RESUMO
Photosensitivity can be due to numerous causes. The photosensitivity associated with deficiency of xeroderma pigmentosum type A (XPA) has been previously shown to be associated with excess levels of the lipid mediator platelet-activating factor (PAF) generated by the keratinocyte. As PAF has been reported to trigger the production of subcellular microvesicle particles (MVP) due to the enzyme acid sphingomyelinase (aSMase), the goal of these studies was to discern if PAF and aSMase could serve as therapeutic targets for the XPA deficiency photosensitivity. HaCaT keratinocytes lacking XPA generated greater levels of MVP in comparison to control cells. Mice deficient in XPA also generated enhanced MVP levels in skin and in plasma in response to UV radiation. Use of a genetic strategy with mice deficient in both XPA and PAF receptors revealed that these mice generated less MVP release as well as decreased skin erythema and cytokine release compared to XPA knockout mice alone. Finally, the aSMase inhibitor imipramine blocked UV-induced MVP release in HaCaT keratinocytes, as well as XPA knockout mice. These studies support the concept that the photosensitivity associated with XPA involves PAF- and aSMase-mediated MVP release and provides a potential pharmacologic target in treating this form of photosensitivity.
Assuntos
Queratinócitos , Camundongos Knockout , Raios Ultravioleta , Xeroderma Pigmentoso , Queratinócitos/efeitos da radiação , Queratinócitos/metabolismo , Animais , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Camundongos , Humanos , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/deficiência , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Transtornos de Fotossensibilidade , Fator de Ativação de Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Imipramina/farmacologiaRESUMO
Background: Interstitial Fibrosis and Tubular Atrophy (IFTA) is the most common cause of long-term graft failure following renal transplant. One of the hallmarks of IFTA is the development of interstitial fibrosis and loss of normal renal architecture. In this study, we evaluated the role of autophagy initiation factor Beclin-1 in protecting against post-renal injury fibrosis. Methods: Adult male wild type (WT) C57BL/6 mice were subjected to Unilateral Ureteral Obstruction (UUO), and kidney tissue samples were harvested at 72-hour, 1- and 3-week post-injury. The UUO-injured and uninjured kidney samples were examined histologically for fibrosis, autophagy flux, inflammation as well activation of the Integrated Stress Response (ISR). We compared WT mice with mice carrying a forced expression of constitutively active mutant form of Beclin-1, Becn1F121A/F121A . Results: In all experiments, UUO injury induces a progressive development of fibrosis and inflammation. These pathological signs were diminished in Becn1F121A/F121A mice. In WT animals, UUO caused a strong blockage of autophagy flux, indicated by continuously increases in LC3II accompanied by an over 3-fold accumulation of p62 1-week post injury. However, increases in LC3II and unaffected p62 level by UUO were observed in Becn1F121A/F121A mice, suggesting an alleviation of disrupted autophagy. Beclin-1 F121A mutation causes a significant decrease in phosphorylation of inflammatory STING signal and limited production of IL6 and IFNγ, but had little effect on TNF-α, in response to UUO. Furthermore, activation of ISR signal cascade was detected in UUO-injured in kidneys, namely the phosphorylation signals of elF2S1 and PERK in addition to the stimulated expression of ISR effector ATF4. However, Becn1F121A/F121A mice did not reveal signs of elF2S1 and PERK activation under the same condition and had a dramatically reduced ATF level at 3-week post injury. Conclusions: The results suggest that UUO causes a insufficient, maladaptive renal autophagy, which triggered downstream activation of inflammatory STING pathway, production of cytokines, and pathological activation of ISR, eventually leading to the development of fibrosis. Enhancing autophagy via Beclin-1 improved renal outcomes with diminished fibrosis, via underlying mechanisms of differential regulation of inflammatory mediators and control of maladaptive ISR.
Assuntos
Traumatismos Abdominais , Obstrução Ureteral , Masculino , Animais , Camundongos , Camundongos Endogâmicos C57BL , Proteína Beclina-1 , Rim , Autofagia , Inflamação , FibroseRESUMO
Solar radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) and other UV photoproducts in the genomic DNA of epidermal keratinocytes. Although CPDs have been detected in urine from UV- and sun-exposed individuals, the pathway by which they arrive there and the mechanisms by which UV-induced DNA damage in the skin has systemic effects throughout the body are not clear. Consistent with previous reports that DNA associates with small extracellular vesicles that are released from a variety of cell types, we observed that a small fraction of CPDs formed in genomic DNA after UVB exposure can later be detected in the culture medium. These extracellular CPDs are found within large fragments of histone-associated DNA and are released in a time- and UVB doseâdependent manner. Moreover, studies with both cultured cells and human skin explants revealed that CPD release into the extracellular environment is blocked by caspase inhibition, which indicates a role for apoptotic signaling in CPD release from UVB-irradiated keratinocytes. Finally, we show that this released CPD-containing DNA can be taken up by other keratinocytes. These results therefore provide possible mechanisms for the export of damaged DNA from UVB-irradiated cells and for systemic effects of UVB exposure throughout the body.
Assuntos
Caspases , Dímeros de Pirimidina , Humanos , Dímeros de Pirimidina/efeitos da radiação , Caspases/metabolismo , Histonas/metabolismo , Raios Ultravioleta/efeitos adversos , Queratinócitos/metabolismo , Dano ao DNA , DNA/metabolismoRESUMO
Smc5/6, like cohesin and condensin, is a structural maintenance of chromosomes complex crucial for genome stability. Unlike cohesin and condensin, Smc5/6 carries an essential Nse2 subunit with SUMO E3 ligase activity. While screening for new DNA replication checkpoint mutants in fission yeast, we have identified two previously uncharacterized mutants in Smc5/6. Characterization of the mutants and a series of previously reported Smc5/6 mutants uncovered that sumoylation of the RecQ helicase Rqh1 by Nse2 facilitates the checkpoint signaling at the replication fork. We found that mutations that eliminate the sumoylation sites or the helicase activity of Rqh1 compromised the checkpoint signaling similar to a nse2 mutant lacking the ligase activity. Surprisingly, introducing a sumoylation site mutation to a helicase-inactive rqh1 mutant promoted cell survival under stress. These findings, together with other genetic data, support a mechanism that sumoylation of Rqh1 by Smc5/6-Nse2 recruits Rqh1 or modulates its helicase activity at the fork to facilitate the checkpoint signaling. Since the Smc5/6 complex, Rqh1, and the replication checkpoint are conserved in eukaryotes, a similar checkpoint mechanism may be operating in human cells.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Proteínas Cromossômicas não Histona/genética , Cromossomos/metabolismo , Dano ao DNA , DNA Helicases/genética , Replicação do DNA , Humanos , Mutação/genética , RecQ Helicases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , SumoilaçãoRESUMO
Schizosaccharomyces pombe is an established yeast model for studying the cellular mechanisms conserved in humans, such as the DNA replication checkpoint. The replication checkpoint deals with replication stress caused by numerous endogenous and exogenous factors that perturb fork movement. If undealt with, perturbed forks collapse, causing chromosomal DNA damage or cell death. Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase (RNR) commonly used in checkpoint studies. It produces replication stress by depleting dNTPs, which slows the movement of ongoing forks and thus activates the replication checkpoint. However, HU also causes side effects such as oxidative stress, particularly under chronic exposure conditions, which complicates the studies. To find a drug that generates replication stress more specifically, we tested three other RNR inhibitors gemcitabine, guanazole and triapine in S. pombe under various experimental conditions. Our results show that guanazole and triapine can produce replication stress more specifically than HU under chronic, not acute drug treatment conditions. Therefore, using the two drugs in spot assay, the method commonly used for testing drug sensitivity in yeasts, should benefit the checkpoint studies in S. pombe and likely the research in other model systems.
Assuntos
Ribonucleotídeo Redutases , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Replicação do DNA , Desoxicitidina/análogos & derivados , Inibidores Enzimáticos/metabolismo , Guanazol , Humanos , Hidroxiureia/farmacologia , Piridinas , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeo Redutases/farmacologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Tiossemicarbazonas , GencitabinaRESUMO
BACKGROUND: We determined the burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in air and on surfaces in rooms of patients hospitalized with coronavirus disease 2019 (COVID-19) and investigated patient characteristics associated with SARS-CoV-2 environmental contamination. METHODS: Nasopharyngeal swabs, surface, and air samples were collected from the rooms of 78 inpatients with COVID-19 at 6 acute care hospitals in Toronto from March to May 2020. Samples were tested for SARS-CoV-2 ribonucleic acid (RNA), cultured to determine potential infectivity, and whole viral genomes were sequenced. Association between patient factors and detection of SARS-CoV-2 RNA in surface samples were investigated. RESULTS: Severe acute respiratory syndrome coronavirus 2 RNA was detected from surfaces (125 of 474 samples; 42 of 78 patients) and air (3 of 146 samples; 3 of 45 patients); 17% (6 of 36) of surface samples from 3 patients yielded viable virus. Viral sequences from nasopharyngeal and surface samples clustered by patient. Multivariable analysis indicated hypoxia at admission, polymerase chain reaction-positive nasopharyngeal swab (cycle threshold ofâ ≤30) on or after surface sampling date, higher Charlson comorbidity score, and shorter time from onset of illness to sampling date were significantly associated with detection of SARS-CoV-2 RNA in surface samples. CONCLUSIONS: The infrequent recovery of infectious SARS-CoV-2 virus from the environment suggests that the risk to healthcare workers from air and near-patient surfaces in acute care hospital wards is likely limited.
Assuntos
COVID-19 , Nasofaringe/virologia , Aerossóis e Gotículas Respiratórios , SARS-CoV-2/isolamento & purificação , Adulto , Idoso , Microbiologia do Ar , COVID-19/epidemiologia , COVID-19/prevenção & controle , COVID-19/transmissão , Teste de Ácido Nucleico para COVID-19 , Canadá/epidemiologia , Exposição Ambiental , Pessoal de Saúde , Humanos , Pacientes Internados , Pessoa de Meia-Idade , Pandemias/prevenção & controle , SARS-CoV-2/genéticaRESUMO
The xeroderma pigmentosum group A (XPA) protein plays an essential role in the removal of UV photoproducts and other bulky lesions from DNA as a component of the nucleotide excision repair (NER) machinery. Using cell lysates prepared from confluent cultures of human cells and from human skin epidermis, we observed an additional XPA antibody-reactive band on immunoblots that was approximately 3-4 kDa smaller than the native, full-length XPA protein. Biochemical studies revealed this smaller molecular weight XPA species to be due to proteolysis at the C-terminus of the protein, which negatively impacted the ability of XPA to interact with the NER protein TFIIH. Further work identified the endopeptidase cathepsin L, which is expressed at higher levels in quiescent cells, as the protease responsible for cleaving XPA during cell lysis. These results suggest that supplementation of lysis buffers with inhibitors of cathepsin L is important to prevent cleavage of XPA during lysis of confluent cells.
Assuntos
Catepsina L/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Catepsina L/antagonistas & inibidores , Células Cultivadas , Reparo do DNA , Humanos , Proteólise , Proteína de Xeroderma Pigmentoso Grupo A/isolamento & purificaçãoRESUMO
DNA replication checkpoint is a cell signaling pathway that is activated in response to perturbed replication. Although it is crucial for maintaining genomic integrity and cell survival, the exact mechanism of the checkpoint signaling remains to be understood. Emerging evidence has shown that RecQ helicases, a large family of helicases that are conserved from bacteria to yeasts and humans, contribute to the replication checkpoint as sensors, adaptors, or regulation targets. Here, we highlight the multiple functions of RecQ helicases in the replication checkpoint in four model organisms and present additional evidence that fission yeast RecQ helicase Rqh1 may participate in the replication checkpoint as a sensor.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , DNA Helicases/genética , Replicação do DNA/genética , RecQ Helicases/genética , Proteínas de Schizosaccharomyces pombe/genética , Humanos , Schizosaccharomyces/genética , Transdução de Sinais/genéticaRESUMO
We enrolled 91 consecutive inpatients with COVID-19 at 6 hospitals in Toronto, Canada, and tested 1 nasopharyngeal swab/saliva sample pair from each patient using real-time RT-PCR for severe acute respiratory syndrome coronavirus 2. Sensitivity was 89% for nasopharyngeal swabs and 72% for saliva (Pâ =â .02). Difference in sensitivity was greatest for sample pairs collected later in illness.
Assuntos
COVID-19 , SARS-CoV-2 , Canadá , Humanos , Nasofaringe , Reação em Cadeia da Polimerase em Tempo Real , SalivaRESUMO
To compare sensitivity of specimens for COVID-19 diagnosis, we tested 151 nasopharyngeal/midturbinate swab pairs from 117 COVID-19 inpatients using reverse-transcriptase polymerase chain reaction (RT-PCR). Sensitivity was 94% for nasopharyngeal and 75% for midturbinate swabs (P = .0001). In 88 nasopharyngeal/midturbinate pairs with matched saliva, sensitivity was 86% for nasopharyngeal swabs and 88% for combined midturbinate swabs/saliva.
Assuntos
COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Humanos , Nasofaringe , Saliva , Manejo de EspécimesRESUMO
MERS-CoV first case was reported on 23rd November 2012 in Saudi Arabia, Since, then MERS has remained on World Health Organization (WHO) Blueprint list and declared pandemic. This study was conducted on MERS lab confirmed cases reported to Ministry of Health, Saudi Arabia and WHO for year 2012-2019. The epidemiology was investigated based on infection rate, death rate, case fatality rate, Gender, Age group, and Medical conditions (Comorbid and Symptomatic). The overall median age of infected male was 58â¯years and of female was 45â¯years. While average mortality age in male was 60â¯years and of female was 65â¯years which is greater than the global average of 50â¯years. The results also report that specially after age of 40â¯years in both men and women, chances of infection are more while comorbidities increase the infection rate. The men are more susceptible to infection than women. In case of asymptomatic distribution trend was vice versa with 69.4% women and 30.6% in men. Second, most infected age group was reduced by 20â¯years in case of men with 47.37% infection for age group of 20-39â¯years. This was also observed in age-group of 20-39â¯years for no comorbid cases (men (50%) & women (79%)). This explains MERS-CoV prevalence in Saudi Arabia, as young and healthy population were infected, and acted as carrier and on coming in contact with vulnerable population (Elderly, chronic and comorbid) transferred the infection. Hence, MERS-CoV outbreak kept on happening from time to time over past years. This finding might very well explain the exponential spread of Novel CoV-19 globally, as initial control measures required older people to stay indoors while younger generation brought infection from outside. Further studies are required for epidemiology analysis based on clusters, travel history and specific disease related mortality.
Assuntos
Infecções por Coronavirus , Coronavírus da Síndrome Respiratória do Oriente Médio , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Coronavirus/epidemiologia , Surtos de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Arábia Saudita/epidemiologia , Viagem , Adulto JovemRESUMO
Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome, looking for mutants defective in response to the replication stress induced by hydroxyurea. In addition to the previously reported mutant with a C-to-Y change at position 307 encoded by tel2 (tel2-C307Y mutant) (Y.-J. Xu, S. Khan, A. C. Didier, M. Wozniak, et al., Mol Cell Biol 39:e00175-19, 2019, https://doi.org/10.1128/MCB.00175-19), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations, except for a start codon mutation, are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect, and heterologous expression of human RECQ1, BLM, and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved, and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.
Assuntos
Quinase do Ponto de Checagem 2/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas de Schizosaccharomyces pombe/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , DNA Helicases/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Instabilidade Genômica , Hidroxiureia/farmacologia , Proteínas Quinases/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE OF REVIEW: Imaging features of lymphoma vary regionally. Awareness of site-specific key imaging characteristics of lymphoma can aid in rapid staging and assist in prompt treatment. FDG PET/CT and conventional MRI are readily available diagnostic modalities with excellent sensitivity and good specificity. Diagnostic specificity can be enhanced using emerging PET radiotracers, e.g., FLT and FET. RECENT FINDINGS: Emerging research has shown higher dimensional analysis (radiomics and radiogenomics) of imaging data can yield information of the underlying genetic aberrations in lymphoma, which can aid in assessing real-time evolution of tumor. CT, PET/CT, MRI, and ultrasound accentuate the intrinsic qualities of lymphoma (e.g., FDG PET/CT for increased metabolic activity, FLT PET/CT for increased proliferation index, and DWI for increased cellularity) and play an essential role in its diagnosis and examination. Advanced radiogenomic analyses use radiomic parameters to deduce genetic variations of lymphoma, providing noninvasive, repeatable, and real-time surveillance of its genetic progression.
Assuntos
Biomarcadores Tumorais/genética , Imagem de Difusão por Ressonância Magnética , Linfoma/diagnóstico por imagem , Imagem Molecular , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Fluordesoxiglucose F18/administração & dosagem , Humanos , Linfoma/genética , Linfoma/terapia , Valor Preditivo dos Testes , Prognóstico , Compostos Radiofarmacêuticos/administração & dosagem , Reprodutibilidade dos TestesRESUMO
Targeted therapy is a new strategy for cancer treatment that targets chemical entities specific to cancer cells than normal ones. One of the features associated with malignancy is the elevated copper which plays an integral role in angiogenesis. Work is in progress in our lab to identify new copper chelators to target elevated copper under targeted therapy for the killing of cancer cells. Recently, a coumarin-based copper chelator, di(2-picolyl)amine-3(bromoacetyl)coumarin hybrid molecule (ligand-L) has been synthesized by us, and also studied its copper-dependent macromolecular damage response in copper overloaded lymphocytes. The present study investigates the anticancer activity of ligand-L and its mode of action in rat model of diethylnitrosamine (DEN) induced hepatocellular carcinoma. It has been found that liver tissue has a marked increase in copper levels in DEN induced hepatocellular carcinoma. Ex vivo results showed that ligand-L inhibited cell viability, induced reactive oxygen species (ROS) generation, DNA damage, loss of mitochondrial membrane potential and caspase-3 activation in isolated hepatocellular carcinoma cells (HCC). All these effects induced by ligand-L were abrogated by neocuproine and N-acetylcysteine (ROS scavenger). Further, ligand-L treatment of animals bearing hepatocellular carcinoma results in an increment in the cellular redox scavengers, lipid peroxidation and DNA breakage in malignant hepatocytes. In vivo studies using ligand-L also showed that ligand-L possesses anticancer properties as evidenced by improvement in liver marker enzymes and liver surface morphology, and reduced alpha-fetoprotein in the treated group compared to untreated cancer-induced group. Overall, this study suggests that copper-ligand-L interaction leads to ROS generation which caused DNA damage and apoptosis in malignant cells. This study provides enough support to establish ligand-L as a clinically relevant lead molecule for the treatment of different malignancies.
Assuntos
Aminocumarinas/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Cobre/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Aminocumarinas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antioxidantes/síntese química , Antioxidantes/química , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cobre/química , Dano ao DNA , Dietilnitrosamina/administração & dosagem , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Injeções Intraperitoneais , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Estrutura Molecular , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/análise , Relação Estrutura-AtividadeRESUMO
Fission yeast Cds1 is responsible for the replication checkpoint activation and helps to protect replication fork collapse in response to hydroxyurea (HU). Here, we investigated the role of histone deacetylase in response to replication fork arrest and observed that in the presence of HU, the survival of cds1Δ cells was improved when the cells were simultaneously treated with histone deacetylase inhibitors. Furthermore, a mutation in the histone deacetylase gene, clr6, also suppresses the growth defect of cds1Δ cells in response to HU indicating a suppressive role of clr6-1 mutation in cds1 deletion background upon HU treatment. Interestingly, in response to HU, phosphorylation of Chk1 kinase and the number of Rad52YFP foci was reduced in cds1Δ clr6-1 double mutant as compared to cds1Δ single mutant indicating a decrease in the level of DNA damage in response to HU. Accordingly, the single-cell gel electrophoresis assay revealed a drastic reduction in the tail length of cds1Δ clr6-1 double mutant as compared to cds1Δ cells in the presence of HU suggesting the suppression of chromosomal defects in the double mutant. Taken together, we proposed that there could be transient suppression of fork collapse in cds1Δ clr6-1 double mutant upon HU treatment due to the delay in mitotic progression that leads to the facilitation of cell growth.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Hidroxiureia/farmacologia , Mutação , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Fosforilação , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidoresRESUMO
Histone Deacetylases (HDACs) play a significant role in the regulation of gene expression by modifying histones and non-histone substrates. Since they are key regulators in the reversible epigenetic mechanism, they are considered as promising drug targets for the treatment of various cancers. In the present study, we have developed a workflow for identification of HDAC1 inhibitors using a multistage virtual screening approach from Maybridge and Chembridge chemical library. Initially, a support vector machine based classification model was generated, followed by generation of a zinc-binding group (ZBG) based pharmacophore model. The hits screened from these models were further subjected to molecular docking. Finally, a set of twenty-three molecules were selected from Maybridge and Chembridge library. The biological evaluation of these hits revealed that three out of the twenty-three tested compounds are showing HDAC1 inhibition along with the moderate anti-proliferative activity. It was found that the identified inhibitors are exerting chromosomal loss effect in growing yeast cells. Further, to extend the activity spectrum of the identified inhibitors, the optimization guidelines were drawn with the hydration site mapping approach by using in silico tool Watermap.Communicated by Ramaswamy H. Sarma.
Assuntos
Inibidores de Histona Desacetilases , Simulação de Acoplamento Molecular , Máquina de Vetores de Suporte , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases , Histonas , Simulação de Dinâmica Molecular , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas PequenasRESUMO
HSA is an important plasma protein responsible for transport of drug molecules. Coumarin derivatives play critical role as anticancer, antidiabetic and antiparkinson agents. In our lab we have synthesized coumarin-based pharmacophore, di(2-picolyl)amine-3(bromoacetyl) coumarin (ligand-L) endowed with anticancer activity. Anticancer agents binding mode of HSA provides valuable pharmacological information and is a structural guidance in synthesizing new drugs with greater efficacy. Thus, binding mechanism of ligand-L with HSA was explored using spectroscopic and molecular docking techniques. UV-Vis spectroscopy demonstrates hyperchromism in the absorbance spectra of HSA on addition of ligand-L suggesting interaction of ligand-L with HSA. Fluorescence spectroscopy indicates quenching in the fluorescence of HSA in the presence of ligand-L confirming the complex formation and this binding follows static mechanism. Steady state fluorescence spectroscopy revealed high binding affinity between ligand-L and HSA with a 1:1 stoichiometry. Thermodynamic parameters obtained by ITC suggest that the interaction between ligand-L and HSA is mainly driven by van der Waals forces and hydrogen bonds, and the negative value of ΔG is an indication of spontaneous binding process. Competitive binding and molecular docking experiments showed that the binding site of ligand-L mainly resides in sub-domain IIA of HSA. CD experiments revealed no significant conformational changes in the secondary structure of HSA on binding of ligand-L. We also found that esterase-like activity of HSA was not affected by ligand-L. In conclusion, this study demonstrates binding mechanism of ligand-L with HSA, and the binding did not induce conformational changes in HSA. This study is likely to provide better understanding of transport and delivery of ligand-L via HSA. Overall, it will provide insights into pharmacokinetic properties of ligand-L and designing new ligand-L based derivatives with greater efficacy.
Assuntos
Aminocumarinas/química , Cumarínicos/química , Modelos Moleculares , Albumina Sérica Humana/química , Análise Espectral , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Esterases/metabolismo , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Carbonilação Proteica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
In response to perturbed DNA replication, ATR (ataxia telangiectasia and Rad3-related) kinase is activated to initiate the checkpoint signaling necessary for maintaining genome integrity and cell survival. To better understand the signaling mechanism, we carried out a large-scale genetic screen in fission yeast looking for mutants with enhanced sensitivity to hydroxyurea. From a collection of â¼370 primary mutants, we found a few mutants in which Rad3 (ATR ortholog)-mediated phospho-signaling was significantly compromised. One such mutant carried an uncharacterized mutation in tel2, a gene encoding an essential and highly conserved eukaryotic protein. Previous studies in various biological models have shown that Tel2 mainly functions in Tel2-Tti1-Tti2 (TTT) complex that regulates the steady-state levels of all phosphatidylinositol 3-kinase-like protein kinases, including ATR. We show here that although the levels of Rad3 and Rad3-mediated phospho-signaling in DNA damage checkpoint were moderately reduced in the tel2 mutant, the phospho-signaling in the DNA replication checkpoint was almost completely eliminated. In addition, the tel2 mutation caused telomere shortening. Since the interactions of Tel2 with Tti1 and Tti2 were significantly weakened by the mutation, destabilization of the TTT complex likely contributes to the observed checkpoint and telomere defects.