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The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5'-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.
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Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA , Trifosfato de Adenosina , Domínio Catalítico , Microscopia Crioeletrônica , DNA/metabolismo , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Células HeLa , Holoenzimas/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Piridazinas , QuinazolinasRESUMO
Ligand binding hotspots are regions of protein surfaces that form particularly favourable interactions with small molecule pharmacophores. Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment-based competitive 19F Ligand Basedâ NMR (LB-NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand-binding hotspots. As a proof of concept, the method was applied to 4'-phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). X-ray crystallographic characterisation of the hits from a 960-member fragment screen identified three ligand-binding hotspots across the PPAT active site. From the fragment hits a collection of 19F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single 19F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive 19Fâ LB-NMR with this suite of 19F reporters recapitulated the binding affinity and site ID assignments made by ITC and X-ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment-based competitive 19Fâ LB-NMR screening platform for hotspot-directed ligand profiling.
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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly growing infectious disease, widely spread with high mortality rates. Since the release of the SARS-CoV-2 genome sequence in March 2020, there has been an international focus on developing target-based drug discovery, which also requires knowledge of the 3D structure of the proteome. Where there are no experimentally solved structures, our group has created 3D models with coverage of 97.5% and characterized them using state-of-the-art computational approaches. Models of protomers and oligomers, together with predictions of substrate and allosteric binding sites, protein-ligand docking, SARS-CoV-2 protein interactions with human proteins, impacts of mutations, and mapped solved experimental structures are freely available for download. These are implemented in SARS CoV-2 3D, a comprehensive and user-friendly database, available at https://sars3d.com/. This provides essential information for drug discovery, both to evaluate targets and design new potential therapeutics.
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Antivirais/farmacologia , COVID-19/virologia , Bases de Dados de Proteínas , Sistemas de Liberação de Medicamentos , Proteoma , SARS-CoV-2/efeitos dos fármacos , Humanos , SARS-CoV-2/isolamento & purificaçãoRESUMO
Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.
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Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , RNA de Transferência/metabolismo , tRNA Metiltransferases/antagonistas & inibidores , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/enzimologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/enzimologia , Ligação Proteica , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismoRESUMO
Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.
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Arginina/deficiência , Mycobacterium tuberculosis/metabolismo , Estresse Oxidativo , Antioxidantes/metabolismo , Antituberculosos/farmacologia , Arginina/metabolismo , Dano ao DNA , Farmacorresistência Bacteriana , Citometria de Fluxo , Perfilação da Expressão Gênica , Técnicas In Vitro , Redes e Vias Metabólicas , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 1040 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an example. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as 'warm spots' for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources. This article is part of the theme issue 'Fifty years of synchrotron science: achievements and opportunities'.
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Descoberta de Drogas/história , Síncrotrons/história , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Ensaios de Triagem em Larga Escala/história , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/tendências , História do Século XX , História do Século XXI , Humanos , Modelos Moleculares , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/metabolismoRESUMO
For over four decades structural biology has been used to understand the mechanisms of disease, and structure-guided approaches have demonstrated clearly that they can contribute to many aspects of early drug discovery, both computationally and experimentally. Structure can also inform our understanding of impacts of mutations in human genetic diseases and drug resistance in cancers and infectious diseases. We discuss the ways that structural insights might be useful in both repurposing off-licence drugs and guide the design of new molecules that might be less susceptible to drug resistance in the future.
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Descoberta de Drogas/métodos , Farmacorresistência Bacteriana/efeitos dos fármacos , Mutação/efeitos dos fármacos , Reposicionamento de Medicamentos , Predisposição Genética para Doença , Humanos , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Native nanoelectrospray ionization mass spectrometry is an underutilized technique for fragment screening. In this study, the first demonstration is provided of the use of native mass spectrometry for screening fragments against a protein-DNA interaction. EthR is a transcriptional repressor of EthA expression in Mycobacterium tuberculosis (Mtb) that reduces the efficacy of ethionamide, a second-line antitubercular drug used to combat multidrug-resistant Mtb strains. A small-scale fragment screening campaign was conducted against the EthR-DNA interaction using native mass spectrometry, and the results were compared with those from differential scanning fluorimetry, a commonly used primary screening technique. Hits were validated by surface plasmon resonance and X-ray crystallography. The screening campaign identified two new fragments that disrupt the EthR-DNA interaction inâ vitro (IC50 =460-610â µm) and bind to the hydrophobic channel of the EthR dimer.
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DNA/química , Espectrometria de Massas/métodos , Mycobacterium tuberculosis/química , Proteínas Repressoras/química , Cristalografia por Raios X , Fluorometria/métodos , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Proteínas/química , Ressonância de Plasmônio de SuperfícieRESUMO
The use of protein crystallography in structure-guided drug discovery allows identification of potential inhibitor-binding sites and optimisation of interactions of hits and lead compounds with a target protein. An early example of this approach was the use of the structure of HIV protease in designing AIDS antivirals. More recently, use of structure-guided design with fragment-based drug discovery, which reduces the size of screening libraries by decreasing complexity, has improved ligand efficiency in drug design. Here, we discuss the use of structure-guided target identification and lead optimisation using fragment-based approaches in the development of new antimicrobials for mycobacterial infections.
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Cristalografia , Descoberta de Drogas/métodos , Proteínas/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Sítios de Ligação , Infecções por HIV/tratamento farmacológico , Humanos , Infecções por Mycobacterium/tratamento farmacológico , Conformação Proteica , Proteínas/química , Proteínas/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The clinical significance of bacteraemia secondary to non-typhoidal Salmonella (NTS) gastroenteritis in hospitalised adults is uncertain. METHODS: Adults admitted to a hospital in Liverpool, UK, with NTS gastroenteritis were identified using hospital discharge data and laboratory records. Patients with known HIV infection were excluded. Risk factors for a complicated or fatal course were determined. RESULTS: Between 1982 and 2006 inclusive, 633 adults were identified. Serovars causing infection included Enteritidis (46.6%), Typhimurium (27.6%) and Virchow (4.9%). A blood culture was taken in 364 (57.5%) patients who were generally sicker than those who were not cultured. Bacteraemia was detected in 63 (17.3%) patients who had blood cultures taken (63/633 (10.0%) of all patients). Bacteraemia was more common in those aged ≥ 65 years (p < 0.001) and in those aged < 65 years who had an underlying chronic disease. A complicated course occurred in 91 (25.0%) patients who had had a blood culture taken (148/633 (23.4%) of all patients). Independent factors associated with a complicated or fatal course among the patients investigated with a blood culture were bacteraemia (Adjusted Odds Ratio 5.34, 95% CI 2.86-9.95); new onset confusion or coma (AOR 4.80, 95% CI 1.91-12.07); prolonged symptoms prior to admission (AOR 2.48, 95% CI 1.44-4.27); dehydration (AOR1.90, 95% CI 1.07-3.38); and absence of fever (AOR 0.56, 95% CI 0.32-0.95). The 30 day attributable case fatality for all patients was 1.5%. CONCLUSIONS: In this study secondary bacteraemia, as well as other clinical factors, was independently associated with a complicated or fatal course in non-HIV infected adults admitted to hospital with NTS gastroenteritis.
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Bacteriemia/microbiologia , Infecções Comunitárias Adquiridas/microbiologia , Gastroenterite/microbiologia , Infecções por Salmonella/microbiologia , Adolescente , Adulto , Idoso , Análise de Variância , Bacteriemia/epidemiologia , Infecções Comunitárias Adquiridas/epidemiologia , Feminino , Gastroenterite/epidemiologia , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Infecções por Salmonella/epidemiologia , Reino Unido/epidemiologiaRESUMO
RNA molecules have the capacity to form a multitude of distinct secondary and tertiary structures, but only the most energetically favorable conformations are adopted at any given time. Formation of such structures strongly depends on the environment and consequently, these structures are highly dynamic and may refold as their surroundings change. Temperature is one of the most direct physical parameters that influence RNA structure dynamics, and in turn, thermosensitive RNA structures can be harnessed by a cell to perceive and respond to its temperature environment. Indeed, many thermosensitive RNA structures with biological function have been identified in prokaryotic organisms, but for a long time such structures remained elusive in eukaryotes. Recent discoveries, however, reveal that thermosensitive RNA structures are also found in plants, where they affect RNA stability, pre-mRNA splicing and translation efficiency in a temperature-dependent manner. In this minireview, we provide a short overview of thermosensitive RNA structures in prokaryotes and eukaryotes, highlight recent advances made in identifying such structures in plants and discuss their similarities and differences to established prokaryotic RNA thermosensors.
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Background. The diagnosis and prognostication of myeloproliferative neoplasm rely on the presence of driver mutations in JAK2, calreticulin (CALR), and MPL mutations. In the past, the presence of these mutations was thought to be mutually exclusive. Since then, there have been multiple reports of the presence of dual mutations. The presence of all three driver mutations in the same patient with myelofibrosis has not been previously described. CASE: A 73-year-old female underwent a hematological workup in our facility after a routine hemogram performed prior to complex ophthalmological surgery revealed severe thrombocytosis. A comprehensive workup including an NGS panel for MPN driver mutations demonstrated that she had a calreticulin type-1 mutation, a JAK2 exon 14 (JAK2L611S) mutation, and an abnormal hotspot variant for MPL with VAF1%. A bone marrow biopsy confirmed a myeloproliferative neoplasm with grade 2 reticulin fibrosis suggesting primary myelofibrosis. Molecular profiling of bone marrow confirmed the previously noted mutations and an MPLW515R mutation. The patient was started on treatment with hydroxyurea and aspirin with improvement in platelet count and resolution of anemia. DISCUSSION: The clinical significance of the presence of multiple driver mutations in the same patient is not well understood at this time. There have been 11 publications between 2014 and 2020 that have described dual mutations of JAK2V617F, MPL, and CALR mutations. The JAK2 exon 14 mutation noted, in this case, is JAK2L611S which has not previously been reported in MPN and only reported in 5 cases in the COSMIC database. The JAK2 exon 14 mutation identified in this case is not an established driver mutation for myeloproliferative neoplasm, and its clinical implication remains unknown. CONCLUSIONS: The above case in addition to recent case reports and case series supports the use of broader NGS sequencing panels for diagnosis and prognostication of MPN. These mutations should not be considered mutually exclusive. The clinical behavior and prognosis of the subgroup with multiple mutations need to be studied in larger series.
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Anti-microbial resistance is a rising global healthcare concern that needs urgent attention as growing number of infections become difficult to treat with the currently available antibiotics. This is particularly true for mycobacterial infections like tuberculosis and leprosy and those with emerging opportunistic pathogens such as Mycobacterium abscessus, where multi-drug resistance leads to increased healthcare cost and mortality. M. abscessus is a highly drug-resistant non-tuberculous mycobacterium which causes life-threatening infections in people with chronic lung conditions such as cystic fibrosis. In this study, we explore M. abscessus phosphopantetheine adenylyl transferase (PPAT), an enzyme involved in the biosynthesis of Coenzyme A, as a target for the development of new antibiotics. We provide structural insights into substrate and feedback inhibitor binding modes of M. abscessus PPAT, thereby setting the basis for further chemical exploration of the enzyme. We then utilize a multi-dimensional fragment screening approach involving biophysical and structural analysis, followed by evaluation of compounds from a previous fragment-based drug discovery campaign against M. tuberculosis PPAT ortholog. This allowed the identification of an early-stage lead molecule exhibiting low micro molar affinity against M. abscessus PPAT (Kd 3.2 ± 0.8 µM) and potential new ways to design inhibitors against this enzyme. The resulting crystal structures reveal striking conformational changes and closure of solvent channel of M. abscessus PPAT hexamer providing novel strategies of inhibition. The study thus validates the ligandability of M. abscessus PPAT as an antibiotic target and identifies crucial starting points for structure-guided drug discovery against this bacterium.
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Mycobacterium abscessus (Mab) has emerged as a challenging threat to individuals with cystic fibrosis. Infections caused by this pathogen are often impossible to treat due to the intrinsic antibiotic resistance leading to lung malfunction and eventually death. Therefore, there is an urgent need to develop new drugs against novel targets in Mab to overcome drug resistance and subsequent treatment failure. In this study, SAICAR synthetase (PurC) from Mab was identified as a promising target for novel antibiotics. An in-house fragment library screen and a high-throughput X-ray crystallographic screen of diverse fragment libraries were explored to provide crucial starting points for fragment elaboration. A series of compounds developed from fragment growing and merging strategies, guided by crystallographic information and careful hit-to-lead optimization, have achieved potent nanomolar binding affinity against the enzyme. Some compounds also show a promising inhibitory effect against Mab and Mtb. This work utilizes a fragment-based design and demonstrates for the first time the potential to develop inhibitors against PurC from Mab.
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Mycobacterium abscessus , Antibacterianos/química , Antibacterianos/farmacologia , Cristalografia por Raios X , Humanos , Peptídeo SintasesRESUMO
OBJECTIVES: To report on the temporal relationship between administration of trimethoprim/sulfamethoxazole to medically immunosuppressed HIV-negative renal patients with Pneumocystis jirovecii pneumonia (PCP) and the development of an acute psychosis. METHODS: We investigated a retrospective case series of renal transplant and immunosuppressed patients with PCP within an ongoing outbreak in the northwest of England since 2009. Four patients with PCP developed psychosis following treatment with trimethoprim/sulfamethoxazole. RESULTS: Four of twenty patients developed acute psychoses following administration of trimethoprim/sulfamethoxazole, including one accidental re-challenge. Symptoms resolved within 24 h of changing the therapy. The striking temporal relationship between the initiation and discontinuation of the drug and the behavioural changes suggests a causal relationship. CONCLUSIONS: With increasing solid organ transplantation and the use of immunosuppressants, vigilance regarding trimethoprim/sulfamethoxazole dose modification is required and the routine use of therapeutic drug monitoring should be considered.
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Transplante de Rim , Transtornos Mentais/induzido quimicamente , Pneumonia por Pneumocystis/tratamento farmacológico , Pneumonia por Pneumocystis/epidemiologia , Transplante , Combinação Trimetoprima e Sulfametoxazol/administração & dosagem , Combinação Trimetoprima e Sulfametoxazol/efeitos adversos , Adulto , Idoso , Inglaterra/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pneumocystis carinii/efeitos dos fármacos , Estudos RetrospectivosRESUMO
Introduction: SARS-CoV-2 antibody detection serves as an important diagnostic marker for past SARS-CoV-2 infection and is essential to determine the spread of COVID-19, monitor potential COVID-19 long-term effects, and to evaluate possible protection from reinfection. A study was conducted across three hospital sites in a large central London NHS Trust in the UK, to evaluate the prevalence and duration of SARS-CoV-2 IgG antibody positivity in healthcare workers. Methods: A matrix equivalence study consisting of 228 participants was undertaken to evaluate the Abbott Panbio™ COVID-19 IgG/IgM rapid test device. Subsequently, 2001 evaluable healthcare workers (HCW), representing a diverse population, were enrolled in a HCW study between June and August 2020. A plasma sample from each HCW was evaluated using the Abbott Panbio™ COVID-19 IgG/IgM rapid test device, with confirmation of IgG-positive results by the Abbott ArchitectTM SARS-CoV-2 IgG assay. 545 participants, of whom 399 were antibody positive at enrolment, were followed up at 3 months. Results: The Panbio™ COVID-19 IgG/IgM rapid test device demonstrated a high concordance with laboratory tests. SARS-CoV-2 antibodies were detected in 506 participants (25.3%) at enrolment, with a higher prevalence in COVID-19 frontline (28.3%) than non-frontline (19.9%) staff. At follow-up, 274/399 antibody positive participants (68.7%) retained antibodies; 4/146 participants negative at enrolment (2.7%) had seroconverted. Non-white ethnicity, older age, hypertension and COVID-19 symptoms were independent predictors of higher antibody levels (OR 1.881, 2.422-3.034, 2.128, and 1.869 respectively), based on Architect™ index quartiles; participants in the first three categories also showed a greater antibody persistence at 3 months. Conclusion: The SARS-CoV-2 anti-nucleocapsid IgG positivity rate among healthcare staff was high, declining by 31.3% during the 3-month follow-up interval. Interestingly, the IgG-positive participants with certain risk factors for severe COVID-19 illness (older age, Black or Asian Ethnicity hypertension) demonstrated greater persistence over time when compared to the IgG-positive participants without these risk factors.
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Viruses often encode proteins that mimic host proteins in order to facilitate infection. Little work has been done to understand the potential mimicry of the SARS-CoV-2, SARS-CoV, and MERS-CoV spike proteins, particularly the receptor-binding motifs, which could be important in determining tropism and druggability of the virus. Peptide and epitope motifs have been detected on coronavirus spike proteins using sequence homology approaches; however, comparing the three-dimensional shape of the protein has been shown as more informative in predicting mimicry than sequence-based comparisons. Here, we use structural bioinformatics software to characterize potential mimicry of the three coronavirus spike protein receptor-binding motifs. We utilize sequence-independent alignment tools to compare structurally known protein models with the receptor-binding motifs and verify potential mimicked interactions with protein docking simulations. Both human and non-human proteins were returned for all three receptor-binding motifs. For example, all three were similar to several proteins containing EGF-like domains: some of which are endogenous to humans, such as thrombomodulin, and others exogenous, such as Plasmodium falciparum MSP-1. Similarity to human proteins may reveal which pathways the spike protein is co-opting, while analogous non-human proteins may indicate shared host interaction partners and overlapping antibody cross-reactivity. These findings can help guide experimental efforts to further understand potential interactions between human and coronavirus proteins.
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The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine. Arginine auxotrophs (argB/argF deletion mutants) of Mycobacterium tuberculosis are rapidly sterilised in mice, while inhibition of ArgJ with Pranlukast was found to clear chronic M. tuberculosis infection in a mouse model. Enzymes in the arginine biosynthetic pathway have therefore emerged as promising targets for anti-tuberculosis drug discovery. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is assessed using a fragment-based approach. We identify several hits against these enzymes validated with biochemical and biophysical assays, as well as X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential for more enzymes in this pathway to be targeted with dedicated drug discovery programmes.
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BACKGROUND: Crimean-Congo hemorrhagic fever epidemics often occur in areas where health services are limited, and result in high case fatality rates. Besides intensive care, ribavirin is often recommended. A solid evidence base for the use of this drug will help justify assuring access to the drug in areas where epidemics are common. METHODS: We carried out a systematic review of observational and experimental studies of people with suspected or confirmed Crimean-Congo hemorrhagic fever that included comparisons between patients given ribavirin and those not. We extracted data on mortality, hospital stay, and adverse events. Risk of bias was assessed using a standard checklist, and data were presented in meta-analytical graphs, stratified by study design, and GRADE tables presented. The risk of bias was summarised using the GRADE method. RESULTS: 21 unique studies, including one randomised controlled trial of ribavirin, were included. Quality of the evidence was very low, with a Down and Black median score of 4 (maximum possible 33). Ribavirin treatment was not shown to be superior to no ribavirin treatment for mortality rate in a single RCT (RR: 1.13, 95%CI: 0.29 to 4.32, 136 participants, GRADE=low quality evidence); but ribavirin was associated with reduced mortality by 44% when compared to no ribavirin treatment in the pooled observational studies (RR: 0.56, 95%CI: 0.35 to 0.90, 955 participants; GRADE=very low quality evidence). Adverse events were more common with the ribavirin patients, but no severe adverse events were reported. No difference in length of hospital stay was reported. CONCLUSIONS: No clear message of benefit is available from the current data on ribavirin as observational data are heavily confounded, and the one trial carried out has limited power. However, ribavirin could potentially have benefits in this condition and these results clearly indicate a pragmatic, randomised controlled trial in the context of good quality supportive care, is urgently needed and ethically justified.
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Antivirais/uso terapêutico , Febre Hemorrágica da Crimeia/tratamento farmacológico , Ribavirina/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antivirais/efeitos adversos , Criança , Pré-Escolar , Feminino , Febre Hemorrágica da Crimeia/mortalidade , Humanos , Lactente , Recém-Nascido , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Ribavirina/efeitos adversos , Resultado do Tratamento , Adulto JovemRESUMO
A young female patient presented with features of ascites and cholecystitis. She was subsequently diagnosed with an acute Epstein-Barr virus infection. This is a rare presentation of a common infection. The patient was managed conservatively and the illness resolved within 6 weeks.