RESUMEN
Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α2δ1. Here, to reveal the mirogabalin recognition mechanisms of α2δ1, we present structures of recombinant human α2δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of α2δ1 to those of the α2δ2, α2δ3, and α2δ4 isoforms, of which α2δ3 and α2δ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in α2δ1 ligand recognition.
Asunto(s)
Canales de Calcio , Gabapentina , Humanos , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Gabapentina/química , Gabapentina/farmacología , LigandosRESUMEN
INTRODUCTION: The mechanisms of fluoroquinolone-resistance of Mycoplasma genitalium were analysed by a new method. METHODS: M. genitalium strains from urinary sediments of patients with urethritis were isolated and examined antimicrobial susceptibilities and the mutations in ParC, GyrA and 23S rRNA. Docking models between gyrase and topoisomerase IV with sitafloxacin showed that two binding modes in which the amine moiety at the C-7 position rotated could be constructed. RESULTS: Among 18 strains, 13 strains had mutations with amino-acid changes at Serine 83 in ParC. The MICs of moxifloxacin or sitafloxacin for three strains with only S83I in ParC were 2, 1 and 8 mg/L (moxifloxacin) or 0.13, 0.13 and 1 mg/L (sitafloxacin), respectively. In contrast, the MICs of moxifloxacin or sitafloxacin for 3 strain with S83N in ParC were 0.25, 0.13 and 0.25 mg/L (moxifloxacin) or 0.06, 0.03, and 0,03 mg/L (sitafloxacin), respectively, not significantly different from wild-type isolates. The docking model of sitafloxacin and topoisomerase IV showed that the oxygen atom at the gamma position of Serine 83 of ParC interacted with the sitafloxacin carboxylate moiety. When the S83I substitution occurs, the isoleucine side chain is lipophilic and the residue hydropathy changes from hydrophilicity to hydrophobicity and important H-bond interactions between serine and the carboxylate moiety are lost. When the serine 83 to asparagine substitution (S83N) occurred, the asparagine side chain is hydrophilic and the residue hydropathy does not change. CONCLUSION: The docking model suggests that Ser83 replacements causes attenuation or loss of activity of fluoroquinolones such as sitafloxacin.
Asunto(s)
Infecciones por Mycoplasma , Mycoplasma genitalium , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Infecciones por Mycoplasma/tratamiento farmacológico , Mycoplasma genitalium/genéticaRESUMEN
Cyclorasinsâ 9A5 and 9A54 are 11-mer cyclic peptides that inhibit the Ras-Raf protein interaction. The peptides share a cell-penetrating peptide (CPP)-like motif; however, only cyclorasinâ 9A5 can permeabilize cells to exhibit strong cell-based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three-dimensional structures of cyclorasinsâ 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasinâ 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasinâ 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino-acid sidechains, Tle-2 and dVal-3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.
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Péptidos Cíclicos/farmacología , Quinasas raf/antagonistas & inhibidores , Proteínas ras/antagonistas & inhibidores , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/química , Conformación Proteica , Quinasas raf/química , Quinasas raf/metabolismo , Proteínas ras/química , Proteínas ras/metabolismoRESUMEN
Cryptic ligand binding sites, which are not evident in the unligated structures, are beneficial in tackling with difficult but attractive drug targets, such as protein-protein interactions (PPIs). However, cryptic sites have thus far not been rationally pursued in the early stages of drug development. Here, we demonstrated by nuclear magnetic resonance that the cryptic site in Bcl-xL exists in a conformational equilibrium between the open and closed conformations under the unligated condition. While the fraction of the open conformation in the unligated wild-type Bcl-xL is estimated to be low, F143W mutation that is distal from the ligand binding site can substantially elevate the population. The F143W mutant showed a higher hit rate in a phage-display peptide screening, and the hit peptide bound to the cryptic site of the wild-type Bcl-xL. Therefore, by controlling the conformational equilibrium in the cryptic site, the opportunity to identify a PPI inhibitor could be improved.
RESUMEN
PURPOSE: Taletrectinib (DS-6051b/AB-106) is an oral, tyrosine kinase inhibitor of ROS1 and NTRK with potent preclinical activity against ROS1 G2032R solvent-front mutation among others. We report the first-in-human U.S. phase I results of taletrectinib. PATIENTS AND METHODS: Patients ≥18 years old with neuroendocrine tumors, with tumor-induced pain, or tumors harboring ROS1/NTRK rearrangements were eligible. Accelerated titration followed by modified continuous reassessment method and escalation with overdose control was used (50-1,200 mg once daily or 400 mg twice daily). Primary objectives were safety/tolerability, and MTD determination. Secondary objectives were food-effect pharmacokinetics and antitumor activity. RESULTS: A total of 46 patients were enrolled. Steady-state peak concentration (C max) and exposure (AUC0-8) increased dose dependently from 50-mg to 800-mg once-daily doses. The ratio of the geometric mean of AUC0-24 between low-fat-diet-fed/fasted state was 123% (90% confidence interval, 104%-149%). Dose-limiting toxicities (grade 3 transaminases increase) occurred in two patients (1,200-mg once-daily dose). MTD was 800 mg once daily. Most common treatment-related adverse events were nausea (47.8%), diarrhea (43.5%), and vomiting (32.6%). Pain score reductions were observed in the 800-mg once-daily dose cohort. Confirmed objective response rate was 33.3% among the six patients with RECIST-evaluable crizotinib-refractory ROS1+ NSCLC. One patient with TPM3-NTRK1 differentiated thyroid cancer achieving a confirmed partial response of 27 months at data cutoff. We identified a cabozantinib-sensitive ROS1 L2086F as an acquired taletrectinib-resistance mutation. CONCLUSIONS: Taletrectinib has manageable toxicities at the MTD of 800 mg daily. Preliminary efficacy was observed in patients with crizotinib-refractory ROS1+ NSCLC.
Asunto(s)
Interacciones Alimento-Droga , Imidazoles/efectos adversos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/efectos adversos , Piridazinas/efectos adversos , Adulto , Anciano , Femenino , Humanos , Imidazoles/administración & dosificación , Imidazoles/farmacocinética , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias/diagnóstico , Neoplasias/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Piridazinas/administración & dosificación , Piridazinas/farmacocinética , Receptor trkA/antagonistas & inhibidores , Criterios de Evaluación de Respuesta en Tumores Sólidos , Adulto JovenRESUMEN
Recently, there has been increasing interest in new modalities such as therapeutic antibodies and gene therapy at a number of pharmaceutical companies. Moreover, in small-molecule drug discovery at such companies, efforts have focused on hard-to-drug targets such as inhibiting protein-protein interactions. Biomolecular NMR spectroscopy has been used in drug discovery in a variety of ways, such as for the reliable detection of binding and providing three-dimensional structural information for structure-based drug design. The advantages of using NMR spectroscopy have been known for decades (Jahnke in J Biomol NMR 39:87-90, (2007); Gossert and Jahnke in Prog Nucl Magn Reson Spectrosc 97:82-125, (2016)). For tackling hard-to-drug targets and increasing the success in discovering drug molecules, in-depth analysis of drug-target protein interactions performed by biophysical methods will be more and more essential. Here, we review the advantages of NMR spectroscopy as a key technology of biophysical methods and also discuss issues such as using cutting-edge NMR spectrometers and increasing the demand of utilizing conformational dynamics information for promoting small-molecule drug discovery.
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Descubrimiento de Drogas/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Rastreo Diferencial de Calorimetría , Cristalografía por Rayos X , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Relación Estructura-Actividad Cuantitativa , Bibliotecas de Moléculas PequeñasRESUMEN
Activation of the mineralocorticoid receptor (MR) has long been considered a risk factor for cardiovascular diseases. It has been reported that the novel MR blocker esaxerenone shows high potency and selectivity for MR in vitro as well as great antihypertensive and renoprotective effects in salt-sensitive hypertensive rats. Here, we determined the cocrystal structure of the MR ligand-binding domain (MR-LBD) with esaxerenone and found that esaxerenone binds to MR-LBD in a unique manner with large side-chain rearrangements, distinct from those of previously published MR antagonists. This structure also displays an antagonist form that has not been observed for MR previously. Such a unique binding mode of esaxerenone provides great insight into the novelty, potency, and selectivity of this novel antihypertensive drug.
Asunto(s)
Antagonistas de Receptores de Mineralocorticoides/química , Dominios Proteicos , Pirroles/química , Receptores de Mineralocorticoides/química , Sulfonas/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Eplerenona/química , Humanos , Ligandos , Modelos Moleculares , Espironolactona/química , Especificidad por SustratoRESUMEN
ROS1 gene rearrangement was observed in around 1-2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1-rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1-rearranged cancers or NTRK-rearranged cancers in vitro and in vivo. Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors.
Asunto(s)
Crizotinib/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Receptor trkB/antagonistas & inhibidores , Aminopiridinas , Benzamidas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desarrollo de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Indazoles/farmacología , Lactamas , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/genética , Mutación/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , PirazolesRESUMEN
The FGFR4/FGF19 signaling axis is overactivated in 20% of liver tumors and currently represents a promising targetable signaling mechanism in this cancer type. However, blocking FGFR4 or FGF19 has proven challenging due to its physiological role in suppressing bile acid synthesis which leads to increased toxic bile acid plasma levels upon FGFR4 inhibition. An FGFR4-targeting antibody, U3-1784, was generated in order to investigate its suitability as a cancer treatment without major side effects.U3-1784 is a high-affinity fully human antibody that was obtained by phage display technology and specifically binds to FGFR4. The antibody inhibits cell signaling by competing with various FGFs for their FGFR4 binding site thereby inhibiting receptor activation and downstream signaling via FRS2 and Erk. The inhibitory effect on tumor growth was investigated in 10 different liver cancer models in vivo The antibody specifically slowed tumor growth of models overexpressing FGF19 by up to 90% whereas tumor growth of models not expressing FGF19 was unaffected. In cynomolgus monkeys, intravenous injection of U3-1784 caused elevated serum bile acid and liver enzyme levels indicating potential liver damage. These effects could be completely prevented by the concomitant oral treatment with the bile acid sequestrant colestyramine, which binds and eliminates bile acids in the gut. These results offer a new biomarker-driven treatment modality in liver cancer without toxicity and they suggest a general strategy for avoiding adverse events with FGFR4 inhibitors.
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Anticuerpos Monoclonales/toxicidad , Anticuerpos Monoclonales/uso terapéutico , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/inmunología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Resina de Colestiramina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Íleon/efectos de los fármacos , Íleon/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Transducción de Señal/efectos de los fármacos , Sorafenib/farmacologíaRESUMEN
Mitogen-activated protein kinase (MAPK)-interacting kinases 1 (Mnk1) and 2 (Mnk2) modulate translation initiation through the phosphorylation of eukaryotic translation initiation factor 4E, which promotes tumorigenesis. However, Mnk1 and Mnk2 are dispensable in normal cells, suggesting that the inhibition of Mnk1 and Mnk2 could be effective in cancer therapy. To provide a structural basis for Mnk1 inhibition, a novel Mnk1 inhibitor was discovered and the crystal structure of Mnk1 in complex with this inhibitor was determined. The crystal structure revealed that the inhibitor binds to the autoinhibited state of Mnk1, stabilizing the Mnk-specific DFD motif in the DFD-out conformation, thus preventing Mnk1 from switching to the active conformation and thereby inhibiting the kinase activity. These results provide a valuable platform for the structure-guided design of Mnk1 inhibitors.
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Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Humanos , Modelos MolecularesRESUMEN
Tumor cells switch glucose metabolism to aerobic glycolysis by expressing the pyruvate kinase M2 isoform (PKM2) in a low active form, providing glycolytic intermediates as building blocks for biosynthetic processes, and thereby supporting cell proliferation. Activation of PKM2 should invert aerobic glycolysis to an oxidative metabolism and prevent cancer growth. Thus, PKM2 has gained attention as a promising cancer therapy target. To obtain novel PKM2 activators, we conducted a high-throughput screening (HTS). Among several hit compounds, a fragment-like hit compound with low potency but high ligand efficiency was identified. Two molecules of the hit compound bound at one activator binding site, and the molecules were linked based on the crystal structure. Since this linkage succeeded in maintaining the original position of the hit compound, the obtained compound exhibited highly improved potency in an in vitro assay. The linked compound also showed PKM2 activating activity in a cell based assay, and cellular growth inhibition of the A549 cancer cell line. Discovery of this novel scaffold and binding mode of the linked compound provides a valuable platform for the structure-guided design of PKM2 activators.
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Antineoplásicos/farmacología , Descubrimiento de Drogas , Piruvato Quinasa/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , TermodinámicaRESUMEN
The thermodynamic properties of a ligand in the bound state affect its binding specificity. Strict binding specificity can be achieved by introducing multiple spatially defined interactions, such as hydrogen bonds and van der Waals interactions, into the ligand-receptor interface. These introduced interactions are characterized by restricted local dynamics and improved surface complementarity in the bound state. In this study, we experimentally evaluated the local dynamics and the surface complementarity of weak-affinity ligands in the receptor-bound state by forbidden coherence transfer analysis in free-bound exchange systems (Ex-FCT), using the interaction between a ligand, a myocyte-enhancer factor 2A (MEF2A) docking peptide, and a receptor, p38α, as a model system. The Ex-FCT analyses successfully provided information for the rational design of a ligand with higher affinity and preferable thermodynamic properties for p38α.
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Termodinámica , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Humanos , Ligandos , Factores de Transcripción MEF2/química , Factores de Transcripción MEF2/metabolismo , Espectroscopía de Resonancia Magnética , Propiedades de Superficie , Proteínas Quinasas p38 Activadas por Mitógenos/químicaRESUMEN
To obtain potent liver X receptor (LXR) agonists, a structure-activity relationship study was performed on a series of tert-butyl benzoate analogs. As the crystal structure analysis suggested applicable interactions between the LXR ligand-binding domain and the ligands, two key functional groups were introduced. The introduction of the hydroxyl group on the C6-position of the benzoate part enhanced the agonistic activity in a cell-based assay, and the carboxyl group in terminal improved the pharmacokinetic profile in mice, respectively. The obtained compound 32b increased blood ABCA1 mRNA expression without plasma TG elevation in both mice and cynomolgus monkeys.
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Benzoatos/farmacología , Descubrimiento de Drogas , Hidrocarburos Fluorados/farmacología , Receptores Nucleares Huérfanos/agonistas , Animales , Benzoatos/administración & dosificación , Benzoatos/química , Relación Dosis-Respuesta a Droga , Humanos , Hidrocarburos Fluorados/administración & dosificación , Hidrocarburos Fluorados/química , Receptores X del Hígado , Ratones , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Numerous small organic compounds exist in equilibrium among monomers, soluble oligomers, and insoluble aggregates in aqueous solution. Compound aggregation is a major reason for false positives in drug screening, and even soluble oligomers can interfere with structural and biochemical analyses. However, an efficient way to manage the equilibrium of aggregation-prone compounds, especially those involved with soluble oligomers, has not been established. In this study, solution NMR spectroscopy was used as a suitable technique to detect compound oligomers in equilibrium, and it was demonstrated that cosolubilization of nondetergent sulfobetaines (NDSBs) can largely suppress compound oligomerization and aggregation by shifting the equilibrium toward the monomers. The rotational correlation time was obtained from the ratio of the selective and nonselective longitudinal NMR relaxation times, which directly and quantitatively reflected the apparent sizes of the compounds in the equilibrium. The rotational correlation time of the aggregation-prone compound SKF86002 (1 mM) was substantially reduced from 0.31 to 0.23 ns by cosolubilization of 100 mM NDSB195. NDSB cosolubilization allowed us to perform successful structural and biochemical experiments with substantially fewer artifacts, which represents a strategy to directly resolve the problematic oligomerization and aggregation of compounds.
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Antiinflamatorios no Esteroideos/química , Betaína/análogos & derivados , Imidazoles/química , Tiazoles/química , Betaína/química , Descubrimiento de Drogas , Espectroscopía de Resonancia Magnética , Solubilidad , SolucionesRESUMEN
While the molecular structures of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT1 receptor, different positions of the AT1 receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT1 receptor, Tyr(113), Tyr(184), Lys(199), His(256) and Gln(257) using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT1 receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr(113) in the AT1 receptor and the hydroxyl group of olmesartan, between Lys(199) and carboxyl or tetrazole groups, and between His(256) or Gln(257) and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys(199), His(256) and Gln(257) of AT1 receptor. Lys(199) in the AT1 receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys(199). The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr(113). On the other hand, the n-butyl group of irbesartan may bind to Tyr(113). In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT1 receptor and for the formation of unique modes of binding.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina/química , Antagonistas de Receptores de Angiotensina/metabolismo , Receptor de Angiotensina Tipo 1/química , Receptor de Angiotensina Tipo 1/metabolismo , Animales , Sitios de Unión , Línea Celular , Fibroblastos/metabolismo , Haplorrinos/metabolismo , Modelos Moleculares , Receptores CXCR4/metabolismo , Programas InformáticosRESUMEN
Small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism or inverse agonism toward a G-protein-coupled receptor (GPCR). Although each ligand may stabilize the receptor conformation in a different way, little is known about the precise conformational differences. We synthesized the angiotensin II type 1 receptor blocker (ARB) olmesartan, R239470 and R794847, which induced inverse agonism, antagonism and agonism, respectively, and then investigated the ligand-specific changes in the receptor conformation with respect to stabilization around transmembrane (TM)3. The results of substituted cysteine accessibility mapping studies support the novel concept that ligand-induced changes in the conformation of TM3 play a role in stabilizing GPCR. Although the agonist-, neutral antagonist and inverse agonist-binding sites in the AT(1) receptor are similar, each ligand induced specific conformational changes in TM3. In addition, all of the experimental data were obtained with functional receptors in a native membrane environment (in situ).
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Imidazoles/farmacología , Receptor de Angiotensina Tipo 1/efectos de los fármacos , Tetrazoles/farmacología , Modelos Moleculares , Conformación Molecular , Receptor de Angiotensina Tipo 1/agonistasRESUMEN
Nuclear magnetic resonance (NMR) is a versatile technique for the pharmaceutical industry. From organic chemistry to MRI, there are a number of applications of NMR. Among them, biomolecular NMR has been used for structure determination of biomolecules and analyzing the interaction between a target protein and its inhibitors. In the context of fragment-based drug discovery (FBDD), NMR has been known as a fragment screening technique, because NMR is good at detecting a weak binding compound in an accurate manner. Generally, the NMR technique for fragment screening is classified into two families: the ligand-based technique and the protein-based technique. The latter technique requires stable isotope labeled protein and also can be applied to a relatively small MW protein target. In the ligand-based technique such as saturation transfer difference (STD) and WaterLOGSY, only the NMR signals of the ligands are observed. The disadvantage of STD and WaterLOGSY is that the non-specific binding is also observed and a competition experiment is required in order to select the specific binding compound. Due to the difference in the consumption of the protein sample, the ligand-based technique has generally been used recently as a primary screening.
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Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Espectroscopía de Resonancia Magnética , Ligandos , ProteínasRESUMEN
Typical CYP2D6 substrates generally contain a basic nitrogen atom that interacts with Asp(301) and/or Glu(216) and an aromatic moiety adjacent to the site of metabolism. Recently, we found novel acidic substrates for CYP2D6, pactimibe, and its indole metabolite, R-125528, that are not protonated but are negatively charged at physiological pH. The K(m) value of R-125528 in CYP2D6-expressing microsomes was determined to be 1.74 microM, which was comparable with those of typical basic CYP2D6 substrates (1-10 microM). Pactimibe has lower affinity than R-125528; however, the K(m) value was comparable with that of metoprolol. Interestingly, their sites of metabolism, the omega-1 position of the N-octyl indoline/indole group, were relatively distant from the aromatic moiety. A pactimibe analog with an N-decyl chain was similarly labile against CYP2D6; however, analogs with N-hexyl or N-dodecyl chains were stable to CYP2D6. An induced fit docking of the ligands with an X-ray crystal structure of substrate-free CYP2D6 (Protein Data Bank code 2F9Q) indicated the involvement of an electrostatic interaction between the carboxyl group and Arg(221) and hydrophobic interaction between the aromatic moiety and Phe(483). The docking model correctly positioned the site of metabolism above the heme. The effect of the N-alkyl chain length of pactimibe analogs on their CYP2D6 metabolic stability was plausibly explained by the docking model. In conclusion, we report herein a novel CYP2D6 binding mode for the acidic substrates pactimibe and R-125528. Further investigation, such as a site-directed mutation, will be necessary to directly demonstrate the involvement of Arg(221) in CYP2D6 binding.