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1.
Nature ; 626(7997): 194-206, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38096902

RESUMEN

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.


Asunto(s)
Endonucleasas , Elementos de Nucleótido Esparcido Largo , ADN Polimerasa Dirigida por ARN , Transcripción Reversa , Humanos , Microscopía por Crioelectrón , Endonucleasas/química , Endonucleasas/genética , Endonucleasas/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , ARN/genética , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Cristalografía por Rayos X , ADN/biosíntesis , ADN/genética , Inmunidad Innata , Interferones/biosíntesis
2.
Proc Natl Acad Sci U S A ; 119(27): e2200260119, 2022 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-35771941

RESUMEN

Human endogenous retroviruses (HERVs) comprise nearly 8% of the human genome and are derived from ancient integrations of retroviruses into the germline. The biology of HERVs is poorly defined, but there is accumulating evidence supporting pathological roles in diverse diseases, such as cancer, autoimmune, and neurodegenerative diseases. Functional proteins are produced by HERV-encoded genes, including reverse transcriptases (RTs), which could be a contributor to the pathology attributed to aberrant HERV-K expression. To facilitate the discovery and development of HERV-K RT potent and selective inhibitors, we expressed active HERV-K RT and determined the crystal structure of a ternary complex of this enzyme with a double-stranded DNA substrate. We demonstrate a range of RT inhibition with antiretroviral nucleotide analogs, while classic nonnucleoside analogs do not inhibit HERV-K RT. Detailed comparisons of HERV-K RT with other known RTs demonstrate similarities to diverse RT families and a striking similarity to the HIV-1 RT asymmetric heterodimer. Our analysis further reveals opportunities for selective HERV-K RT inhibition.


Asunto(s)
Antirretrovirales , Descubrimiento de Drogas , Retrovirus Endógenos , ADN Polimerasa Dirigida por ARN , Inhibidores de la Transcriptasa Inversa , Antirretrovirales/química , Antirretrovirales/farmacología , Retrovirus Endógenos/enzimología , Retrovirus Endógenos/genética , Genes Virales , Transcriptasa Inversa del VIH/química , Humanos , Multimerización de Proteína , ADN Polimerasa Dirigida por ARN/química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología
3.
Bioorg Med Chem Lett ; 25(9): 1856-63, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25845281

RESUMEN

Glycogen synthase kinase-3 (GSK-3) has been proposed to play a crucial role in the pathogenesis of many diseases including cancer, stroke, bipolar disorders, diabetes and neurodegenerative diseases. GSK-3 inhibition has been a major area of pharmaceutical interest over the last two decades. A plethora of reports appeared recently on selective inhibitors and their co-crystal structures in GSK-3ß. We identified several series of promising new GSK-3ß inhibitors from a coherent design around a pyrrolopyridinone core structure. A systematic exploration of the chemical space around the central spacer led to potent single digit and sub-nanomolar GSK-3ß inhibitors. When dosed orally in a transgenic mouse model of Alzheimer's disease (AD), an exemplary compound showed significant lowering of Tau phosphorylation at one of the GSK-3 phosphorylating sites, Ser396. X-ray crystallography greatly aided in validating the binding hypotheses.


Asunto(s)
Aminopiridinas/farmacología , Descubrimiento de Drogas , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/química , Pirroles/química , Aminopiridinas/administración & dosificación , Aminopiridinas/química , Animales , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad
4.
Pharmacoepidemiol Drug Saf ; 24(1): 45-51, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25408418

RESUMEN

PURPOSE: To compare probabilistic and deterministic algorithms for linking mothers and infants within electronic health records (EHRs) to support pregnancy outcomes research. METHODS: The study population was women enrolled in Group Health (Washington State, USA) delivering a liveborn infant from 2001 through 2008 (N = 33,093 deliveries) and infant members born in these years. We linked women to infants by surname, address, and dates of birth and delivery using deterministic and probabilistic algorithms. In a subset previously linked using "gold standard" identifiers (N = 14,449), we assessed each approach's sensitivity and positive predictive value (PPV). For deliveries with no "gold standard" linkage (N = 18,644), we compared the algorithms' linkage proportions. We repeated our analyses in an independent test set of deliveries from 2009 through 2013. We reviewed medical records to validate a sample of pairs apparently linked by one algorithm but not the other (N = 51 or 1.4% of discordant pairs). RESULTS: In the 2001-2008 "gold standard" population, the probabilistic algorithm's sensitivity was 84.1% (95% CI, 83.5-84.7) and PPV 99.3% (99.1-99.4), while the deterministic algorithm had sensitivity 74.5% (73.8-75.2) and PPV 95.7% (95.4-96.0). In the test set, the probabilistic algorithm again had higher sensitivity and PPV. For deliveries in 2001-2008 with no "gold standard" linkage, the probabilistic algorithm found matched infants for 58.3% and the deterministic algorithm, 52.8%. On medical record review, 100% of linked pairs appeared valid. CONCLUSIONS: A probabilistic algorithm improved linkage proportion and accuracy compared to a deterministic algorithm. Better linkage methods can increase the value of EHRs for pregnancy outcomes research.


Asunto(s)
Algoritmos , Registros Electrónicos de Salud/normas , Bienestar del Lactante , Bienestar Materno , Registro Médico Coordinado/normas , Adolescente , Adulto , Atención a la Salud/normas , Atención a la Salud/estadística & datos numéricos , Registros Electrónicos de Salud/estadística & datos numéricos , Femenino , Humanos , Bienestar del Lactante/estadística & datos numéricos , Recién Nacido , Bienestar Materno/estadística & datos numéricos , Madres , Embarazo , Adulto Joven
5.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 1): 134-43, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24419386

RESUMEN

The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR leverages prior knowledge from earlier structures to facilitate ligand placement in the current structure.


Asunto(s)
Cristalografía por Rayos X/métodos , Diseño de Fármacos , Proteasa del VIH/química , Proteasa del VIH/metabolismo , VIH-1/enzimología , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
6.
Proc Natl Acad Sci U S A ; 108(37): 15366-71, 2011 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-21896751

RESUMEN

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.


Asunto(s)
Antivirales/farmacología , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Orthomyxoviridae/fisiología , Bibliotecas de Moléculas Pequeñas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Ensayos Analíticos de Alto Rendimiento , Hidrodinámica , Ratones , Modelos Moleculares , Nucleoproteínas/ultraestructura , Orthomyxoviridae/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Soluciones
7.
Pharmacoepidemiol Drug Saf ; 22(8): 834-41, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23554109

RESUMEN

PURPOSE: This study aimed to develop Natural Language Processing (NLP) approaches to supplement manual outcome validation, specifically to validate pneumonia cases from chest radiograph reports. METHODS: We trained one NLP system, ONYX, using radiograph reports from children and adults that were previously manually reviewed. We then assessed its validity on a test set of 5000 reports. We aimed to substantially decrease manual review, not replace it entirely, and so, we classified reports as follows: (1) consistent with pneumonia; (2) inconsistent with pneumonia; or (3) requiring manual review because of complex features. We developed processes tailored either to optimize accuracy or to minimize manual review. Using logistic regression, we jointly modeled sensitivity and specificity of ONYX in relation to patient age, comorbidity, and care setting. We estimated positive and negative predictive value (PPV and NPV) assuming pneumonia prevalence in the source data. RESULTS: Tailored for accuracy, ONYX identified 25% of reports as requiring manual review (34% of true pneumonias and 18% of non-pneumonias). For the remainder, ONYX's sensitivity was 92% (95% CI 90-93%), specificity 87% (86-88%), PPV 74% (72-76%), and NPV 96% (96-97%). Tailored to minimize manual review, ONYX classified 12% as needing manual review. For the remainder, ONYX had sensitivity 75% (72-77%), specificity 95% (94-96%), PPV 86% (83-88%), and NPV 91% (90-91%). CONCLUSIONS: For pneumonia validation, ONYX can replace almost 90% of manual review while maintaining low to moderate misclassification rates. It can be tailored for different outcomes and study needs and thus warrants exploration in other settings.


Asunto(s)
Procesamiento de Lenguaje Natural , Farmacoepidemiología , Neumonía/diagnóstico , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Humanos , Lactante , Modelos Logísticos , Persona de Mediana Edad , Neumonía/diagnóstico por imagen , Neumonía/epidemiología , Valor Predictivo de las Pruebas , Prevalencia , Radiografía , Adulto Joven
8.
Protein Expr Purif ; 79(1): 102-10, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21664975

RESUMEN

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.


Asunto(s)
Receptores Nicotínicos/genética , Receptores Nicotínicos/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/metabolismo , Unión Proteica , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ultracentrifugación , Receptor Nicotínico de Acetilcolina alfa 7
9.
J Chem Inf Model ; 51(8): 1931-41, 2011 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-21736376

RESUMEN

The method of conserved core substructure matching (CSM) for the overlay of protein-ligand complexes is described. The method relies upon distance geometry to align structurally similar substructures without regard to sequence similarity onto substructures from a reference protein empirically selected to include key determinants of binding site location and geometry. The error in ligand position is reduced in reoriented ensembles generated with CSM when compared to other overlay methods. Since CSM can only succeed when the selected core substructure is geometrically conserved, misalignments only rarely occur. The method may be applied to reliably overlay large numbers of protein-ligand complexes in a way that optimizes ligand position at a specific binding site or subsite or to align structures from large and diverse protein families where the conserved binding site is localized to only a small portion of either protein. Core substructures may be complex and must be chosen with care. We have created a database of empirically selected core substructures to demonstrate the utility of CSM alignment of ligand binding sites in important drug targets. A Web-based interface can be used to apply CSM to align large collections of protein-ligand complexes for use in drug design using these substructures or to evaluate the use of alternative core substructures that may then be shared with the larger user community. Examples show the benefit of CSM in the practice of structure-based drug design.


Asunto(s)
Química Farmacéutica/métodos , Descubrimiento de Drogas/métodos , Preparaciones Farmacéuticas/análisis , Proteínas Quinasas/análisis , Programas Informáticos , Estaurosporina/análisis , Sitios de Unión , Dominio Catalítico , Simulación por Computador , Minería de Datos , Bases de Datos de Proteínas , Diseño de Fármacos , Humanos , Ligandos , Modelos Moleculares , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Unión Proteica , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Alineación de Secuencia , Bibliotecas de Moléculas Pequeñas , Estaurosporina/química , Estaurosporina/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato
10.
Blood Adv ; 4(18): 4538-4549, 2020 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-32956453

RESUMEN

B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiological conditions using healthy human whole blood, teclistamab mediated dose-dependent lysis of H929 cells and activation of T cells. Antitumor activity of teclistamab was also observed in 2 BCMA+ MM murine xenograft models inoculated with human T cells (tumor inhibition with H929 model and tumor regression with the RPMI 8226 model) compared with vehicle and antibody controls. The specific and potent activity of teclistamab against BCMA-expressing cells from MM cell lines, patient samples, and MM xenograft models warrant further evaluation of this bispecific antibody for the treatment of MM. Phase 1 clinical trials (monotherapy, #NCT03145181; combination therapy, #NCT04108195) are ongoing for patients with relapsed/refractory MM.


Asunto(s)
Anticuerpos Biespecíficos , Mieloma Múltiple , Animales , Anticuerpos Biespecíficos/farmacología , Antígeno de Maduración de Linfocitos B , Humanos , Activación de Linfocitos , Ratones , Mieloma Múltiple/tratamiento farmacológico , Linfocitos T
11.
J Neurosurg Spine ; 26(6): 744-750, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28362214

RESUMEN

OBJECTIVE Systematic multidisciplinary approaches to improving quality and safety in complex surgical care have shown promise. Complication rates from complex spine surgery range from 10% to 90% for all surgeries, and the overall mortality rate is 1%-4%. These rates suggest the need for improved perioperative complex spine surgery processes designed to minimize risk and improve quality. METHODS The Group Health Research Institute and Virginia Mason Medical Center implemented a systematic multidisciplinary protocol, the Seattle Spine Team Protocol, in 2010. This protocol involves the following elements: 1) a comprehensive multidisciplinary conference including clinicians from neurosurgery, anesthesia, orthopedics, internal medicine, behavioral health, and nursing, collaboratively deciding on each patient's suitability for surgery; 2) a mandatory patient education course that reviews the risks of surgery, preparation for the surgery, and postoperative care; 3) a dual-attending-surgeon approach involving 1 neurosurgeon and 1 orthopedic spine surgeon; 4) a dedicated specialist complex spine anesthesia team; and 5) rigorous intraoperative monitoring of a patient's blood loss and coagulopathy. The authors identified 71 patients who underwent complex spine surgery involving fusion of 6 or more levels before implementation of the protocol (surgery between 2008 and 2010) and 69 patients who underwent complex spine surgery after the implementation of the protocol (2010 and 2012). All patient demographic variables, including age, sex, body mass index, smoking status, diagnosis of diabetes and/or osteoporosis, previous surgery, and the nature of the spinal deformity, were comprehensively assessed. Also comprehensively assessed were surgical variables, including operative time, number of levels fused, and length of stay. The authors assessed overall complication rates at 30 days and 1 year and detailed deaths, cardiovascular events, infections, instrumentation failures, and CSF leaks. Chi-square and Wilcoxon rank-sum tests were used to assess differences in patient characteristics for patients with a procedure in the preimplementation period from those in the postimplementation period under a Poisson distribution model. RESULTS Patients who underwent surgery after implementation of the Seattle Spine Team Protocol had a statistically significant reduction (relative risk 0.49 [95% CI 0.30-0.78]) in all measured complications, including cardiovascular events, wound infections, other perioperative infections, and implant failures within 30 days after surgery; the analysis was adjusted for age and Charlson comorbidity score. A trend toward fewer deaths in this group was also found. CONCLUSIONS This type of systematic quality improvement strategy can improve quality and patient safety and might be applicable to other complex surgical disciplines. Implementation of these strategies in the treatment of adult spinal deformity will likely lead to better patient outcomes.


Asunto(s)
Complicaciones Posoperatorias/prevención & control , Escoliosis/cirugía , Anciano , Protocolos Clínicos , Medicina Basada en la Evidencia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Selección de Paciente , Complicaciones Posoperatorias/epidemiología , Mejoramiento de la Calidad , Estudios Retrospectivos , Riesgo , Escoliosis/epidemiología , Fusión Vertebral/efectos adversos , Columna Vertebral/cirugía
12.
Genome Med ; 7(1): 67, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26221186

RESUMEN

BACKGROUND: In an effort to return actionable results from variant data to electronic health records (EHRs), participants in the Electronic Medical Records and Genomics (eMERGE) Network are being sequenced with the targeted Pharmacogenomics Research Network sequence platform (PGRNseq). This cost-effective, highly-scalable, and highly-accurate platform was created to explore rare variation in 84 key pharmacogenetic genes with strong drug phenotype associations. METHODS: To return Clinical Laboratory Improvement Amendments (CLIA) results to our participants at the Group Health Cooperative, we sequenced the DNA of 900 participants (61 % female) with non-CLIA biobanked samples. We then selected 450 of those to be re-consented, to redraw blood, and ultimately to validate CLIA variants in anticipation of returning the results to the participant and EHR. These 450 were selected using an algorithm we designed to harness data from self-reported race, diagnosis and procedure codes, medical notes, laboratory results, and variant-level bioinformatics to ensure selection of an informative sample. We annotated the multi-sample variant call format by a combination of SeattleSeq and SnpEff tools, with additional custom variables including evidence from ClinVar, OMIM, HGMD, and prior clinical associations. RESULTS: We focused our analyses on 27 actionable genes, largely driven by the Clinical Pharmacogenetics Implementation Consortium. We derived a ranking system based on the total number of coding variants per participant (75.2±14.7), and the number of coding variants with high or moderate impact (11.5±3.9). Notably, we identified 11 stop-gained (1 %) and 519 missense (20 %) variants out of a total of 1785 in these 27 genes. Finally, we prioritized variants to be returned to the EHR with prior clinical evidence of pathogenicity or annotated as stop-gain for the following genes: CACNA1S and RYR1 (malignant hyperthermia); SCN5A, KCNH2, and RYR2 (arrhythmia); and LDLR (high cholesterol). CONCLUSIONS: The incorporation of genetics into the EHR for clinical decision support is a complex undertaking for many reasons including lack of prior consent for return of results, lack of biospecimens collected in a CLIA environment, and EHR integration. Our study design accounts for these hurdles and is an example of a pilot system that can be utilized before expanding to an entire health system.

13.
J Mol Biol ; 427(4): 924-942, 2015 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-25579995

RESUMEN

The human pregnane X receptor (PXR) is a promiscuous nuclear receptor that functions as a sensor to a wide variety of xenobiotics and regulates expression of several drug metabolizing enzymes and transporters. We have generated "Adnectins", derived from 10th fibronectin type III domain ((10)Fn3), that target the PXR ligand binding domain (LBD) interactions with the steroid receptor co-activator-1 (SRC-1) peptide, displacing SRC-1 binding. Adnectins are structurally homologous to the immunoglobulin superfamily. Three different co-crystal structures of PXR LBD with Adnectin-1 and CCR1 (CC chemokine receptor-1) antagonist Compound-1 were determined. This structural information was used to modulate PXR affinity for a related CCR1 antagonist compound that entered into clinical trials for rheumatoid arthritis. The structures of PXR with Adnectin-1 reveal specificity of Adnectin-1 in not only targeting the interface of the SRC-1 interactions but also engaging the same set of residues that are involved in binding of SRC-1 to PXR. Substituting SRC-1 with Adnectin-1 does not alter the binding conformation of Compound-1 in the ligand binding pocket. The structure also reveals the possibility of using Adnectins as crystallization chaperones to generate structures of PXR with compounds of interest.


Asunto(s)
Coactivador 1 de Receptor Nuclear/química , Receptores CCR1/antagonistas & inhibidores , Receptores de Esteroides/química , Urea/análogos & derivados , Valina/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Lignanos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Receptor X de Pregnano , Unión Proteica , Estructura Terciaria de Proteína , Receptores CCR1/metabolismo , Alineación de Secuencia , Resonancia por Plasmón de Superficie , Urea/química , Urea/metabolismo , Urea/farmacología , Valina/química , Valina/metabolismo , Valina/farmacología
14.
Drug Discov Today ; 17(17-18): 935-41, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22406695

RESUMEN

Recent treatments of computational knowledge worker productivity have focused upon the value the discipline brings to drug discovery using positive anecdotes. While this big picture approach provides important validation of the contributions of these knowledge workers, the impact accounts do not provide the granular detail that can help individuals and teams perform better. I suggest balancing the impact-focus with quantitative measures that can inform the development of scientists. Measuring the quality of work, analyzing and improving processes, and the critical evaluation of communication can provide immediate performance feedback. The introduction of quantitative measures can complement the longer term reporting of impacts on drug discovery. These metric data can document effectiveness trends and can provide a stronger foundation for the impact dialogue.


Asunto(s)
Diseño Asistido por Computadora , Diseño de Fármacos , Industria Farmacéutica/métodos , Humanos
15.
J Biol Chem ; 281(26): 18193-200, 2006 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-16638752

RESUMEN

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 A resolution. The structural similarity of this polymerase to other alpha polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.


Asunto(s)
ADN Polimerasa Dirigida por ADN/química , Exodesoxirribonucleasas/química , Herpesvirus Humano 1/enzimología , Proteínas Virales/química , Aciclovir/química , Antivirales/química , Sitios de Unión , Cristalografía , Diseño de Fármacos , Farmacorresistencia Viral , Herpesvirus Humano 1/efectos de los fármacos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Quinolinas/química
16.
Anal Biochem ; 309(2): 186-95, 2002 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-12413450

RESUMEN

Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)'s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.


Asunto(s)
Inhibidores Enzimáticos/análisis , Hepacivirus/enzimología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Calorimetría/métodos , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , ADN/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Cinética , Ligandos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Desnaturalización Proteica , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Especificidad por Sustrato
17.
J Biol Chem ; 277(34): 31163-71, 2002 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-12048187

RESUMEN

The first crystal structure of Class II peptide deformylase has been determined. The enzyme from Staphylococcus aureus has been overexpressed and purified in Escherichia coli and the structure determined by x-ray crystallography to 1.9 A resolution. The purified iron-enriched form of S. aureus peptide deformylase enzyme retained high activity over many months. In contrast, the iron-enriched form of the E. coli enzyme is very labile. Comparison of the two structures details many differences; however, there is no structural explanation for the dramatic activity differences we observed. The protein structure of the S. aureus enzyme reveals a fold similar, but not identical to, the well characterized E. coli enzyme. The most striking deviation of the S. aureus from the E. coli structure is the unique conformation of the C-terminal amino acids. The distinctive C-terminal helix of the latter is replaced by a strand in S. aureus which wraps around the enzyme, terminating near the active site. Although there are no differences at the amino acid level near the active site metal ion, significant changes are noted in the peptide binding cleft which may play a role in the design of general peptide deformylase inhibitors.


Asunto(s)
Amidohidrolasas , Aminopeptidasas/química , Proteínas Bacterianas/química , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/antagonistas & inhibidores , Cristalización , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína
18.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 12): 2153-6, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12454484

RESUMEN

In bacteria the biosynthesis of all nascent polypeptides begins with N-formylmethionine. The post-translational removal of the N-formyl group is carried out by peptide deformylase (PDF). Processing of the N-formyl group from critical bacterial proteins is required for cell survival. This formylation/deformylation cycle is unique to eubacteria and is not utilized in eucaryotic cytosolic protein biosynthesis. Thus, inhibition of PDF would halt bacterial growth, spare host cell-function, and would be a novel mechanism for a new class of antibiotic. Diffraction-quality Se-met crystals of S. aureus PDF were prepared that belong to space group C222(1) with unit cell parameters of a = 94.1 b = 121.9 c = 47.6 A. Multiple anomalous dispersion data were collected at the Advanced Photon Source 17-ID beamline and used to solve the PDF structure to 1.9 A resolution. Crystals were also prepared with three PDF inhibitors: thiorphan, actinonin and PNU-172550. The thiorphan and actinonin co-crystals belong to space group C222(1) with similar unit-cell dimensions. Repeated attempts to generate a complex structure of PDF with PNU-172550 from the orthorhombic space group were unsuccessful. Crystallization screening identified an alternate C2 crystal form with unit-cell dimensions of a = 93.4 b = 42.5 c = 104.1 A, beta = 93 degrees.


Asunto(s)
Amidohidrolasas , Aminopeptidasas/química , Inhibidores Enzimáticos/química , Staphylococcus aureus/enzimología , Aminopeptidasas/antagonistas & inhibidores , Cristalización , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Conformación Proteica
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