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The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2ß2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the ß-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.
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Acilcoenzima A , Proteínas Bacterianas , Dominio Catalítico , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimología , Cristalografía por Rayos X , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Acilcoenzima A/metabolismo , Acilcoenzima A/química , Especificidad por Sustrato , Sitios de Unión , Modelos Moleculares , Enoil-CoA Hidratasa/metabolismo , Enoil-CoA Hidratasa/química , Unión Proteica , 3-Hidroxiacil-CoA Deshidrogenasas/química , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismoRESUMEN
KDM4 histone demethylases became an exciting target for inhibitor development as the evidence linking them directly to tumorigenesis mounts. In this study, we set out to better understand the binding cavity using an X-ray crystallographic approach to provide a detailed landscape of possible interactions within the under-investigated region of KDM4. Our design strategy was based on utilizing known KDM binding motifs, such as nicotinic acid and tetrazolylhydrazides, as core motifs that we decided to enrich with flexible tails to map the distal histone binding site. The resulting X-ray structures of the novel compounds bound to KDM4D, a representative of the KDM4 family, revealed the interaction pattern with distal residues in the histone-binding site. The most prominent protein rearrangement detected upon ligand binding is the loop movement that blocks the accessibility to the histone binding site. Apart from providing new sites that potential inhibitors can target, the novel compounds may prove helpful in exploring the capacity of ligands to bind in sites distal to the cofactor-binding site of other KDMs or 2-oxoglutarate (2OG)-dependent oxygenases. The case study proves that combining a strong small binding motif with flexible tails to probe the binding pocket will facilitate lead discovery in classical drug-discovery campaigns, given the ease of accessing X-ray quality crystals.
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Histonas , Histona Demetilasas con Dominio de Jumonji , Piridinas , Tetrazoles , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/química , Tetrazoles/química , Tetrazoles/farmacología , Tetrazoles/metabolismo , Tetrazoles/síntesis química , Piridinas/química , Piridinas/farmacología , Piridinas/metabolismo , Piridinas/síntesis química , Humanos , Sitios de Unión , Cristalografía por Rayos X , Relación Estructura-Actividad , Histonas/metabolismo , Histonas/química , Estructura Molecular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Modelos Moleculares , Relación Dosis-Respuesta a DrogaRESUMEN
BACKGROUND: ICU nurses are most frequently at the patient's bedside, providing care for both patients and family members. They perform an essential role and are involved in decision-making. Despite this, research suggests that nurses have a limited role in the end-of-life decision-making process and are occasionally not involved. OBJECTIVE: Explore global ICU nurse involvement in end of life decisions based on the physician's perceptions and sub-analyses from the ETHICUS-2 study. DESIGN: This is a secondary analysis of a prospective multinational, observational study of the ETHICUS-2 study. SETTING: End of life decision-making processes in ICU patients were studied during a 6-month period between Sept 1, 2015, and Sept 30, 2016, in 199 ICUs in 36 countries. INTERVENTION: None. METHODS: The ETHICUS II study instrument contained 20 questions. This sub-analysis addressed the four questions related to nurse involvement in end-of-life decision-making: Who initiated the end-of-life discussion? Was withholding or withdrawing treatment discussed with nurses? Was a nurse involved in making the end-of-life decision? Was there agreement between physicians and nurses? These 4 questions are the basis for our analysis. Global regions were compared. RESULTS: Physicians completed 91.8â¯% of the data entry. A statistically significant difference was found between regions (pâ¯<â¯0.001) with Northern Europe and Australia/New Zealand having the most discussion with nurses and Latin America, Africa, Asia and North America the least. The percentages of end-of-life decisions in which nurses were involved ranged between 3 and 44â¯%. These differences were statistically significant. Agreement between physicians and nurses related to decisions resulted in a wide range of responses (27-86â¯%) (pâ¯<â¯0.001). There was a wide range of those who replied "not applicable" to the question of agreement between physicians and nurses on EOL decisions (0-41â¯%). CONCLUSION: There is large variability in nurse involvement in end-of-life decision-making in the ICU. The most concerning findings were that in some regions, according to physicians, nurses were not involved in EOL decisions and did not initiate the decision-making process. There is a need to develop the collaboration between nurses and physicians. Nurses have valuable contributions for best possible patient-centered decisions and should be respected as important parts of the interdisciplinary team. TWEETABLE ABSTRACT: Wide global differences were found in nurse end of life decision involvement, with low involvement in North and South America and Africa and higher involvement in Europe and Australia/New Zealand.
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Unidades de Cuidados Intensivos , Cuidado Terminal , Estudios Prospectivos , Humanos , Toma de Decisiones , Rol de la Enfermera , Personal de Enfermería en Hospital/psicologíaRESUMEN
CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.
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Listeria monocytogenes , Listeria monocytogenes/enzimología , Cristalografía por Rayos X/métodos , Sitios de Unión , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Fosfatos de Dinucleósidos/metabolismo , Fosfatos de Dinucleósidos/química , Antibacterianos/farmacología , Humanos , Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/metabolismo , Conformación ProteicaRESUMEN
A 1H-isoindol-3-amine was identified as suitable P1 group for the proprotein convertase furin using a crystallographic screening with a set of 20 fragments known to occupy the S1 pocket of trypsin-like serine proteases. Its binding mode is very similar to that observed for the P1 group of benzamidine-derived peptidic furin inhibitors suggesting an aminomethyl substitution of this fragment to obtain a couplable P1 residue for the synthesis of substrate-analogue furin inhibitors. The obtained inhibitors possess a slightly improved picomolar inhibitory potency compared to their benzamidine-derived analogues. The crystal structures of two inhibitors in complex with furin revealed that the new P1 group is perfectly suited for incorporation in peptidic furin inhibitors. Selected inhibitors were tested for antiviral activity against respiratory syncytial virus (RSV) and a furin-dependent influenza A virus (SC35M/H7N7) in A549 human lung cells and demonstrated an efficient inhibition of virus activation and replication at low micromolar or even submicromolar concentrations. First results suggest that the Mas-related G-protein coupled receptor GPCR-X2 could be a potential off-target for certain benzamidine-derived furin inhibitors.
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Antivirales , Diseño de Fármacos , Furina , Furina/antagonistas & inhibidores , Furina/metabolismo , Humanos , Antivirales/farmacología , Antivirales/síntesis química , Antivirales/química , Relación Estructura-Actividad , Células A549 , Virus de la Influenza A/efectos de los fármacos , Cristalografía por Rayos X , Indoles/farmacología , Indoles/química , Indoles/síntesis química , Estructura Molecular , Modelos Moleculares , Virus Sincitiales Respiratorios/efectos de los fármacos , Relación Dosis-Respuesta a DrogaRESUMEN
The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
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Descubrimiento de Drogas , SARS-CoV-2 , Descubrimiento de Drogas/métodos , SARS-CoV-2/metabolismo , Dominio Catalítico , Espectroscopía de Resonancia Magnética , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/metabolismo , Antivirales/farmacología , Simulación del Acoplamiento MolecularRESUMEN
The Shigella pathogenicity factor IpgC belongs to the class II of type III secretion system chaperones, whose members are characterized by a tetratricopeptide repeat (TPR) domain consisting of three and a half TPR motifs. Since IpgC is essential for Shigella virulence, we determined a high-resolution crystal structure of this chaperone to facilitate its use as a target for the structure-based design of anti-shigellosis compounds. The crystal structure revealed two possible homodimer assemblies, which strongly differ from the homodimer architectures so far known for IpgC and orthologues thereof. Through crystallographic fragment screening, we identified 10 small molecules that bind to IpgC and, therefore, are available for expansion to generate larger, more potent binders. A follow-up compound, based on one of our fragment hits, binds to a strictly conserved site, which overlaps with the binding site of the chaperone's substrates, IpaB and IpaC. Therefore, it constitutes a promising starting point for the design of functional IpgC inhibitors.
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Cases of thromboembolic events in 2021 flared up the discussion about the safety of Astra Zeneca's AZD1222 vaccine. We hereby report three cases of pulmonary embolism (PE), one case of extended portal vein thrombosis, and one case of combined portal vein thrombosis and PE within 2 weeks after vaccination with the Astra Zeneca AZD1222 vaccine in a 60-year-old, a 50-year old, a 33-year-old, a 30-year old, and a 40-year-old male in that year. All patients were healthy before. In three patients, we observed thrombocytopenia and to some extent unusually low antibody levels for the Spike Protein (S-protein), while the other two had normal thrombocyte counts. Only one patient had anti-platelet factor 4 (PF4)-antibodies detectable as it has been described in the "heparin-induced thrombocytopenia (HIT)-like" disease of "vaccine-induced prothrombotic immune thrombocytopenia" (VIPIT) and we therefore assume that heterogeneous mechanisms led to PE. Therefore, we advise to collect and report more cases, in order to determine the age-related risks of vaccination balanced against the benefits of immunity to SARS-COV-2 for the AZD1222 vaccine in order to gain knowledge for the next pandemic.
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COVID-19 , Tromboembolia , Trombosis , Masculino , Humanos , Adulto , Persona de Mediana Edad , ChAdOx1 nCoV-19 , Vacunas contra la COVID-19/efectos adversos , COVID-19/prevención & control , SARS-CoV-2 , Anticuerpos , Factores Inmunológicos , Tromboembolia/etiología , Factor Plaquetario 4RESUMEN
The increasing number of people dying from tuberculosis and the existence of extensively drug-resistant strains has led to an urgent need for new antituberculotic drugs with alternative modes of action. As part of the thioredoxin system, thioredoxin reductase (TrxR) is essential for the survival of Mycobacterium tuberculosis (Mtb) and shows substantial differences from human TrxR, making it a promising and most likely selective target. As a model organism for Mtb, crystals of Mycobacterium smegmatis TrxR that diffracted to high resolution were used in crystallographic fragment screening to discover binding fragments and new binding sites. The application of the 96 structurally diverse fragments from the F2X-Entry Screen revealed 56 new starting points for fragment-based drug design of new TrxR inhibitors. Over 200 crystal structures were analyzed using FragMAXapp, which includes processing and refinement by largely automated software pipelines and hit identification via the multi-data-set analysis approach PanDDA. The fragments are bound to 11 binding sites, of which four are positioned at binding pockets or important interaction sites and therefore show high potential for possible inhibition of TrxR.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Reductasa de Tiorredoxina-Disulfuro/química , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Mycobacterium tuberculosis/metabolismo , Sitios de Unión , Diseño de FármacosRESUMEN
Secretory proteins are used by pathogenic bacteria to manipulate the host systems and compete with other microorganisms, thereby enabling their survival in their host. Similar to other bacteria, secretory proteins of Mycobacterium tuberculosis also play a pivotal role in evading immune response within hosts, thereby leading to acute and latent tuberculosis infection. Prokaryotes have several classes of bacterial secretory systems out of which the Sec and Tat pathways are the most conserved in Mtb to transport proteins across the cytoplasmic membrane. Here, we report the crystal structure of a secretory protein, Rv0398c determined to 1.9 Å resolution. The protein comprises a core of antiparallel ß sheets surrounded by α helices adopting a unique ß sandwich fold. Structural comparison with other secretory proteins in Mtb and other pathogenic bacteria reveals that Rv0398c may be secreted via the Sec pathway. Our structural and in silico analyses thus provide mechanistic insights into the pathway adopted by Mtb to transport out secretory protein, Rv0398c which will facilitate the invasion to the host immune system.
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Proteínas Bacterianas , Mycobacterium tuberculosis , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Portadoras/metabolismo , Transporte BiológicoRESUMEN
The ephrin type-A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under-examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP-BHG712 and triazine-based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X-ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low-nanomolar regime (KD <10â nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.
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Neoplasias Colorrectales , Pirazoles , Humanos , Pirazoles/química , Pirimidinas/farmacología , Pirimidinas/química , Línea Celular , Neoplasias Colorrectales/tratamiento farmacológico , Línea Celular TumoralRESUMEN
The positive side of the Covid-19 pandemic is that it has provided a stimulation for innovations. As an example, we discuss the Pragmatic Controlled Trial (PCT), a twin study that can be used to confirm the real-world effectiveness (RWE) of health services under the non-standardized conditions of everyday care. Proof of the RWE could generally be important for health services with conditional approval. The PCT uses the Bayesian principle instead of randomization. It enables care under non-standardized everyday conditions and, by describing the endpoint-specific risks of each individual patient and by classifying the interventions, creates an unbiased evaluation in a non-experimental, but risk-stratified and controlled study. Patients expect a timely solution to their health problems. Until now, science has required sufficiently well-founded results before approving an innovation. The Covid-19 pandemic has shown that the demand for immediate and best possible care can be met through the conditional approval of a new care principle. With the PCT, we describe a procedure with which, after conditional approval, the missing data can be collected in order to successfully complete the approval process.
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COVID-19 , Atención a la Salud , Humanos , Teorema de Bayes , COVID-19/epidemiología , Alemania , Pandemias/prevención & control , Proyectos de Investigación , Investigación BiomédicaRESUMEN
TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structure-function relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.
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Hematological and hemorheological parameters are known to be altered in COVID-19; however, the value of combined monitoring in order to deduce disease severity is only scarcely examined. A total of 44 acute SARS-CoV-2-infected patients (aCOV) and 44 age-matched healthy controls (Con) were included. Blood of aCOV was sampled at admission (T0), and at day 2 (T2), day 5 (T5), day 10 (T10), and day 30 (T30) while blood of Con was only sampled once. Inter- and intra-group differences were calculated for hematological and hemorheological parameters. Except for mean cellular volume and mean cellular hemoglobin, all blood cell parameters were significantly different between aCOV and Con. During the acute disease state (T0-T5), hematological and hemorheological parameters were highly altered in aCOV; in particular, anemic conditions and increased immune cell response/inflammation, oxidative/nitrosative stress, decreased deformability, as well as increased aggregation, were observed. During treatment and convalescence until T30, almost all abnormal values of aCOV improved towards Con values. During the acute state of the COVID-19 disease, the hematological, as well as the hemorheological system, show fast and potentially pathological changes that might contribute to the progression of the disease, but changes appear to be largely reversible after four weeks. Measuring RBC deformability and aggregation, as well as oxidative stress induction, may be helpful in monitoring critically ill COVID-19 patients.
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COVID-19 , Hematología , Humanos , Hemorreología , SARS-CoV-2 , Índices de Eritrocitos , Enfermedad Crítica , Agregación EritrocitariaRESUMEN
BACKGROUNDResults of many randomized trials on COVID-19 convalescent plasma (CCP) have been reported, but information on long-term outcome after CCP treatment is limited. The objectives of this extended observation of the randomized CAPSID trial are to assess long-term outcome and disease burden in patients initially treated with or without CCP.METHODSOf 105 randomized patients, 50 participated in the extended observation. Quality of life (QoL) was assessed by questionnaires and a structured interview. CCP donors (n = 113) with asymptomatic to moderate COVID-19 were included as a reference group.RESULTSThe median follow-up of patients was 396 days, and the estimated 1-year survival was 78.7% in the CCP group and 60.2% in the control (P = 0.08). The subgroup treated with a higher cumulative amount of neutralizing antibodies showed a better 1-year survival compared with the control group (91.5% versus 60.2%, P = 0.01). Medical events and QoL assessments showed a consistent trend for better results in the CCP group without reaching statistical significance. There was no difference in the increase in neutralizing antibodies after vaccination between the CCP and control groups.CONCLUSIONThe trial demonstrated a trend toward better outcome in the CCP group without reaching statistical significance. A predefined subgroup analysis showed a significantly better outcome (long-term survival, time to discharge from ICU, and time to hospital discharge) among those who received a higher amount of neutralizing antibodies compared with the control group. A substantial long-term disease burden remains after severe COVID-19.Trial registrationEudraCT 2020-001310-38 and ClinicalTrials.gov NCT04433910.FundingBundesministerium für Gesundheit (German Federal Ministry of Health).
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COVID-19 , Humanos , COVID-19/terapia , COVID-19/etiología , SARS-CoV-2 , Calidad de Vida , Cápside , Estudios de Seguimiento , Inmunización Pasiva/efectos adversos , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
The identification of starting points for compound development is one of the key steps in early-stage drug discovery. Information-rich techniques such as crystallographic fragment screening can potentially increase the efficiency of this step by providing the structural information of the binding mode of the ligands in addition to the mere binding information. Here, we present the crystallographic screening of our 1000-plus-compound F2X-Universal Library against the complex of the yeast spliceosomal Prp8 RNaseH-like domain and the snRNP assembly factor Aar2. The observed 269 hits are distributed over 10 distinct binding sites on the surface of the protein-protein complex. Our work shows that hit clusters from large-scale crystallographic fragment screening campaigns identify known interaction sites with other proteins and suggest putative additional interaction sites. Furthermore, the inherent binding pose validation within the hit clusters may accelerate downstream compound optimization.
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Descubrimiento de Drogas , Proteínas , Cristalografía por Rayos X , Ligandos , Descubrimiento de Drogas/métodos , Sitios de Unión , Unión ProteicaRESUMEN
Objectives: To determine the profile of cytokines in patients with severe COVID-19 who were enrolled in a trial of COVID-19 convalescent plasma (CCP). Methods: Patients were randomized to receive standard treatment and 3 CCP units or standard treatment alone (CAPSID trial, ClinicalTrials.gov NCT04433910). The primary outcome was a dichotomous composite outcome (survival and no longer severe COVID-19 on day 21). Time to clinical improvement was a key secondary endpoint. The concentrations of 27 cytokines were measured (baseline, day 7). We analyzed the change and the correlation between serum cytokine levels over time in different subgroups and the prediction of outcome in receiver operating characteristics (ROC) analyses and in multivariate models. Results: The majority of cytokines showed significant changes from baseline to day 7. Some were strongly correlated amongst each other (at baseline the cluster IL-1ß, IL-2, IL-6, IL-8, G-CSF, MIP-1α, the cluster PDGF-BB, RANTES or the cluster IL-4, IL-17, Eotaxin, bFGF, TNF-α). The correlation matrix substantially changed from baseline to day 7. The heatmaps of the absolute values of the correlation matrix indicated an association of CCP treatment and clinical outcome with the cytokine pattern. Low levels of IP-10, IFN-γ, MCP-1 and IL-1ß on day 0 were predictive of treatment success in a ROC analysis. In multivariate models, low levels of IL-1ß, IFN-γ and MCP-1 on day 0 were significantly associated with both treatment success and shorter time to clinical improvement. Low levels of IP-10, IL-1RA, IL-6, MCP-1 and IFN-γ on day 7 and high levels of IL-9, PDGF and RANTES on day 7 were predictive of treatment success in ROC analyses. Low levels of IP-10, MCP-1 and high levels of RANTES, on day 7 were associated with both treatment success and shorter time to clinical improvement in multivariate models. Conclusion: This analysis demonstrates a considerable dynamic of cytokines over time, which is influenced by both treatment and clinical course of COVID-19. Levels of IL-1ß and MCP-1 at baseline and MCP-1, IP-10 and RANTES on day 7 were associated with a favorable outcome across several endpoints. These cytokines should be included in future trials for further evaluation as predictive factors.
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COVID-19 , Citocinas , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-17 , Quimiocina CCL3 , Factor de Necrosis Tumoral alfa , Interleucina-6 , Interleucina-4 , Cápside , COVID-19/terapia , Becaplermina , Quimiocina CXCL10 , Interleucina-2 , Interleucina-8 , Interleucina-9 , Factor Estimulante de Colonias de Granulocitos , Sueroterapia para COVID-19RESUMEN
In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, they claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.
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Proteínas , Cristalografía por Rayos X , Ligandos , Proteínas/químicaRESUMEN
Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MDâ¢GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.
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ADP-Ribosilación , Adenosina/análogos & derivados , Inhibidores de Proteasa de Coronavirus , Poli(ADP-Ribosa) Polimerasas , SARS-CoV-2 , ADP-Ribosilación/efectos de los fármacos , Adenosina/química , Adenosina/farmacología , Adenosina Difosfato Ribosa/química , Inhibidores de Proteasa de Coronavirus/química , Inhibidores de Proteasa de Coronavirus/farmacología , Humanos , Poli(ADP-Ribosa) Polimerasas/química , Unión Proteica , Dominios Proteicos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimologíaRESUMEN
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.