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1.
Viruses ; 13(10)2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34696498

RESUMO

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is an excellent target for developing small-molecule inhibitors. The current strategy for developing HuNoV protease inhibitors is by targeting the enzyme's active site and designing inhibitors that bind to the substrate-binding pockets located near the active site. However, subtle differential conformational flexibility in response to the different substrates in the polyprotein and structural differences in the active site and substrate-binding pockets across different genogroups, hamper the development of effective broad-spectrum inhibitors. A comparative analysis of the available HuNoV protease structures may provide valuable insight for identifying novel strategies for the design and development of such inhibitors. The goal of this review is to provide such analysis together with an overview of the current status of the design and development of HuNoV protease inhibitors.

2.
Front Microbiol ; 12: 708480, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335544

RESUMO

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40 T . We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

3.
Biopolymers ; 112(8): e23465, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34242395

RESUMO

The efficient and low-cost way for gene mutation detection and identification are conducive for the detection of disease. Here, we report the electronic characteristics of the gene of breast cancer 1 in four common mutation types: duplication, single nucleotide variant, deletion, and indel. The electronic characteristics are investigated by the combination of density functional theory and non-equilibrium Green's function formulation with decoherence. The magnitude of conductance of these DNA molecules and mutational changes are found to be detectable experimentally. In this study, we also find the significant mutation type dependent on the change of conductance. Hence these mutations are expected to be identifiable. We find deletion type mutation shows the largest change in relative conductance (~97%), whereas the indel mutation shows the smallest change in relative conductance (~27%). Therefore, this work presents a possibility of electronic detection and identification of mutations in DNA, which could be an efficient method as compared to the conventional methods.

4.
Nat Commun ; 12(1): 4158, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230497

RESUMO

Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible ß-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.


Assuntos
Cicloexenos/síntese química , Cicloexenos/metabolismo , Alcaloides Indólicos/síntese química , Alcaloides Indólicos/metabolismo , Vias Biossintéticas/genética , Catálise , Técnicas de Química Sintética , Compostos de Epóxi , Fermentação , Proteínas Fúngicas/genética , Modelos Moleculares , Estrutura Molecular , Oxigenases , Penicillium/genética , Penicillium/metabolismo
5.
Adv Ther ; 38(5): 2323-2338, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33754300

RESUMO

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disorder characterized by a high level of low-density lipoprotein cholesterol (LDL-C) and is an important cause for premature cardiovascular disease. Because of underdiagnoses, an acute event is often the first clinical manifestation of FH. There are limited data on the prevalence and treatment of FH among adults admitted for treatment of acute cardiovascular events in Bulgaria. Our objective was to assess the proportion and management of FH patients from those admitted to hospital for treatment of acute symptomatic acute atherosclerotic cardiovascular events (ASCVD), the achievement of LDL-C targets of European Society of Cardiology/European Atherosclerosis Society guidelines and related public healthcare resources. OBJECTIVE: Digitalized healthcare records for patients admitted for treatment of symptomatic ASCVD acute events between August 2018 and August 2019 were used for the analysis. Five cardiology hospitals provided data for hospitalizations, laboratory tests, and ambulatory follow-ups up to February 2020. Patients' hospital and ambulatory records were linked, and medical histories were extracted via a specifically developed algorithm, and analyzed. Outcomes included the proportion of patients classified as FH as defined by the Dutch Lipid Network Criteria (DLNC), use of lipid-lowering therapy, LDL-C achieved by 1, 3, 6, and 12 months post-index event, and public resources spent on hospital and ambulatory treatment. RESULTS: We reviewed 11,090 hospital records of patients admitted for treatment of acute events in the period August 2018-August 2019 with ICD codes for ASCVD (Supplementary Table S3). FH was identified in 731 (6.6%) patients, with DLNC score ≥ 3, (682 with coronary artery disease, 32 with cerebrovascular disease, and 17 with peripheral artery disease). We did not find the criteria for FH in 5797 patients. The remaining 4562 records were inconclusive due to lack of data in the hospital dossier. Less than half of FH patients (274/731, 37%) were discharged on high-intensity statin therapy prescribed (34/731, 5%) with combination therapy. The vast majority (96.2% with LDL-C ≥ 1.8 mmol/l) had poorly controlled LDL-C during the first year after discharge. Patients with a probable/definite DLNC score ≥ 6 points and those with recurrent events contributed to the higher cost paid both by the healthcare system and the patients themselves. CONCLUSION: These findings reinforce the need for more aggressive lipid-lowering therapy, and underline the efficiency of using an electronic medical records search tool to support physicians in improving early FH diagnosis, aiming to minimize residual and future ASCVD events among FH patients and their family members. Supplementary file1 (MP4 21838 KB).


Assuntos
Doenças Cardiovasculares , Hiperlipoproteinemia Tipo II , Adulto , Algoritmos , Bulgária/epidemiologia , Eletrônica , Hospitais , Humanos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Hiperlipoproteinemia Tipo II/epidemiologia , Fatores de Risco
6.
Mol Cell Biochem ; 476(3): 1599-1603, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33405086

RESUMO

DNA hydroxymethylation plays a very important role in some biological processes, such as DNA methylation process. In addition, its presence can also cause some diseases. In this paper, the electrical properties of cytosine hydroxymethylated (Chm) DNA sequences are studied. The density functional theory (DFT) and Landauer-Büttiker framework are used to study the decoherence conductance and transmission of the Chm strands in different configurations, which provides a theoretical basis for the detection of Chm. The results show that the conductance of the hydroxymethylated DNA strand is smaller than that of the native and methylated strands. The length dependence of the Chm strands is also studied. With the length increasing, the conductance becomes larger. This study shows that DNA methylation can be detected electrically.


Assuntos
Citosina/metabolismo , Metilação de DNA , DNA/química , 5-Metilcitosina , Biofísica , Eletrofisiologia , Epigênese Genética , Conformação Molecular , Solventes/química , Temperatura
7.
Indoor Air ; 31(3): 769-782, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33108019

RESUMO

Household humidification is widely practiced to combat dry indoor air. While the benefits of household humidification are widely perceived, its implications to the indoor air have not been critically appraised. In particular, ultrasonic humidifiers are known to generate fine particulate matter (PM). In this study, we first conducted laboratory experiments to investigate the size, quantity, and chemical composition of PM generated by an ultrasonic humidifier. The mass of PM generated showed a correlation with the total alkalinity of charge water, suggesting that CaCO3 is likely making a major contribution to PM. Ion chromatography analysis revealed a large amount of SO4 2- in PM, representing a previously unrecognized indoor source. Preliminary results of organic compounds being present in humidifier PM are also presented. A whole-house experiment was further conducted at an actual residential house, with five low-cost sensors (AirBeam) monitoring PM in real time. Operation of a single ultrasonic humidifier resulted in PM2.5 concentrations up to hundreds of µg m-3 , and its influence extended across the entire household. The transport and loss of PM2.5 depended on the rate of air circulation and ventilation. This study emphasizes the need to further investigate the impact of humidifier operation, both on human health and on the indoor atmospheric chemistry, for example, partitioning of acidic and basic compounds.


Assuntos
Poluição do Ar em Ambientes Fechados/estatística & dados numéricos , Umidificadores , Material Particulado , Poluentes Atmosféricos , Monitoramento Ambiental , Humanos , Compostos Orgânicos , Tamanho da Partícula , Ultrassom , Água
8.
Nat Commun ; 11(1): 6039, 2020 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247101

RESUMO

Crystalline solids exhibiting glass-like thermal conductivity have attracted substantial attention both for fundamental interest and applications such as thermoelectrics. In most crystals, the competition of phonon scattering by anharmonic interactions and crystalline imperfections leads to a non-monotonic trend of thermal conductivity with temperature. Defect-free crystals that exhibit the glassy trend of low thermal conductivity with a monotonic increase with temperature are desirable because they are intrinsically thermally insulating while retaining useful properties of perfect crystals. However, this behavior is rare, and its microscopic origin remains unclear. Here, we report the observation of ultralow and glass-like thermal conductivity in a hexagonal perovskite chalcogenide single crystal, BaTiS3, despite its highly symmetric and simple primitive cell. Elastic and inelastic scattering measurements reveal the quantum mechanical origin of this unusual trend. A two-level atomic tunneling system exists in a shallow double-well potential of the Ti atom and is of sufficiently high frequency to scatter heat-carrying phonons up to room temperature. While atomic tunneling has been invoked to explain the low-temperature thermal conductivity of solids for decades, our study establishes the presence of sub-THz frequency tunneling systems even in high-quality, electrically insulating single crystals, leading to anomalous transport properties well above cryogenic temperatures.

9.
PLoS Pathog ; 16(9): e1008851, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986782

RESUMO

Enteroaggregative Escherichia coli (EAEC) is a significant cause of acute and chronic diarrhea, foodborne outbreaks, infections of the immunocompromised, and growth stunting in children in developing nations. There is no vaccine and resistance to antibiotics is rising. Unlike related E. coli pathotypes that are often associated with acute bouts of infection, EAEC is associated with persistent diarrhea and subclinical long-term colonization. Several secreted virulence factors have been associated with EAEC pathogenesis and linked to disease in humans, less certain are the molecular drivers of adherence to the intestinal mucosa. We previously established human intestinal enteroids (HIEs) as a model system to study host-EAEC interactions and aggregative adherence fimbriae A (AafA) as a major driver of EAEC adherence to HIEs. Here, we report a large-scale assessment of the host response to EAEC adherence from all four segments of the intestine across at least three donor lines for five E. coli pathotypes. The data demonstrate that the host response in the duodenum is driven largely by the infecting pathotype, whereas the response in the colon diverges in a patient-specific manner. Major pathways altered in gene expression in each of the four enteroid segments differed dramatically, with responses observed for inflammation, apoptosis and an overwhelming response to different mucin genes. In particular, EAEC both associated with large mucus droplets and specific mucins at the epithelial surface, binding that was ameliorated when mucins were removed, a process dependent on AafA. Pan-screening for glycans for binding to purified AafA identified the human ligand as heparan sulfate proteoglycans (HSPGs). Removal of HSPG abrogated EAEC association with HIEs. These results may mean that the human intestine responds remarkably different to distinct pathobionts that is dependent on the both the individual and intestinal segment in question, and uncover a major role for surface heparan sulfate proteoglycans as tropism-driving factor in adherence and/or colonization.


Assuntos
Aderência Bacteriana/fisiologia , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Adesinas de Escherichia coli/genética , Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Fatores de Virulência/metabolismo
10.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461314

RESUMO

Many RNA viruses replicate in cytoplasmic compartments (virus factories or viroplasms) composed of viral and cellular proteins, but the mechanisms required for their formation remain largely unknown. Rotavirus (RV) replication in viroplasms requires interactions between virus nonstructural proteins NSP2 and NSP5, which are associated with components of lipid droplets (LDs). We previously identified two forms of NSP2 in RV-infected cells, a cytoplasmically dispersed form (dNSP2) and a viroplasm-specific form (vNSP2), which interact with hypophosphorylated and hyperphosphorylated NSP5, respectively, indicating that a coordinated phosphorylation cascade controls viroplasm assembly. The cellular kinase CK1α phosphorylates NSP2 on serine 313, triggering the localization of vNSP2 to sites of viroplasm assembly and its association with hyperphosphorylated NSP5. Using reverse genetics, we generated a rotavirus with a phosphomimetic NSP2 (S313D) mutation to directly evaluate the role of CK1α NSP2 phosphorylation in viroplasm formation. Recombinant rotavirus NSP2 S313D (rRV NSP2 S313D) is significantly delayed in viroplasm formation and in virus replication and interferes with wild-type RV replication in coinfection. Taking advantage of the delay in viroplasm formation, the NSP2 phosphomimetic mutant was used as a tool to observe very early events in viroplasm assembly. We show that (i) viroplasm assembly correlates with NSP5 hyperphosphorylation and (ii) vNSP2 S313D colocalizes with RV-induced LDs without NSP5, suggesting that vNSP2 phospho-S313 is sufficient for interacting with LDs and may be the virus factor required for RV-induced LD formation. Further studies with the rRV NSP2 S313D virus are expected to reveal new aspects of viroplasm and LD initiation and assembly.IMPORTANCE Reverse genetics was used to generate a recombinant rotavirus with a single phosphomimetic mutation in nonstructural protein 2 (NSP2 S313D) that exhibits delayed viroplasm formation, delayed replication, and an interfering phenotype during coinfection with wild-type rotavirus, indicating the importance of this amino acid during virus replication. Exploiting the delay in viroplasm assembly, we found that viroplasm-associated NSP2 colocalizes with rotavirus-induced lipid droplets prior to the accumulation of other rotavirus proteins that are required for viroplasm formation and that NSP5 hyperphosphorylation is required for viroplasm assembly. These data suggest that NSP2 phospho-S313 is sufficient for interaction with lipid droplets and may be the virus factor that induces lipid droplet biogenesis in rotavirus-infected cells. Lipid droplets are cellular organelles critical for the replication of many viral and bacterial pathogens, and thus, understanding the mechanism of NSP2-mediated viroplasm/lipid droplet initiation and interaction will lead to new insights into this important host-pathogen interaction.


Assuntos
Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/virologia , Proteínas de Ligação a RNA/metabolismo , Rotavirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Animais , Linhagem Celular , Cricetinae , Fosforilação , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/genética
11.
Cell Rep ; 30(12): 3951-3963.e4, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209458

RESUMO

Rationally designing drugs that last longer in the face of biological evolution is a critical objective of drug discovery. However, this goal is thwarted by the diversity and stochasticity of evolutionary trajectories that drive uncertainty in the clinic. Although biophysical models can qualitatively predict whether a mutation causes resistance, they cannot quantitatively predict the relative abundance of resistance mutations in patient populations. We present stochastic, first-principle models that are parameterized on a large in vitro dataset and that accurately predict the epidemiological abundance of resistance mutations across multiple leukemia clinical trials. The ability to forecast resistance variants requires an understanding of their underlying mutation biases. Beyond leukemia, a meta-analysis across prostate cancer, breast cancer, and gastrointestinal stromal tumors suggests that resistance evolution in the adjuvant setting is influenced by mutational bias. Our analysis establishes a principle for rational drug design: when evolution favors the most probable mutant, so should drug design.


Assuntos
Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Estudos Epidemiológicos , Alelos , Animais , Desenvolvimento de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Evolução Molecular , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Camundongos , Modelos Biológicos , Mutação/genética , Proteínas Proto-Oncogênicas c-abl/genética , Sais/química , Processos Estocásticos
12.
PLoS Comput Biol ; 15(10): e1007467, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658270

RESUMO

The somatic co-evolution of tumors and the cellular immune responses that combat them drives the diversity of immune-tumor interactions. This includes tumor mutations that generate neo-antigenic epitopes that elicit cytotoxic T-cell activity and subsequent pressure to select for genetic loss of antigen presentation. Most studies have focused on how tumor missense mutations can drive tumor immunity, but frameshift mutations have the potential to create far greater antigenic diversity. However, expression of this antigenic diversity is potentially regulated by Nonsense Mediated Decay (NMD) and NMD has been shown to be of variable efficiency in cancers. Here we studied how mutational changes influence global NMD and cytolytic immune responses. Using TCGA datasets, we derived novel patient-level metrics of 'NMD burden' and interrogated how different mutation and most importantly NMD burdens influence cytolytic activity using machine learning models and survival outcomes. We find that NMD is a significant and independent predictor of immune cytolytic activity. Different indications exhibited varying dependence on NMD and mutation burden features. We also observed significant co-alteration of genes in the NMD pathway, with a global increase in NMD efficiency in patients with NMD co-alterations. Finally, NMD burden also stratified patient survival in multivariate regression models in subset of cancer types. Our work suggests that beyond selecting for mutations that elicit NMD in tumor suppressors, tumor evolution may react to the selective pressure generated by inflammation to globally enhance NMD through coordinated amplification and/or mutation.


Assuntos
Citotoxicidade Imunológica/genética , Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Evolução Biológica , Simulação por Computador , Citosol/metabolismo , Bases de Dados Genéticas , Evolução Molecular , Mutação da Fase de Leitura/genética , Humanos , Aprendizado de Máquina , Mutação/genética , Mutação de Sentido Incorreto/genética
13.
JCO Clin Cancer Inform ; 3: 1-9, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31577448

RESUMO

PURPOSE: A substantial portion of medical data is unstructured. Extracting data from unstructured text presents a barrier to advancing clinical research and improving patient care. In addition, ongoing studies have been focused predominately on the English language, whereas inflected languages with non-Latin alphabets (such as Slavic languages with a Cyrillic alphabet) present numerous linguistic challenges. We developed deep-learning-based natural language processing algorithms for automatically extracting biomarker status of patients with breast cancer from three oncology centers in Bulgaria. METHODS: We used dual embeddings for English and Bulgarian languages, encoding both syntactic and polarity information for the words. The embeddings were subsequently aligned so that they were in the same vector space. The embeddings were used as input to convolutional or recurrent neural networks to derive the biomarker status of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. RESULTS: We showed that we can resolve ambiguity in highly variable medical text containing both Latin and Cyrillic text. Final models incorporating both English and Bulgarian syntax and polarity embeddings achieved F1 scores of 0.90 or higher for all estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 biomarkers. The models were robust against human errors originally found in the training set. In addition, such models can be extended for analyzing text containing words not seen during training. CONCLUSION: By using several techniques that incorporate dual-word embeddings encoding syntactic and polarity information in two languages followed by deep neural network architectures, we show that researchers can extract and normalize parameters within medical data. The principles described here can be used to analyze Cyrillic or Latin mixed medical text and extract other parameters.


Assuntos
Aprendizado Profundo , Registros Eletrônicos de Saúde , Informática Médica/métodos , Processamento de Linguagem Natural , Algoritmos , Área Sob a Curva , Biomarcadores Tumorais , Neoplasias da Mama/etiologia , Bulgária , Feminino , Humanos , Redes Neurais de Computação , Reprodutibilidade dos Testes
14.
J Clin Invest ; 129(9): 3839-3851, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31403468

RESUMO

We previously generated 32 rotavirus-specific (RV-specific) recombinant monoclonal antibodies (mAbs) derived from B cells isolated from human intestinal resections. Twenty-four of these mAbs were specific for the VP8* fragment of RV VP4, and most (20 of 24) were non-neutralizing when tested in the conventional MA104 cell-based assay. We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells including ileal enteroids and HT-29 cells. Most (18 of 20) of the "non-neutralizing" VP8* mAbs efficiently neutralized human RV in HT-29 cells or enteroids. Serum RV neutralization titers in adults and infants were significantly higher in HT-29 than MA104 cells and adsorption of these sera with recombinant VP8* lowered the neutralization titers in HT-29 but not MA104 cells. VP8* mAbs also protected suckling mice from diarrhea in an in vivo challenge model. X-ray crystallographic analysis of one VP8* mAb (mAb9) in complex with human RV VP8* revealed that the mAb interaction site was distinct from the human histo-blood group antigen binding site. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, these findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Células Epiteliais/imunologia , Intestinos/citologia , Infecções por Rotavirus/imunologia , Adsorção , Animais , Antígenos Virais/imunologia , Linfócitos B/imunologia , Linfócitos B/virologia , Sítios de Ligação , Células CACO-2 , Linhagem Celular , Cristalografia por Raios X , Células Epiteliais/virologia , Genótipo , Haplorrinos , Humanos , Imunoglobulina G/química , Índia , Lactente , Recém-Nascido , Intestinos/virologia , Camundongos , Testes de Neutralização , Polissacarídeos/química , Conformação Proteica , Proteínas Recombinantes/imunologia , Estados Unidos
15.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626675

RESUMO

Human noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substrate-binding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pH-dependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs.IMPORTANCE Human noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.


Assuntos
Arginina/metabolismo , Domínio Catalítico/genética , Histidina/metabolismo , Norovirus/metabolismo , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Infecções por Caliciviridae/metabolismo , Domínio Catalítico/fisiologia , Variação Genética/genética , Genótipo , Humanos , Concentração de Íons de Hidrogênio , Norovirus/genética , Filogenia , Proteólise
16.
Sci Rep ; 6: 36198, 2016 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-27819268

RESUMO

Recent drug discovery and development efforts have created a large arsenal of targeted and chemotherapeutic drugs for precision medicine. However, drug resistance remains a major challenge as minor pre-existing resistant subpopulations are often found to be enriched at relapse. Current drug design has been heavily focused on initial efficacy, and we do not fully understand the effects of drug selective pressure on long-term drug resistance potential. Using a minimal two-population model, taking into account subpopulation proportions and growth/kill rates, we modeled long-term drug treatment and performed parameter sweeps to analyze the effects of each parameter on therapeutic efficacy. We found that drugs with the same overall initial kill may exert differential selective pressures, affecting long-term therapeutic outcome. We validated our conclusions experimentally using a preclinical model of Burkitt's lymphoma. Furthermore, we highlighted an intrinsic tradeoff between drug-imposed overall selective pressure and rate of adaptation. A principled approach in understanding the effects of distinct drug selective pressures on short-term and long-term tumor response enables better design of therapeutics that ultimately minimize relapse.


Assuntos
Linfoma de Burkitt/metabolismo , Resistencia a Medicamentos Antineoplásicos , Modelos Biológicos , Neoplasias Experimentais/metabolismo , Animais , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia
17.
PLoS Genet ; 12(6): e1006081, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27304678

RESUMO

The identification of biologically significant variants in cancer genomes is critical to therapeutic discovery, but it is limited by the statistical power needed to discern driver from passenger. Independent biological data can be used to filter cancer exomes and increase statistical power. Large genetic databases for inherited diseases are uniquely suited to this task because they contain specific amino acid alterations with known pathogenicity and molecular mechanisms. However, no rigorous method to overlay this information onto the cancer exome exists. Here, we present a computational methodology that overlays any variant database onto the somatic mutations in all cancer exomes. We validate the computation experimentally and identify novel associations in a re-analysis of 7362 cancer exomes. This analysis identified activating SOS1 mutations associated with Noonan syndrome as significantly altered in melanoma and the first kinase-activating mutations in ACVR1 associated with adult tumors. Beyond a filter, significant variants found in both rare cancers and rare inherited diseases increase the unmet medical need for therapeutics that target these variants and may bootstrap drug discovery efforts in orphan indications.


Assuntos
Receptores de Ativinas Tipo I/genética , Colágeno Tipo III/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Neoplasias do Endométrio/genética , Predisposição Genética para Doença , Neoplasias Pulmonares/genética , Melanoma/genética , Síndrome de Noonan/genética , Proteína SOS1/genética , Sequência de Aminoácidos , Linhagem Celular , Exoma/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Variação Genética/genética , Células HEK293 , Humanos , Mutação/genética , Fosforilação
18.
Cell ; 165(1): 234-246, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-26924578

RESUMO

The prevailing approach to addressing secondary drug resistance in cancer focuses on treating the resistance mechanisms at relapse. However, the dynamic nature of clonal evolution, along with potential fitness costs and cost compensations, may present exploitable vulnerabilities-a notion that we term "temporal collateral sensitivity." Using a combined pharmacological screen and drug resistance selection approach in a murine model of Ph(+) acute lymphoblastic leukemia, we indeed find that temporal and/or persistent collateral sensitivity to non-classical BCR-ABL1 drugs arises in emergent tumor subpopulations during the evolution of resistance toward initial treatment with BCR-ABL1-targeted inhibitors. We determined the sensitization mechanism via genotypic, phenotypic, signaling, and binding measurements in combination with computational models and demonstrated significant overall survival extension in mice. Additional stochastic mathematical models and small-molecule screens extended our insights, indicating the value of focusing on evolutionary trajectories and pharmacological profiles to identify new strategies to treat dynamic tumor vulnerabilities.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Modelos Biológicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Camundongos , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcr/análise , Proteínas Proto-Oncogênicas c-bcr/genética
19.
Trends Cancer ; 2(3): 144-158, 2016 03.
Artigo em Inglês | MEDLINE | ID: mdl-28435907

RESUMO

Cancer is a clonal evolutionary process. This presents challenges for effective therapeutic intervention, given the constant selective pressure towards drug resistance. Mathematical modeling from population genetics, evolutionary dynamics, and engineering perspectives are being increasingly employed to study tumor progression, intratumoral heterogeneity, drug resistance, and rational drug scheduling and combinations design. In this review, we discuss promising opportunities these inter-disciplinary approaches hold for advances in cancer biology and treatment. We propose that quantitative modeling perspectives can complement emerging experimental technologies to facilitate enhanced understanding of disease progression and improved capabilities for therapeutic drug regimen designs.


Assuntos
Evolução Clonal , Desenho de Fármacos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Animais , Combinação de Medicamentos , Heterogeneidade Genética , Humanos
20.
Oncotarget ; 6(32): 32646-55, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26360609

RESUMO

Although targeted therapies have revolutionized cancer treatment, overcoming acquired resistance remains a major clinical challenge. EZH2 inhibitors (EZH2i), EPZ-6438 and GSK126, are currently in the early stages of clinical evaluation and the first encouraging signs of efficacy have recently emerged in the clinic. To anticipate mechanisms of resistance to EZH2i, we used a forward genetic platform combining a mutagenesis screen with next generation sequencing technology and identified a hotspot of secondary mutations in the EZH2 D1 domain (Y111 and I109). Y111D mutation within the WT or A677G EZH2 allele conferred robust resistance to both EPZ-6438 and GSK126, but it only drove a partial resistance within the Y641F allele. EZH2 mutants required histone methyltransferase (HMT) catalytic activity and the polycomb repressive complex 2 (PRC2) components, SUZ12 and EED, to drive drug resistance. Furthermore, D1 domain mutations not only blocked the ability of EZH2i to bind to WT and A677G mutant, but also abrogated drug binding to the Y641F mutant. These data provide the first cellular validation of the mechanistic model underpinning the oncogenic function of WT and mutant EZH2. Importantly, our findings suggest that acquired-resistance to EZH2i may arise in WT and mutant EZH2 patients through a single mutation that remains targetable by second generation EZH2i.


Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Piridonas/farmacologia , Antineoplásicos/metabolismo , Benzamidas/metabolismo , Compostos de Bifenilo , Relação Dose-Resposta a Droga , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Indóis/metabolismo , Terapia de Alvo Molecular , Morfolinas , Proteínas de Neoplasias , Neoplasias/enzimologia , Neoplasias/patologia , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Piridonas/metabolismo , Interferência de RNA , Fatores de Tempo , Fatores de Transcrição , Transfecção
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