producing CMC/PVA aqueous-based nanofibers loaded with flufenamic acid (FFA) as a drug containing amine groups. The CMC/PVA solutions with 90/10, 80/20, 70/30, 60/40 and 50/50 ratios were considered for electrospinning. Two integration methods were studied for loading FFA on the nanofibers during the electrospinning process. The characterization techniques of SEM, AFM, fluorescence microscopy and FT-IR spectroscopy were used to study the produced nanofibers, indicating a uniform distribution of FFA throughout the samples. The resulting nanofibers were formed in a diameter range of 176-285 nm and exhibited a 5 h degradation time in the PBS buffer solution. A standard diagram of drug loading was obtained for the samples. The drug release pattern was examined using a dialysis tube method. UV-visible spectroscopy revealed a time-dependent drug release behavior in CMC/PVA/FFA nanofibers where a sharp release occurred over the first 20 min. However, a prolonged release time of 10 h was achieved using a cross-linker (EDC).
Asunto(s)
Carboximetilcelulosa de Sodio/química , Sistemas de Liberación de Medicamentos , Ácido Flufenámico/farmacología , Nanofibras/química , Carboximetilcelulosa de Sodio/farmacología , Ácido Flufenámico/química , Humanos , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Nanofibras/uso terapéutico , Nanofibras/ultraestructura , Nanotecnología/tendencias , Alcohol Polivinílico/química , Alcohol Polivinílico/uso terapéutico , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The present study demonstrates the solubility and dissolution of flufenamic acid (FLF)/ß-cyclodextrin (ß-CD)/Soluplus® supramolecular ternary inclusion complex. The binary and ternary inclusion complexes were prepared using solvent evaporation and the microwave irradiation method. The prepared inclusion complexes were evaluated for physicochemical characterization and anti-inflammatory activity using a murine paw edema mol. The phase solubility studies demonstrated 4.59-fold and 17.54-fold enhancements in FLF solubility with ß-CD alone and ß-CD:Soluplus® combination compared with pure FLF, respectively. The in vitro drug release results revealed a significant improvement (P < 0.05) in the release pattern compared with pure FLF. Maximum release was found with flufenamic acid binary and ternary complexes prepared using the microwave irradiation method, i.e., 75.23 ± 3.12% and 95.36 ± 3.23% in 60 min, respectively. The physicochemical characterization results showed complex formation and conversion of the crystalline form of FLF to an amorphous form. The SEM study revealed the presence of a more agglomerated and amorphous structure of the solid particles, which confirmed the formation of complexes. The anti-inflammatory effect of the complex was higher than pure FLF. Therefore, the FLF:ß-CD:Soluplus® inclusion complex may be a very valuable formulation with improved solubility, dissolution, and anti-inflammatory effect.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Ácido Flufenámico/química , Ácido Flufenámico/farmacología , Polietilenglicoles/química , Polivinilos/química , beta-Ciclodextrinas/química , Animales , Rastreo Diferencial de Calorimetría , Carragenina , Cristalización , Composición de Medicamentos , Edema/inducido químicamente , Edema/patología , Excipientes , Masculino , Microondas , Ratas , Ratas Wistar , Solubilidad , beta-Ciclodextrinas/farmacologíaRESUMEN
Flufenamic acid (FFA) is a problem drug that has up to eight different polymorphs and shows poor solubility. Variability in bioavailability has been reported in the past resulting in limited use of FFA in the oral solid dosage form. The goal of this article was to investigate the polymorphism and amorphization behavior of FFA in non-heated and heated mixtures with high surface area nanocellulose, i.e., Cladophora cellulose (CLAD). As a benchmark, low surface area microcrystalline cellulose (MCC) was used. The solid-state properties of mixtures were characterized with X-ray diffraction, Fourier-transform infrared spectroscopy, and differential scanning calorimetry. The dissolution behavior of mixtures was studied in three biorelevant media, i.e., fasted state simulated gastric fluid, fasted state simulated intestinal fluid, and fed state simulated intestinal fluid. Additional thermal analysis and dissolution tests were carried out following 4 months of storage at 75% RH and room temperature. Heated mixtures of FFA with CLAD resulted in complete amorphization of the drug, whereas that with MCC produced a mixture of up to four different polymorphs. The amorphous FFA mixture with CLAD exhibited rapid and invariable fasted/fed state dissolution in simulated intestinal fluids, whereas that of MCC mixtures was highly dependent on the biorelevant medium. The storage of the heated FFA-CLAD mixture did not result in recrystallization or changes in dissolution profile, whereas heated FFA-MCC mixture showed polymorphic changes. The straightforward dry powder formulation strategy presented here bears great promise for reformulating a number of problem drugs to enhance their dissolution properties and reduce the fasted/fed state variability.
Asunto(s)
Antiinflamatorios/química , Celulosa/química , Ácido Flufenámico/química , Nanoestructuras/química , Materiales Biomiméticos/química , Química Farmacéutica , Estabilidad de Medicamentos , Jugo Gástrico/química , Calor , Humanos , SolubilidadRESUMEN
Here, we show that the development of nuclei and subsequent growth of a molecular organic crystal system can be induced by electron beam irradiation by exploiting the radiation chemistry of the carrier solvent. The technique of Liquid Cell Electron Microscopy was used to probe the crystal growth of flufenamic acid; a current commercialised active pharmaceutical ingredient. This work demonstrates liquid phase electron microscopy analysis as an essential tool for assessing pharmaceutical crystal growth in their native environment while giving insight into polymorph identification of nano-crystals at their very inception. Possible mechanisms of crystal nucleation due to the electron beam with a focus on radiolysis are discussed along with the innovations this technique offers to the study of pharmaceutical crystals and other low contrast materials.
Asunto(s)
Ácido Flufenámico/química , Microscopía Electrónica de Transmisión , Nanopartículas/química , Nanopartículas/ultraestructura , Cristalización , Tamaño de la PartículaRESUMEN
The molecular interactions between the surfaces of cocrystals [i.e., flufenamic acid and theophylline (FFA-TP), flufenamic acid and nicotinamide (FFA-NIC), and carbamazepine and nicotinamide (CBZ-NIC)] and the polymers [i.e., polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and copolymer of vinylpyrrolidone (60%)/vinyl acetate (40%) (PVP-VA)] were investigated through combined experimental and molecular dynamics simulation approaches to resolve the mechanisms of cocrystal dissolution and precipitation. It was found that adsorption of the polymers on the surfaces of cocrystals might prevent the precipitation of the parent drug and alter the dissolution rate. The effect of polymers on precipitation could be determined by the cocrystal dissolution rate, the interactions of polymers with the surfaces of cocrystals, the characters of the noncovalent bonds formed between the polymers and the cocrystal surfaces, and the mobility and conformation of the polymers. The etching experiments of single cocrystals revealed that FFA-NIC and CBZ-NIC appeared as surface precipitation cocrystals while FFA-TP could lead to bulk precipitation. Both PVP and PVP-VA were good precipitation inhibitors for FFA-NIC, and they could completely inhibit the recrystallization of FFA III on the surfaces of dissolving cocrystals. In addition, as the adsorption of the polymer was slower than dissolution rate of the cocrystals, PVP and PVP-VA could only partially inhibit the recrystallization of CBZ dihydrate on the surface of CBZ-NIC. While PEG had no inhibitory effect on the surface crystallization of FFA-NIC and CBZ-NIC, due to its weak interactions with the surfaces of the cocrystals, it enhanced the dissolution performance of FFA-TP. In contrast, PVP and PVP-VA reduced the dissolution rate of FFA-TP and subsequently undermined the performance of cocrystals. Taken together, the approach of combining experimental and molecular dynamics simulation provided insights into the mechanisms of cocrystal dissolution as well as the polymers acting as inhibitory excipients for precipitation/recrystallization, making contribution to the development of novel formulations.
Asunto(s)
Carbamazepina/química , Ácido Flufenámico/química , Niacinamida/química , Polietilenglicoles/química , Povidona/química , Pirrolidinas/química , Teofilina/química , Compuestos de Vinilo/química , Adsorción , Precipitación Química , Cristalización , Composición de Medicamentos/métodos , Liberación de Fármacos , Excipientes/química , Simulación de Dinámica Molecular , SolubilidadRESUMEN
Flufenamic acid is a nonsteroidal anti-inflammatory drug characterized by a low solubility and a variable oral bioavailability. Flufenamic acid is present in the commercial products in two polymorphic enantiotropic forms (Form I and III). Bioinequivalence was observed for commercial solid dosage forms due to the different dissolution rate of batches. Aim of this work is the full characterization of the solid state properties of flufenamic acid in order to evidence reasons of its variable dissolution properties. Two different batches of pure drug obtained by different suppliers were fully characterized. In order to evaluate the effect of the technological processes used for tablet production, the powders were submitted to grinding, kneading, and compression. Thermal analysis and X-ray diffraction studies proved that the drug was provided by both suppliers as Form I, Form III is obtained by recrystallization from ethanol or ethanol/water of both batches and no changes were observed after the different mechanical treatments. No difference was observed between the two forms in terms of equilibrium solubility values. Dissolution rate studies evidenced a difference between the two batches due to their different particle size, which disappeared after sieving. Interestingly, a significant difference in terms of intrinsic dissolution rate and surface wettability of the two compacted powders was observed, even after sieving, probably related to a different behavior of the two powder samples under compaction. These results should be taken into account, during a tablet formulation, in order to obtain a reproducible dissolution performance of the drug, regardless of its original supplier.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Ácido Flufenámico/química , Transición de Fase , Tecnología Farmacéutica/métodos , Administración Oral , Disponibilidad Biológica , Cristalización , Tamaño de la Partícula , Polvos , Solubilidad , Equivalencia Terapéutica , HumectabilidadRESUMEN
Novel sensors based on carbon paste assorted with amberlite XAD-4 resin and silver-doped titanium dioxide/amberlite XAD-4 resin (Ag-TiO2/XAD) were designed and employed for sensitive detection of flufenamic acid (FFA). The main objective of this study is to develop novel electrochemical sensors for more sensitive and selective detection of FFA and to propose its oxidation mechanism. In this perspective, we have developed modified electrodes using amberlite XAD-4 matrix and Ag-TiO2/XAD-4 mixture. These sensors when used in conjunction with different voltammetric techniques to observe enhanced outcome on FFA electrode reaction in PBS of pH 7.0. Our data confirmed exceptional stability, sensitivity, and quick responses for FFA. The effect of modifier, analyte concentration, pH of buffer solution, pre-concentration time, and sweep rates on electrochemical behavior of FFA was investigated. The current intensities were linearly proportional to the concentration of FFA. From the calibration plot of different concentrations of FFA, the detection limit of 3.6 nM at XAD-CPE and 1.2 nM, respectively at AgTiO2/XAD-CPE were obtained. The estimation of FFA in biological as well as clinical samples was achieved using the chemically modified electrodes.
Asunto(s)
Carbono/química , Técnicas Electroquímicas , Ácido Flufenámico/química , Poliestirenos/química , Polivinilos/química , Plata/química , Titanio/química , Electrodos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Recent advances to utilize two or more nanoparticles for developing novel sensors with superior sensitivity have spurred advanced detection limits even at low concentrations. In this research, a blend of rutheniumdoped TiO2 (Ru-TiO2) nanoparticles and multiwalled carbon nanotubes (MWCNTs) loaded into carbon paste matrix to fabricate a novel Ru-TiO2/MWCNTs-CPE sensor was used for the detection and quantification of flufenamic acid (FFA) and mefenamic acid (MFA) drugs. The surface morphology of Ru-TiO2 was assessed by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), X-ray diffraction (XRD) and atomic force microscopy (AFM). Sensitivity and selectivity of the electrode was improved at the Ru-TiO2/MWCNTs modified CPE compared to nascent CPE due to the amazing surface distinctive characteristics of the modifier at pH 5.0. The effect of concentration of the modifier, pH, pre-concentration time, sweep rate and concentration on signal enhancement of FFA and MFA was studied. The square wave voltammetry (SWV) currents are linearly related in the concentration range of 0.01⯵M-0.9⯵M with the detection limit values of 0.68â¯nM for FFA and 0.45â¯nM for MFA, respectively. The developed electrode assembly was used for the quantification of both the drug analytes in human urine samples.
Asunto(s)
Electroquímica/instrumentación , Ácido Flufenámico/análisis , Ácido Mefenámico/análisis , Nanopartículas/química , Nanotubos de Carbono/química , Rutenio/química , Titanio/química , Ácido Flufenámico/química , Ácido Flufenámico/orina , Humanos , Ácido Mefenámico/química , Ácido Mefenámico/orina , Factores de TiempoRESUMEN
Anisotropic features with other crystallographic properties like d-spacing, and attachment energy (Eatt) can predict material performance during the secondary pharmaceutical processing. A newly developed state-of-the-art compression cell lodged in a powder X-ray diffractometer was used to measure anisotropic Young's moduli (YM) of flufenamic acid (FFA) polymorphs in this study. Methodology is based on the generation of a single crystal deformation in this cell, which reflects as a change in the d-spacing in the PXRD pattern. Anisotropic YM was calculated from such information gathered along different FFA planes. Measured FFA crystallographic molecular features were concatenated to understand macroscopic compaction (Heckel and Shapirao's parameters) and tableting performance. Block shaped crystals of FFA form I, and III after initial characterization with SEM, DSC, PXRD, and FTIR were compressed normal to X, Y, and Z-planes, identified from calculated PXRD pattern using the reported single crystal structure. YM of X and Y planes of form I was significantly higher than corresponding planes of form III. Z plane of form III showed significantly higher YM than that for form I. Low YM of form III can be attributed to its large d-spacing regardless of their high Eatt than form I, as well as orientation of supramolecular acid dimer (OHâ¯O) homosynthon chains in the FFA planes. FFA form I stiffness was further confirmed with lower densification and higher yield pressure of deformation than form III. Clearly, form III exhibited better compressibility, compactibility, and tableting performance than form I due to favorable molecular and macroscopic features. Thus, developed anisotropic measurement approach can be used to distinguish material performance in the early development stage of the pharmaceutical processes.
Asunto(s)
Antiinflamatorios/administración & dosificación , Química Farmacéutica/métodos , Composición de Medicamentos/métodos , Ácido Flufenámico/administración & dosificación , Anisotropía , Antiinflamatorios/química , Cristalización , Módulo de Elasticidad , Ácido Flufenámico/química , Microscopía Electrónica de Rastreo , Presión , Espectroscopía Infrarroja por Transformada de Fourier , Comprimidos , Difracción de Rayos XRESUMEN
The development of a myriad of nanoparticles types has opened new possibilities for the diagnostics and treatment of many diseases, especially for cancer. However, most of the researches done so far do not focus on the protection of normal cells surrounding a tumor from irradiation bystander effects that might lead to cancer recurrence. Gap-junctions are known to be involved in this process, which leads to genomic instability of neighboring normal cells, and flufenamic acid (FFA) is included in a new group of gap-junction blockers recently discovered. The present work explores the use of mesoporous silica nanoparticles MCM-41 functionalized with 3-Aminopropyltriethoxysilane (APTES) for anchoring the flufenamic acid for its prolonged and controlled release and protection from radiation bystander effects. MCM-41 and functionalized samples were structurally and chemically characterized with multiple techniques. The biocompatibility of all samples was tested in a live/dead assay performed in cultured MRC-5 and HeLa cells. HeLa cells cultured were exposed to 50 Gy of gamma-rays and the media transferred to fibroblast cells cultured separately. Our results show that MCM-41 and functionalized samples have high biocompatibility with MCR-5 and HeLa cells, and most importantly, the FFA delivered by these NPs was able to halt apoptosis, one of main bystander effects.
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Efecto Espectador/efectos de la radiación , Ácido Flufenámico/química , Ácido Flufenámico/farmacología , Nanopartículas/química , Dióxido de Silicio/química , Dióxido de Silicio/farmacología , Efecto Espectador/efectos de los fármacos , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Rayos gamma/efectos adversos , Humanos , Ensayo de Materiales , Microscopía Electrónica de RastreoRESUMEN
The dissolution and permeation of the cocrystals, flufenamic acid-nicotinamide (FFA-NIC) and flufenamic acid-theophylline (FFA-TP), have been investigated in the presence of two polymers, polyvinylpyrrolidone (PVP) and copolymer of vinylpyrrolidone/vinyl acetate (PVP-VA), using a dissolution/permeation (D/P) system. It showed that the types and concentrations of the polymers and their interactions with the coformers had significant effects on the dissolution and permeation of the FFA cocrystals. The role of PVP as a stabilizing agent was not altered in spite of its interaction with the coformer of NIC or TP, which was supported by the proportional flux rate of FFA to the dissolution performance parameter (DPP). With an appropriate PVP concentration, the maximal flux rate of FFA could be obtained for a given FFA cocrystal. The situation was complicated in the presence of PVP-VA. The role of PVP-VA could change because of its association with the coformers, i.e., from a stabilizing agent to a solubilization agent. In addition, PVP-VA reduced the flux rate of FFA, in contrast to its DPP for FFA cocrystals. Finally, 1H NMR provided evidence regarding the molecular interactions between FFA, coformers, and polymers at the atomic level and gave insight into the mechanism underlying the supersaturated solution and subsequent permeation behavior of the cocrystals.
Asunto(s)
Ácido Flufenámico/química , Polímeros/química , Espectroscopía de Resonancia Magnética , Povidona/química , Solubilidad , Compuestos de Vinilo/químicaRESUMEN
The intermolecular interaction of flufenamic acid (Hfluf) with two model proteins i.e., hemoglobin and lysozyme was explored using fluorescence, UV-vis, circular dichroism, DLS, and molecular docking techniques. The corroborative spectroscopic techniques suggested efficient binding of Hfluf to both the proteins. The S-V plot in Hb-Hfluf system showed positive deviation highlighting the presence of both static and dynamic quenching. Hence, ground state complex model and sphere of action quenching model were used for the study. In Lyz-Hfluf system, a linear S-V plot was obtained indicating the presence of a single quenching mechanism. FRET study suggested a high probability of energy transfer from Hb/Lyz to Hfluf. Our thermodynamic results revealed that binding reaction in both the systems was exothermic and spontaneous. The UV-vis spectroscopy demonstrated that the binding of Hfluf affected the globin, Soret and oxy-bands of Hb along with globin band and polypeptide backbone of Lyz. CD spectra revealed the enhancement of É-helicity in Lyz and decrease in case of Hb whereas the Rh values of proteins from DLS experiment corroborated the CD findings. 3-D fluorescence spectra highlighted the conformational changes upon binding whereas docking studies predicted the active binding site of both the proteins as the binding site of Hfluf.
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Ácido Flufenámico/química , Hemoglobinas/química , Simulación del Acoplamiento Molecular , Muramidasa/química , Animales , Sitios de Unión , Bovinos , PollosRESUMEN
The oncoprotein YAP (Yes-associated protein) requires the TEAD family of transcription factors for the up-regulation of genes important for cell proliferation. Disrupting YAP-TEAD interaction is an attractive strategy for cancer therapy. Targeting TEADs using small molecules that either bind to the YAP-binding pocket or the palmitate-binding pocket is proposed to disrupt the YAP-TEAD interaction. There is a need for methodologies to facilitate robust and reliable identification of compounds that occupy either YAP-binding pocket or palmitate-binding pocket. Here, using NMR spectroscopy, we validated compounds that bind to these pockets and also identify the residues in mouse TEAD4 (mTEAD4) that interact with these compounds. Flufenamic acid (FA) was used as a positive control for validation of palmitate-binding pocket-occupying compounds by NMR. Furthermore, we identify a hit from a fragment screen and show that it occupies a site close to YAP-binding pocket on the TEAD surface. Our results also indicate that purified mTEAD4 can catalyze autopalmitoylation. NMR studies on mTEAD4 revealed that exchanges exist in TEAD as NMR signal broadening was observed for residues close to the palmitoylation site. Mutating the palmitoylated cysteine (C360S mutant) abolished palmitoylation, while no significant changes in the NMR spectrum were observed for the mutant which still binds to YAP. We also show that FA inhibits TEAD autopalmitoylation. Our studies highlight the utility of NMR spectroscopy in identifying small molecules that bind to TEAD pockets and reinforce the notion that both palmitate-binding pocket and YAP-binding pocket are targetable.
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Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Unión al ADN/química , Proteínas Musculares/química , Fosfoproteínas/química , Factores de Transcripción/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ácido Flufenámico/química , Lipoilación , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Dominios Proteicos , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Cocrystallization is a technique for modifying the physicochemical and pharmacokinetic properties of an active pharmaceutical ingredient (API) embodying the concept of supramolecular synthon. Most of the examples cited in the literature are of cocrystals formed between an API and a coformer chosen from the generally recognized as safe (GRAS) substance list; however few examples exist where a cocrystal consists of two or more APIs. These cocrystals are commonly known as multi API, multi-drug or drug- drug cocrystals. The formation of such cocrystals is feasible by virtue of non covalent interactions between the APIs, which help them in retaining their activity. In addition, drugdrug cocrystals also offer potential solution to the limitations such as solubility, stability differences and chemical interaction between the APIs which is often faced during the traditional combination therapy. Cocrystallization of two or more APIs can be employed for delivery of combination drugs for the better and efficacious management of many complex disorders where existing monotherapies do not furnish the desired therapeutic effect. This review on the existing drug-drug cocrystals is to gain an insight for better designing of multi API cocrystals with improved physicochemical and pharmacokinetic profile and its application in multiple target therapy.
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Preparaciones Farmacéuticas/química , Asma/tratamiento farmacológico , Asma/patología , Química Farmacéutica , Cristalización , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/patología , Quimioterapia Combinada , Ácido Flufenámico/química , Ácido Flufenámico/uso terapéutico , Gliclazida/química , Gliclazida/uso terapéutico , Humanos , Metformina/química , Metformina/uso terapéutico , Teofilina/química , Teofilina/uso terapéuticoRESUMEN
The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. Recently, we employed a scaffold hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative rounds of drug design, synthesis and biological evaluation, several compounds were discovered to target AKR1C3 in a selective manner. The most promising compound of series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2 isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained modulating the best example of hydroxylated triazoles we previously presented. In cell-based assays, the most promising compounds of both series reduced the cell proliferation, prostate specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate cancer cells and showed synergistic effect when assayed in combination with abiraterone and enzalutamide. Structure determination of AKR1C3 co-crystallized with one representative compound from each of the two series clearly identified both compounds in the androstenedione binding site, hence supporting the biochemical data.
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Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Benzoxazoles/farmacología , Inhibidores Enzimáticos/farmacología , Ácido Flufenámico/farmacología , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzoxazoles/síntesis química , Benzoxazoles/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ácido Flufenámico/síntesis química , Ácido Flufenámico/química , Humanos , Estructura Molecular , Antígeno Prostático Específico/antagonistas & inhibidores , Antígeno Prostático Específico/metabolismo , Relación Estructura-Actividad , Testosterona/antagonistas & inhibidores , Testosterona/biosíntesisRESUMEN
The human epidermal growth factor receptor 2 (HER2) is overexpressed in about a third of breast cancer patients, with a strong involvement of the cyclooxygenase-2 (COX-2) enzyme in the tumor progress. HER2 and COX-2 are consequently potential targets for inhibiting carcionogenesis. Herceptin (trastuzumab) is an antibody that partially blocks HER2-positive cancers at their initial stage. Unfortunately, the overall response rate to the single treatment with this antibody is still modest, and therefore, it needs to be improved by combining several chemotherapeutic agents. On the other hand, nonsteroidal anti-inflammatory drugs (NSAIDs) are designed to halt COX-2 functionality, so they might also exhort an anticancer activity. In this contribution, dual Herceptin-NSAID drugs are designed using theoretical tools. More specifically, blind docking, molecular dynamics, and quantum calculations are performed to assess the stability of 14 NSAIDs embedded inside Herceptin. Our calculations reveal the feasibility of improving the antitumor activity of the parent Herceptin by designing a dual HER2-targeting with Etofenamate. That coupling mode might be used to further rationalize new clinical strategies beyond classical antibody dosages.