RESUMEN
BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structureactivity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.
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Antiinflamatorios no Esteroideos , Antineoplásicos , Anticonceptivos Masculinos , Descubrimiento de Drogas , Proteínas Nucleares , Bibliotecas de Moléculas Pequeñas , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/aislamiento & purificación , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Anticonceptivos Masculinos/química , Anticonceptivos Masculinos/aislamiento & purificación , Anticonceptivos Masculinos/farmacología , ADN/genética , Humanos , Masculino , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Dominios Proteicos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of cis PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and cis Pro enhancement at both PEST sites lead to a 5-10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The cis-populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.
Asunto(s)
Peptidilprolil Isomerasa de Interacción con NIMA , Proteína Homeótica Nanog , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Proteína Homeótica Nanog/metabolismo , Proteína Homeótica Nanog/genética , Fosforilación , Humanos , Estabilidad Proteica , Unión Proteica , EstereoisomerismoRESUMEN
Ammonium perfluorooctanoate (APFOA) was used as a surfactant for the separation of free unsaturated C18 fatty acids by micellar electrokinetic chromatography. A simple background electrolyte of 50 mM APFOA water/methanol (90:10, v/v) at pH = 10 enabled the repeatable separation of oleic acid, elaidic acid, linoleic acid, and alpha-linolenic acid in less than 20 min. Separation conditions were optimized regarding various parameters (organic solvent, counterion, APFOA concentration, and pH). Because the repulsive interactions between fluorocarbon chains and hydrogenated chains are known to lead to segregation and phase separation, the choice of perfluorinated micelles to separate such perhydrogenated long-chain acids could appear astonishing. Therefore, the critical micelle concentration, the charge density, and the mobility of the micelles have been determined, resulting in a first description of the separation process.
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Cromatografía Capilar Electrocinética Micelar , Micelas , Cromatografía Capilar Electrocinética Micelar/métodos , Tensoactivos/química , CaprilatosRESUMEN
Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFß) to determine the structure of the (A3G-Vif-CRL5-CBFß) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFß complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes.
Asunto(s)
Desaminasa APOBEC-3G/química , Subunidad beta del Factor de Unión al Sitio Principal/química , Proteínas Cullin/química , Ubiquitina-Proteína Ligasas/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Espectrometría de Masas , Modelos MolecularesRESUMEN
Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Estructuras R-Loop , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , ADN/metabolismo , ARN/metabolismo , CohesinasRESUMEN
Capillary electrophoresis coupled to LED-induced fluorescence detection is a robust and sensitive technique used for amino acids (AA) analysis in biological media, after labeling with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA). We wanted to quantitate in plasma tryptophan (Trp), tyrosine (Tyr), valine (Val), and isoleucine (Ile). Among the different labeled AA-CBQCA, Trp has the lowest fluorescence yield, which makes its detection and quantification very difficult in biological samples such as plasma. We tried to improve Trp analysis by CE/LED-induced fluorescence detection to its maximal sensitivity by using large volume sample stacking as a preconcentration step in our analytical protocol. At pH 9.5, this step caused a drop in resolution during the separation of the four AAs and it was therefore necessary to work at pH 10. We have found that Tyr, Val, Ile, and Trp are detected and well separated from the other AAs, but Trp cannot be quantified in plasma samples, mainly because of the low fluorescence yield of the Trp-CBQCA derivative. The recorded LOD is 0.18 µM for Trp-CBQCA in standard solution with a resolution between Trp and Tyr of 1.2, while the LOD is 6 µM in plasma with the same resolution. Trp, Tyr, Val, and Ile are, however, efficiently quantified when using a 3 M acetic acid electrolyte and CE associated with capacitively coupled contactless conductivity detection, which also has the advantage of not requiring derivatization or large volume sample stacking. This article demonstrates, for the CE user, that quantitative analysis of these four AA in mouse plasma can be performed by CE-fluorescence after CBQCA labeling, with the exception of Trp. It can be advantageously replaced by CE/capacitively coupled contactless conductivity detection, the only efficient one for Trp, Tyr, Val, and Ile quantification. In this case, the LOD for Trp is 2 µM. The four AAs are separated with resolution with neighbors above 1.5.
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Aminoácidos/sangre , Animales , Isoleucina , Ratones , Quinolinas , Triptófano , Tirosina , ValinaRESUMEN
The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.
Asunto(s)
Antivirales/farmacología , Citosina Desaminasa/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/inmunología , Desaminasas APOBEC , Antivirales/química , Proteínas Cullin/química , Proteínas Cullin/metabolismo , Citidina Desaminasa , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/terapia , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Ubiquitina/metabolismo , Ubiquitinación , Ensamble de Virus , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Human immunodeficiency virus-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 (A3) antiviral proteins. Vif folds when it binds Cullin-RING E3 ligase 5 and the transcription cofactor CBF-ß. A five-protein complex containing the substrate receptor (Vif, CBF-ß, Elongin-B, Elongin-C (VCBC)) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use molecular dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and an A3 protein bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, whereas VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation-optimized spectroscopy NMR data, which indicates that the VCBC complex without CUL5 is dynamic on the µs-ms timescale. Our NMR data also show that the VCBC complex is more conformationally restricted when bound to the antiviral APOBEC3F (one of the A3 proteins), consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjunction with the global dynamics of the complex. Like other substrate receptors, VCBC can exist alone or in complex with CUL5 and other proteins in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.
Asunto(s)
Proteínas Cullin/química , Citidina Desaminasa/química , Simulación de Dinámica Molecular , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Desaminasas APOBEC , Subunidad beta del Factor de Unión al Sitio Principal/química , Cristalización , Elonguina/química , Humanos , Cinética , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
Lipids are naturally occurring organic compounds that can be classified into a number of types based on their solubility in nonpolar organic solvents, and are generally insoluble in water. The great structural variety of these various types of lipids has led them to be components of many different biological substances such as oils, waxes, cellular membranes, tissues and biological fluids. The use of capillary electrophoresis (CE) for the study of lipids during the past 30 years has been relatively rare when compared to its use for other classes of biomolecules, primarily due to their insolubility in water. However, a number of interesting studies have been conducted, and as part of this review, we will present the different approaches that were used, which mainly consist of micellar kinetic chromatography and non-aqueous CE. The main advantages of the use of these techniques compared to GC is the very simple sample preparation that is required and, compared to LC, the very robust and quick separations that can be obtained. In this review, we present the various methods that have been reported in the literature that have been used for the study of fatty acids, phospholipids, glycerides, eicosanoids and sterols, with the inclusion of various tables presenting descriptions of the CE methods used as well as the methods of detection, including UV absorbance, fluorescence, mass spectrometry, and conductivity. This review aims to demonstrate that CE can be easily used for the analysis of lipids.
Asunto(s)
Electroforesis Capilar/métodos , Lípidos/análisis , Conductividad Eléctrica , Lípidos/química , Análisis EspectralRESUMEN
Tryptophane (Trp) labelled by 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) is very difficult to identify using CE and fluorescence detection (480 nm). Why in this article some mass spectrometry experiments show that Trp is really labelled by CBQCA as Leucine (Leu)? If the maximum of UV absorption (λmax ) is the same between Leu-CBQCA and Trp-CBQCA, the molar extinction coefficient is around 2 fold higher for Trp-CBQCA. The fluorescence of the Leu-CBQCA derivative is 50 times more important than for Trp-CBQCA. The addition of 7.5 mM of ß-cyclodextrin (ß-CD) was found to be a good mean to improve 2.1 fold the sensitivity of the Trp-CBQCA fluorescence. Using a buffer containing SDS and ß-CD in CE, a LOD of 0.7 µM of L-Trp can be reached and the ratio of the intensities between Leu, Isoleucine, Valine, Trp is 100, 21, 15, 1. Negative ESI/ MS and MS/MS of the labeled amino acids show that a loss of the carboxylate function takes place. In the presence of two enantiomers of Trp-CBQCA, we have shown that this decarboxylation is not due to the derivatization process in the solution but rather occurs in the source of the mass spectrometer.
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Benzoatos/química , Electroforesis Capilar/métodos , Colorantes Fluorescentes/química , Quinolinas/química , Espectrometría de Fluorescencia/métodos , Triptófano , Leucina/análisis , Leucina/química , Estereoisomerismo , Espectrometría de Masas en Tándem , Triptófano/análisis , Triptófano/químicaRESUMEN
In the postprandial state, glucose homeostasis is challenged by macronutrient intake, including proteins that trigger insulin secretion and provide glucose precursors. However, little is known about the postprandial response of gluconeogenesis to a protein meal. We aimed to quantify the evolution of fractional gluconeogenesis after a meat meal. Thirteen healthy subjects received oral doses of D2O. After fasting overnight, they ingested a steak (120 g). Glycemia, insulinemia, and 2H enrichments in glucose and plasma water were measured for 8 h after the meal. Fractional gluconeogenesis was assessed using the average method. Glucose was stable for 5 h and then decreased. There was a slight increase of insulin 1 h after the meal. 2H enrichment in the carbon 5 position of glucose (C5) increased after 2 h, whereas it decreased in plasma water. Consequently, fractional gluconeogenesis increased from 68.2 ± 7.2% before the meal to 75.5 ± 5.8% 8 h after the meal, the latter corresponding to 22 h without a glucose supply. These values are consistent with the exhaustion of glycogen stores after 24 h but represent the highest among values in the literature. The impact of methodological conditions is discussed.
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Glucemia/metabolismo , Agua Corporal/metabolismo , Proteínas en la Dieta , Gluconeogénesis/fisiología , Insulina/metabolismo , Periodo Posprandial/fisiología , Carne Roja , Adulto , Óxido de Deuterio , Ayuno , Femenino , Voluntarios Sanos , Humanos , Masculino , Plasma/metabolismo , Factores de Tiempo , Adulto JovenRESUMEN
Men or mice with homozygous serine/threonine kinase 33 (STK33) mutations are sterile owing to defective sperm morphology and motility. To chemically evaluate STK33 for male contraception with STK33-specific inhibitors, we screened our multibillion-compound collection of DNA-encoded chemical libraries, uncovered potent STK33-specific inhibitors, determined the STK33 kinase domain structure bound with a truncated hit CDD-2211, and generated an optimized hit CDD-2807 that demonstrates nanomolar cellular potency (half-maximal inhibitory concentration = 9.2 nanomolar) and favorable metabolic stability. In mice, CDD-2807 exhibited no toxicity, efficiently crossed the blood-testis barrier, did not accumulate in brain, and induced a reversible contraceptive effect that phenocopied genetic STK33 perturbations without altering testis size. Thus, STK33 is a chemically validated, nonhormonal contraceptive target, and CDD-2807 is an effective tool compound.
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Anticoncepción , Anticonceptivos Masculinos , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Bibliotecas de Moléculas Pequeñas , Animales , Humanos , Masculino , Ratones , Barrera Hematotesticular/metabolismo , Anticonceptivos Masculinos/química , Anticonceptivos Masculinos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Testículo/efectos de los fármacos , Anticoncepción/métodos , Relación Estructura-ActividadRESUMEN
The specificity of proteases for the residues in and length of substrates is key to understanding their regulatory mechanism, but little is known about length selectivity. Crystal structure analyses of the bacterial aminopeptidase PepS, combined with functional and single-molecule FRET assays, have elucidated a molecular basis for length selectivity. PepS exists in open and closed conformations. Substrates can access the binding hole in the open conformation, but catalytic competency is only achieved in the closed conformation by formation of the S1 binding pocket and proximal movement of Glu343, a general base, to the cleavage site. Hence, peptides longer than the depth of the binding hole block the transition from the open to the closed conformation, and thus length selection is a prerequisite for catalytic activation. A triple-sieve interlock mechanism is proposed featuring the coupling of length selectivity with residue specificity and active-site positioning.
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Aminopeptidasas/química , Aminopeptidasas/metabolismo , Aminopeptidasas/genética , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Conformación Proteica , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/genética , Relación Estructura-Actividad , Especificidad por Sustrato/genéticaRESUMEN
The receptor activator of nuclear factor-κB (RANK) and its ligand RANKL, which belong to the tumor necrosis factor (TNF) receptor-ligand family, mediate osteoclastogenesis. The crystal structure of the RANKL ectodomain (eRANKL) in complex with the RANK ectodomain (eRANK) combined with biochemical assays of RANK mutants indicated that three RANK loops (Loop1, Loop2, and Loop3) bind to the interface of a trimeric eRANKL. Loop3 is particularly notable in that it is structurally distinctive from other TNF-family receptors and forms extensive contacts with RANKL. The disulfide bond (C125-C127) at the tip of Loop3 is important for determining the unique topology of Loop3, and docking E126 close to RANKL, which was supported by the inability of C127A or E126A mutants of RANK to bind to RANKL. Inhibitory activity of RANK mutants, which contain loops of osteoprotegerin (OPG), a soluble decoy receptor to RANKL, confirmed that OPG shares the similar binding mode with RANK and OPG. Loop3 plays a key role in RANKL binding. Peptide inhibitors designed to mimic Loop3 blocked the RANKL-induced differentiation of osteoclast precursors, suggesting that they could be developed as therapeutic agents for the treatment of osteoporosis and bone-related diseases. Furthermore, some of the RANK mutations associated with autosomal recessive osteopetrosis (ARO) resulted in reduced RANKL-binding activity and failure to induce osteoclastogenesis. These results, together with structural interpretation of eRANK-eRANKL interaction, provided molecular understanding for pathogenesis of ARO.
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Huesos/metabolismo , Modelos Moleculares , Oligopéptidos/farmacología , Osteopetrosis/metabolismo , Osteoprotegerina/metabolismo , Péptidos Cíclicos/farmacología , Ligando RANK/química , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Cristalografía , Ratones , Mutagénesis Sitio-Dirigida , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteopetrosis/genética , Ligando RANK/antagonistas & inhibidores , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
BACKGROUND: Postherpetic neuralgia (PHN) is the most common and bearable complication of herpes zoster (HZ). This pain may have negative impact on the patient's all aspects of daily life and health-related quality of life (HRQOL). Despite numerous advances in treatment, many patients remain resistant to the current therapy options. It is the first time subcutaneous injection of methylprednisolone acetate and lidocaine has been used to treat refractory PHN. We report the results of this treatment evaluating pain relief and HRQOL improvement in this disorder. METHODS: A total of 43 patients with refractory PHN was enrolled in the observational study. All patients received daily subcutaneous injection of methylprednisolone acetate and lidocaine for 10 consecutive days. The severity of pain was assessed by using Visual Analog Scale (VAS), and 36-Item Short Form Survey (SF-36) was applied to evaluate HRQOL. Assessment of the pain and HRQOL was carried out at baseline and posttreatment at 4 weeks as well as 6 and 12 months. RESULTS: At baseline, all patients experienced severe PHN with average VAS scores of 8.44 ± 0.85 (minimum 7; maximum 10). At 4 weeks, 6 months, and 12 months after treatment, the pain had significantly decreased (P < .001), and all subjects showed significant improvement in all eight domains of HRQOL. No major adverse events associated with the subcutaneous injection were observed. CONCLUSIONS: Our results indicate that subcutaneous injection of methylprednisolone acetate and lidocaine can be an effective and safe treatment for PHN.
RESUMEN
Mannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme in Staphylococcus aureus mannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH from S. aureus USA300, a strain of community-associated methicillin-resistant S. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target. In vitro cell viability assessment of ΔmtlD knockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor of S. aureus M1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of SaM1PDH at the atomic level, the crystal structure of SaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the SaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose SaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors of SaM1PDH based on structure-function studies.IMPORTANCE Due to the shortage of effective antibiotics against drug-resistant Staphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase of S. aureus USA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using SaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with a SaM1PDH inhibitor or knockout of the gene encoding SaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of SaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of SaM1PDH. Our results strongly support the concept that targeting of SaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.
Asunto(s)
Antibacterianos/farmacología , Manitol/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Deshidrogenasas del Alcohol de Azúcar/química , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Animales , Femenino , Macrófagos/microbiología , Masculino , Staphylococcus aureus Resistente a Meticilina/enzimología , Staphylococcus aureus Resistente a Meticilina/genética , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Mutación , Células RAW 264.7 , Infecciones Estafilocócicas/microbiología , Deshidrogenasas del Alcohol de Azúcar/genética , VirulenciaRESUMEN
The receptor activator of nuclear factor κB (RANK) and its ligand RANKL are key regulators of osteoclastogenesis and well-recognized targets in developing treatments for bone disorders associated with excessive bone resorption, such as osteoporosis. Our previous work on the structure of the RANK-RANKL complex revealed that Loop3 of RANK, specifically the non-canonical disulfide bond at the tip, performs a crucial role in specific recognition of RANKL. It also demonstrated that peptide mimics of Loop3 were capable of interfering with the function of RANKL in osteoclastogenesis. Here, we reported the structure-based design of a smaller peptide with enhanced inhibitory efficiency. The kinetic analysis and osteoclast differentiation assay showed that in addition to the sharp turn induced by the disulfide bond, two consecutive arginine residues were also important for binding to RANKL and inhibiting osteoclastogenesis. Docking and molecular dynamics simulations proposed the binding mode of the peptide to the RANKL trimer, showing that the arginine residues provide electrostatic interactions with RANKL and contribute to stabilizing the complex. These findings provided useful information for the rational design of therapeutics for bone diseases associated with RANK/RANKL function.
Asunto(s)
Osteogénesis/efectos de los fármacos , Peptidomiméticos/síntesis química , Peptidomiméticos/farmacología , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/química , Animales , Arginina/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Diseño de Fármacos , Humanos , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Peptidomiméticos/química , Unión Proteica , Relación Estructura-ActividadRESUMEN
Streptococcus pneumoniae Sp1610, a Class-I fold S-adenosylmethionine (AdoMet)-dependent methyltransferase, is a member of the COG2384 family in the Clusters of Orthologous Groups database, which catalyzes the methylation of N(1)-adenosine at position 22 of bacterial tRNA. We determined the crystal structure of Sp1610 in the ligand-free and the AdoMet-bound forms at resolutions of 2.0 and 3.0 A, respectively. The protein is organized into two structural domains: the N-terminal catalytic domain with a Class I AdoMet-dependent methyltransferase fold, and the C-terminal substrate recognition domain with a novel fold of four alpha-helices. Observations of the electrostatic potential surface revealed that the concave surface located near the AdoMet binding pocket was predominantly positively charged, and thus this was predicted to be an RNA binding area. Based on the results of sequence alignment and structural analysis, the putative catalytic residues responsible for substrate recognition are also proposed.