RESUMEN
BACKGROUND: The effect of a liberal transfusion strategy as compared with a restrictive strategy on outcomes in critically ill patients with traumatic brain injury is unclear. METHODS: We randomly assigned adults with moderate or severe traumatic brain injury and anemia to receive transfusion of red cells according to a liberal strategy (transfusions initiated at a hemoglobin level of ≤10 g per deciliter) or a restrictive strategy (transfusions initiated at ≤7 g per deciliter). The primary outcome was an unfavorable outcome as assessed by the score on the Glasgow Outcome Scale-Extended at 6 months, which we categorized with the use of a sliding dichotomy that was based on the prognosis of each patient at baseline. Secondary outcomes included mortality, functional independence, quality of life, and depression at 6 months. RESULTS: A total of 742 patients underwent randomization, with 371 assigned to each group. The analysis of the primary outcome included 722 patients. The median hemoglobin level in the intensive care unit was 10.8 g per deciliter in the group assigned to the liberal strategy and 8.8 g per deciliter in the group assigned to the restrictive strategy. An unfavorable outcome occurred in 249 of 364 patients (68.4%) in the liberal-strategy group and in 263 of 358 (73.5%) in the restrictive-strategy group (adjusted absolute difference, restrictive strategy vs. liberal strategy, 5.4 percentage points; 95% confidence interval, -2.9 to 13.7). Among survivors, a liberal strategy was associated with higher scores on some but not all the scales assessing functional independence and quality of life. No association was observed between the transfusion strategy and mortality or depression. Venous thromboembolic events occurred in 8.4% of the patients in each group, and acute respiratory distress syndrome occurred in 3.3% and 0.8% of patients in the liberal-strategy and restrictive-strategy groups, respectively. CONCLUSIONS: In critically ill patients with traumatic brain injury and anemia, a liberal transfusion strategy did not reduce the risk of an unfavorable neurologic outcome at 6 months. (Funded by the Canadian Institutes of Health Research and others; HEMOTION ClinicalTrials.gov number, NCT03260478.).
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Anemia , Lesiones Traumáticas del Encéfalo , Transfusión de Eritrocitos , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Anemia/sangre , Anemia/etiología , Anemia/terapia , Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/diagnóstico , Lesiones Traumáticas del Encéfalo/terapia , Enfermedad Crítica , Depresión/etiología , Transfusión de Eritrocitos/efectos adversos , Transfusión de Eritrocitos/métodos , Escala de Consecuencias de Glasgow , Hemoglobinas/análisis , Calidad de VidaRESUMEN
Spinal Muscular Atrophy is caused by partial loss of survival of motoneuron (SMN) protein expression. The numerous interaction partners and mechanisms influenced by SMN loss result in a complex disease. Current treatments restore SMN protein levels to a certain extent, but do not cure all symptoms. The prolonged survival of patients creates an increasing need for a better understanding of SMA. Although many SMN-protein interactions, dysregulated pathways, and organ phenotypes are known, the connections among them remain largely unexplored. Monogenic diseases are ideal examples for the exploration of cause-and-effect relationships to create a network describing the disease-context. Machine learning tools can utilize such knowledge to analyze similarities between disease-relevant molecules and molecules not described in the disease so far. We used an artificial intelligence-based algorithm to predict new genes of interest. The transcriptional regulation of 8 out of 13 molecules selected from the predicted set were successfully validated in an SMA mouse model. This bioinformatic approach, using the given experimental knowledge for relevance predictions, enhances efficient targeted research in SMA and potentially in other disease settings.
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Inteligencia Artificial , Biología Computacional , Modelos Animales de Enfermedad , Atrofia Muscular Espinal , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Animales , Ratones , Humanos , Biología Computacional/métodos , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Aprendizaje Automático , Algoritmos , Regulación de la Expresión Génica/genéticaRESUMEN
We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4'-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity.
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Benzoatos , Pirazinas , Relación Estructura-Actividad , Pirazinas/farmacología , Antivirales/farmacologíaRESUMEN
A structure-activity relationship study performed on 1H-pyrrolo[3,2-g]isoquinoline scaffold identified new haspin inhibitors with nanomolar potencies and selectivity indices (SI) over 6 (inhibitory potency evaluated against 8 protein kinases). Compound 22 was the most active of the series (haspin IC50 = 76 nM). Cellular evaluation of 22 confirmed its activity for endogenous haspin in U-2 OS cells and its anti-proliferative activity against various cell lines. In addition, the binding mode of analog 22 in complex with haspin was determined by X-ray crystallography.
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Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Pirroles , Inhibidores de Proteínas Quinasas/química , Pirroles/química , Relación Estructura-Actividad , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Isoquinolinas/química , Isoquinolinas/farmacologíaRESUMEN
BACKGROUND: Near-infrared spectroscopy regional cerebral oxygen saturation (rSO2) has gained interest as a raw parameter and as a basis for measuring cerebrovascular reactivity (CVR) due to its noninvasive nature and high spatial resolution. However, the prognostic utility of these parameters has not yet been determined. This study aimed to identify threshold values of rSO2 and rSO2-based CVR at which outcomes worsened following traumatic brain injury (TBI). METHODS: A retrospective multi-institutional cohort study was performed. The cohort included TBI patients treated in four adult intensive care units (ICU). The cerebral oxygen indices, COx (using rSO2 and cerebral perfusion pressure) as well as COx_a (using rSO2 and arterial blood pressure) were calculated for each patient. Grand mean thresholds along with exposure-based thresholds were determined utilizing sequential chi-squared analysis and univariate logistic regression, respectively. RESULTS: In the cohort of 129 patients, there was no identifiable threshold for raw rSO2 at which outcomes were found to worsen. For both COx and COx_a, an optimal grand mean threshold value of 0.2 was identified for both survival and favorable outcomes, while percent time above - 0.05 was uniformly found to have the best discriminative value. CONCLUSIONS: In this multi-institutional cohort study, raw rSO2was found to contain no significant prognostic information. However, rSO2-based indices of CVR, COx and COx_a, were found to have a uniform grand mean threshold of 0.2 and exposure-based threshold of - 0.05, above which clinical outcomes markedly worsened. This study lays the groundwork to transition to less invasive means of continuously measuring CVR.
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Lesiones Traumáticas del Encéfalo , Espectroscopía Infrarroja Corta , Adulto , Humanos , Estudios de Cohortes , Pronóstico , Estudios Retrospectivos , Espectroscopía Infrarroja Corta/métodos , Saturación de Oxígeno , Canadá , Lesiones Traumáticas del Encéfalo/diagnóstico por imagenRESUMEN
Electroencephalography is an accessible, portable, noninvasive and safe means of evaluating a patient's brain activity. It can aid in diagnosis and management decisions for post-cardiac arrest patients with seizures, myoclonus and other non-epileptic movements. It also plays an important role in a multimodal approach to neuroprognostication predicting both poor and favorable outcomes. Individuals ordering, performing and interpreting these tests, regardless of the indication, should understand the supporting evidence, logistical considerations, limitations and impact the results may have on postarrest patients and their families as outlined herein.
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The cavity-creating p53 cancer mutation Y220C is an ideal paradigm for developing small-molecule drugs based on protein stabilization. Here, we have systematically analyzed the structural and stability effects of all oncogenic Tyr-to-Cys mutations (Y126C, Y163C, Y205C, Y220C, Y234C, and Y236C) in the p53 DNA-binding domain (DBD). They were all highly destabilizing, drastically lowering the melting temperature of the protein by 8-17 °C. In contrast, two non-cancerous mutations, Y103C and Y107C, had only a moderate effect on protein stability. Differential stabilization of the mutants upon treatment with the anticancer agent arsenic trioxide and stibogluconate revealed an interesting proximity effect. Crystallographic studies complemented by MD simulations showed that two of the mutations, Y234C and Y236C, create internal cavities of different size and shape, whereas the others induce unique surface lesions. The mutation-induced pockets in the Y126C and Y205C mutant were, however, relatively small compared with that of the already druggable Y220C mutant. Intriguingly, our structural studies suggest a pronounced plasticity of the mutation-induced pocket in the frequently occurring Y163C mutant, which may be exploited for the development of small-molecule stabilizers. We point out general principles for reactivating thermolabile cancer mutants and highlight special cases where mutant-specific drugs are needed for the pharmacological rescue of p53 function in tumors.
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Mutación , Neoplasias , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Humanos , Mutación/genética , Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/metabolismo , Trióxido de Arsénico/farmacología , Simulación de Dinámica Molecular , Estabilidad Proteica/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/químicaRESUMEN
OBJECTIVE: The incidence of spondylodiscitis is rising across Europe, but the ideal treatment approach remains controversial. The choice between conservative and surgical therapies is ambiguous due to a lack of consensus. This European survey aimed to explore prevailing treatment paradigms for primary spondylodiscitis. METHODS: Spine neurosurgeons were invited through the European Association of Neurosurgical Societies Spine Section's mailing list to participate in an online survey featuring 7 spondylodiscitis case vignettes. Along with general management queries, specific patient treatment questions were posed. Data analysis was performed using R software (version 4.0.4). The index of qualitative variation (IQV) was calculated to quantify the variability in responses. RESULTS: A total of 130 responses were collected, comprising 86.9% board-certified neurosurgeons and 13.1% neurosurgeons in training, with an average of 11 years of practice. Most respondents performed 50-100 spine surgeries annually, with 66.7% specializing in spine surgery. An epidural empyema causing pronounced neurological deficits influenced 95.4% toward a surgical intervention, and mild neurological deficits and challenges in pathogen identification prompted 72.3% and 80%, respectively, to consider a surgical approach. Vertebral body destruction and spinal deformity directed 60% and 66.2%, respectively, toward surgery, whereas advanced age and comorbidities had a much smaller impact-5.4% and 9.2%, respectively. Clinical vignettes highlighted a predominant preference for conservative treatment in specific cases, with statistical significance (p < 0.05). The IQV values evaluated for each question ranged from 0.88 to 0.99, indicating low agreement across all questions among respondents. When examining the average IQV by country, intercountry variations in IQV were substantial, as illustrated by the diverse range of overall mean IQV values (0.15-0.85). CONCLUSIONS: The findings reveal a significant variability in the treatment of spondylodiscitis among European neurosurgeons, with most neurosurgeons opting for conservative treatment. These diverse strategies, both between and within countries, highlight an imperative for evidence-backed guidelines and consensus statements for this grave condition.
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Discitis , Procedimientos Neuroquirúrgicos , Humanos , Discitis/cirugía , Europa (Continente) , Masculino , Neurocirujanos , Femenino , Persona de Mediana Edad , Encuestas y Cuestionarios , Pautas de la Práctica en Medicina/estadística & datos numéricos , Adulto , Sociedades Médicas , AncianoRESUMEN
Casein kinase-2 (CK2) are serine/threonine kinases with dual co-factor (ATP and GTP) specificity, that are involved in the regulation of a wide variety of cellular functions. Small molecules targeting CK2 have been described in the literature targeting different binding pockets of the kinase with a focus on type I inhibitors such as the recently published chemical probe SGC-CK2-1. In this study, we investigated whether known allosteric inhibitors binding to a pocket adjacent to helix αD could be combined with ATP mimetic moieties defining a novel class of ATP competitive compounds with a unique binding mode. Linking both binding sites requires a chemical linking moiety that would introduce a 90-degree angle between the ATP mimetic ring system and the αD targeting moiety, which was realized using a sulfonamide. The synthesized inhibitors were highly selective for CK2 with binding constants in the nM range and low micromolar activity. While these inhibitors need to be further improved, the present work provides a structure-based design strategy for highly selective CK2 inhibitors.
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Quinasa de la Caseína II , Inhibidores de Proteínas Quinasas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Humanos , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Adenosina Trifosfato/metabolismo , Sitios de UniónRESUMEN
TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and ferroptosis. It contains a PRYSPRY domain that specifically recognizes degron sequences containing C-terminal glutamine. Ligands that bind to the TRIM7 PRYSPRY domain may have applications in the treatment of viral infections, as modulators of inflammation, and in the design of a new class of PROTACs (PROteolysis TArgeting Chimeras) that mediate the selective degradation of therapeutically relevant proteins (POIs). Here, we developed an assay toolbox for the comprehensive evaluation of TRIM7 ligands. Using TRIM7 degron sequences together with a structure-based design, we developed the first series of peptidomimetic ligands with low micromolar affinity. The terminal carboxylate moiety was required for ligand activity but prevented cell penetration. A prodrug strategy using an ethyl ester resulted in enhanced permeability, which was evaluated using confocal imaging.
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Ubiquitina-Proteína Ligasas , Ligandos , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Peptidomiméticos/química , Peptidomiméticos/farmacología , Proteolisis , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/química , Profármacos/química , Profármacos/farmacología , DegronesRESUMEN
MST1, MST2, MST3, MST4, and YSK1 are conserved members of the mammalian sterile 20-like serine/threonine (MST) family that regulate cellular functions such as proliferation and migration. The MST3 isozyme plays a role in regulating cell growth and apoptosis, and its dysregulation has been linked to high-grade tumors. To date, there are no isoform-selective inhibitors that could be used for validating the role of MST3 in tumorigenesis. We designed a series of 3-aminopyrazole-based macrocycles based on the structure of a promiscuous inhibitor. By varying the moieties targeting the solvent-exposed region and optimizing the linker, macrocycle JA310 (21c) was synthesized. JA310 exhibited high cellular potency for MST3 (EC50 = 106 nM) and excellent kinome-wide selectivity. The crystal structure of the MST3-JA310 complex provided intriguing insights into the binding mode, which is associated with large-scale structural rearrangements. In summary, JA310 demonstrates the utility of macrocyclization for the design of highly selective inhibitors and presents the first chemical probe for MST3.
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Apoptosis , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Fosforilación , Mamíferos/metabolismoRESUMEN
Mammalian STE20-like (MST) kinases 1-4 play key roles in regulating the Hippo and autophagy pathways, and their dysregulation has been implicated in cancer development. In contrast to the well-studied MST1/2, the roles of MST3/4 are less clear, in part due to the lack of potent and selective inhibitors. Here, we re-evaluated literature compounds, and used structure-guided design to optimize the p21-activated kinase (PAK) inhibitor G-5555 (8) to selectively target MST3/4. These efforts resulted in the development of MR24 (24) and MR30 (27) with good kinome-wide selectivity and high cellular potency. The distinct cellular functions of closely related MST kinases can now be elucidated with subfamily-selective chemical tool compounds using a combination of the MST1/2 inhibitor PF-06447475 (2) and the two MST3/4 inhibitors developed. We found that MST3/4-selective inhibition caused a cell-cycle arrest in the G1 phase, whereas MST1/2 inhibition resulted in accumulation of cells in the G2/M phase.
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Proteínas Serina-Treonina Quinasas , Quinasas p21 Activadas , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Mamíferos/metabolismoRESUMEN
Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here, we developed a 2-aminopyrimidine-based macrocyclic dual EPHA2/GAK kinase inhibitor as a chemical tool to study the role of these two kinases in viral entry and assembly. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55 as a potent dual EPHA2/GAK inhibitor. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells, mainly via its EPHA2 activity, and is therefore a promising candidate for further optimization of its activity against dengue virus.
RESUMEN
Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here we have optimized 2-aminopyrimidine-based macrocycles to use these compounds as chemical tools for the ephrin kinase family. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55, a potent inhibitor of EPHA2/A4 and GAK. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/A4 and GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells. However, further investigations are needed to determine whether this was a compound-specific effect or target-related.
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Inhibidores de Proteínas Quinasas , Pirimidinas , Receptor EphA2 , Humanos , Línea Celular Tumoral , Cristalografía por Rayos X , Virus del Dengue/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Compuestos Macrocíclicos/síntesis química , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis química , Receptor EphA2/antagonistas & inhibidores , Receptor EphA2/metabolismo , Relación Estructura-Actividad , MorfolinasRESUMEN
The pyrazolo[1,5-a]pyrimidine scaffold is a promising scaffold to develop potent and selective CSNK2 inhibitors with antiviral activity against ß-coronaviruses. Herein, we describe the discovery of a 1,2,4-triazole group to substitute a key amide group for CSNK2 binding present in many potent pyrazolo[1,5-a]pyrimidine inhibitors. Crystallographic evidence demonstrates that the 1,2,4-triazole replaces the amide in forming key hydrogen bonds with Lys68 and a water molecule buried in the ATP-binding pocket. This isosteric replacement improves potency and metabolic stability at a cost of solubility. Optimization for potency, solubility, and metabolic stability led to the discovery of the potent and selective CSNK2 inhibitor 53. Despite excellent in vitro metabolic stability, rapid decline in plasma concentration of 53 in vivo was observed and may be attributed to lung accumulation, although in vivo pharmacological effect was not observed. Further optimization of this novel chemotype may validate CSNK2 as an antiviral target in vivo.
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Antivirales , Quinasa de la Caseína II , Pirimidinas , Triazoles , Replicación Viral , Triazoles/farmacología , Triazoles/química , Triazoles/síntesis química , Pirimidinas/farmacología , Pirimidinas/química , Pirimidinas/síntesis química , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Animales , Humanos , Replicación Viral/efectos de los fármacos , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Pirazoles/farmacología , Pirazoles/química , Pirazoles/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Amidas/química , Amidas/farmacología , Amidas/síntesis química , Relación Estructura-Actividad , Ratones , Ratas , SARS-CoV-2/efectos de los fármacos , Descubrimiento de Drogas , MasculinoRESUMEN
Machine learning plays an important and growing role in molecular simulation. The newest version of the OpenMM molecular dynamics toolkit introduces new features to support the use of machine learning potentials. Arbitrary PyTorch models can be added to a simulation and used to compute forces and energy. A higher-level interface allows users to easily model their molecules of interest with general purpose, pretrained potential functions. A collection of optimized CUDA kernels and custom PyTorch operations greatly improves the speed of simulations. We demonstrate these features in simulations of cyclin-dependent kinase 8 (CDK8) and the green fluorescent protein chromophore in water. Taken together, these features make it practical to use machine learning to improve the accuracy of simulations with only a modest increase in cost.
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Simulación de Dinámica Molecular , Agua , Aprendizaje AutomáticoRESUMEN
We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4'-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity.