RESUMO
We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Contenção de Riscos Biológicos/métodos , Temperatura Alta , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Manejo de Espécimes/métodos , Inativação de Vírus , Animais , COVID-19/virologia , Linhagem Celular , Humanos , Camundongos , Vírus da Hepatite Murina/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.
Assuntos
SARS-CoV-2RESUMO
Pulmonary hypertension (PH) is a disease of hyperplasia of pulmonary vascular cells. The pentose phosphate pathway (PPP)-a fundamental glucose metabolism pathway-is vital for cell growth. Because treatment of PH is inadequate, our goal was to determine whether inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, prevents maladaptive gene expression that promotes smooth muscle cell (SMC) growth, reduces pulmonary artery remodeling, and normalizes hemodynamics in experimental models of PH. PH was induced in mice by exposure to 10% oxygen (Hx) or weekly injection of vascular endothelial growth factor receptor blocker [Sugen5416 (SU); 20 mg kg-1] during exposure to hypoxia (Hx + SU). A novel G6PD inhibitor (N-[(3ß,5α)-17-oxoandrostan-3-yl]sulfamide; 1.5 mg kg-1) was injected daily during exposure to Hx. We measured right ventricle (RV) pressure and left ventricle pressure-volume relationships and gene expression in lungs of normoxic, Hx, and Hx + SU and G6PD inhibitor-treated mice. RV systolic and end-diastolic pressures were higher in Hx and Hx + SU than normoxic control mice. Hx and Hx + SU decreased expression of epigenetic modifiers (writers and erasers), increased hypomethylation of the DNA, and induced aberrant gene expression in lungs. G6PD inhibition decreased maladaptive expression of genes and SMC growth, reduced pulmonary vascular remodeling, and decreased right ventricle pressures compared with untreated PH groups. Pharmacologic inhibition of G6PD activity, by normalizing activity of epigenetic modifiers and DNA methylation, efficaciously reduces RV pressure overload in Hx and Hx + SU mice and preclinical models of PH and appears to be a safe pharmacotherapeutic strategy. SIGNIFICANCE STATEMENT: The results of this study demonstrated that inhibition of a metabolic enzyme efficaciously reduces pulmonary hypertension. For the first time, this study shows that a novel inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the fundamental pentose phosphate pathway, modulates DNA methylation and alleviates pulmonary artery remodeling and dilates pulmonary artery to reduce pulmonary hypertension.
Assuntos
Cardiomegalia/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Glucosefosfato Desidrogenase/antagonistas & inibidores , Hipertensão Pulmonar/tratamento farmacológico , Animais , Pressão Sanguínea , Cardiomegalia/etiologia , Hipóxia Celular , Células Cultivadas , Metilação de DNA , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hipertensão Pulmonar/etiologia , Indóis/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Pirróis/toxicidade , Função Ventricular DireitaRESUMO
Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2'-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.
Assuntos
Anemia Falciforme , Hemoglobina Fetal , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Animais , Azacitidina/farmacologia , Metilação de DNA , Hemoglobina Fetal/genética , Camundongos , gama-Globinas/genéticaRESUMO
The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.
Assuntos
DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerases/genética , Processamento de Proteína Pós-Traducional , Reparo de DNA por Recombinação , Telômero/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestrutura , Dano ao DNA , DNA de Neoplasias/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fase G2 , Glicosídeo Hidrolases/metabolismo , Células HeLa , Chaperonas de Histonas/antagonistas & inibidores , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Telômero/ultraestrutura , Homeostase do Telômero , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismoRESUMO
A combination of focused library and virtual screening, hit expansion, and rational design has resulted in the development of a series of inhibitors of RETV804M kinase, the anticipated drug-resistant mutant of RET kinase. These agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET inhibitors currently undergoing clinical evaluation.
RESUMO
Metabolic reprogramming is considered important in the pathogenesis of the occlusive vasculopathy observed in pulmonary hypertension (PH). However, the mechanisms that link reprogrammed metabolism to aberrant expression of genes, which modulate functional phenotypes of cells in PH, remain enigmatic. Herein, we demonstrate that, in mice, hypoxia-induced PH was prevented by glucose-6-phosphate dehydrogenase deficiency (G6PDDef), and further show that established severe PH in Cyp2c44-/- mice was attenuated by knockdown with G6PD shRNA or by G6PD inhibition with an inhibitor (N-ethyl-N'-[(3ß,5α)-17-oxoandrostan-3-yl]urea, NEOU). Mechanistically, G6PDDef, knockdown and inhibition in lungs: 1) reduced hypoxia-induced changes in cytoplasmic and mitochondrial metabolism, 2) increased expression of Tet methylcytosine dioxygenase 2 (Tet2) gene, and 3) upregulated expression of the coding genes and long noncoding (lnc) RNA Pint, which inhibits cell growth, by hypomethylating the promoter flanking region downstream of the transcription start site. These results suggest functional TET2 is required for G6PD inhibition to increase gene expression and to reverse hypoxia-induced PH in mice. Furthermore, the inhibitor of G6PD activity (NEOU) decreased metabolic reprogramming, upregulated TET2 and lncPINT, and inhibited growth of control and diseased smooth muscle cells isolated from pulmonary arteries of normal individuals and idiopathic-PAH patients, respectively. Collectively, these findings demonstrate a previously unrecognized function for G6PD as a regulator of DNA methylation. These findings further suggest that G6PD acts as a link between reprogrammed metabolism and aberrant gene regulation and plays a crucial role in regulating the phenotype of cells implicated in the pathogenesis of PH, a debilitating disorder with a high mortality rate.
Assuntos
Metilação de DNA/genética , Glucosefosfato Desidrogenase/genética , Hipertensão Pulmonar/genética , Hipóxia/genética , Animais , Proliferação de Células/genética , Família 2 do Citocromo P450/genética , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Pulmão/metabolismo , Masculino , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Artéria Pulmonar/metabolismo , RNA Longo não Codificante/genética , Regulação para Cima/genéticaRESUMO
There is mounting evidence of androgen receptor signaling inducing genome instability and changing DNA repair capacity in prostate cancer cells. Expression of genes associated with base excision repair (BER) is increased with prostate cancer progression and correlates with poor prognosis. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are key enzymes in BER that elongate and degrade PAR polymers on target proteins. While PARP inhibitors have been tested in clinical trials and are a promising therapy for prostate cancer patients with TMPRSS2-ERG fusions and mutations in DNA repair genes, PARG inhibitors have not been evaluated. We show that PARG is a direct androgen receptor (AR) target gene. AR is recruited to the PARG locus and induces PARG expression. Androgen ablation combined with PARG inhibition synergistically reduces BER capacity in independently derived LNCaP and LAPC4 prostate cancer cell lines. A combination of PARG inhibition with androgen ablation or with the DNA damaging drug, temozolomide, significantly reduces cellular proliferation and increases DNA damage. PARG inhibition alters AR transcriptional output without changing AR protein levels. Thus, AR and PARG are engaged in reciprocal regulation suggesting that the success of androgen ablation therapy can be enhanced by PARG inhibition in prostate cancer patients.
Assuntos
Inibidores de Glicosídeo Hidrolases/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicosídeo Hidrolases/metabolismo , Humanos , Masculino , Terapia de Alvo MolecularRESUMO
Pulmonary hypertension (PH) is a multicellular and progressive disease with a high mortality rate. Among many cell types, hematopoietic stem cells (HSCs) are incriminated in the pathogenesis of PH. However, our understanding of the mechanisms that increase HSCs in blood and lungs of hypertensive animals or patients and the role played by HSCs in the pathogenesis of PH remains elusive. Studies suggest that glycolysis is critical for the survival and growth of HSCs. In various cell types from hypertensive lungs of animals and patients, glycolysis and the glucose-6-phosphate dehydrogenase (G6PD) activity are increased. Herein, we demonstrated in mice that chronic hypoxia increased HSCs (CD34+, CD117+, CD133+, CD34+/CD117+, and CD34+/CD133+) in bone marrow and blood and around hypertensive pulmonary arteries in a time-dependent manner. Intriguingly, we found fewer CD133+ cells in the bone marrow of C57BL/6 mice compared with Sv129J mice, and C57BL mice developed less severe chronic hypoxia-elicited PH and heart failure than Sv129J mice. Similarly, the numbers of CD34+ and CD117+ cells in blood of patients with pulmonary arterial hypertension (PAH) were higher (>3-fold) compared with healthy individuals. By allogeneic bone marrow transplantation, we found that GFP+ bone marrow cells infiltrated the lungs and accumulated around the pulmonary arteries in lungs of hypoxic mice, and these cells contributed to increased α-adrenergic receptor-mediated contraction of the pulmonary artery cultured in hypoxia. Inhibition of G6PD activity with (3ß,5α)-3,21-dihydroxypregnan-20-one, a novel and potent G6PD inhibitor, decreased HSCs in bone marrow, blood, and lungs of hypoxic mice and reduced α-agonist-induced contraction of the pulmonary artery and established hypoxia-induced PH. We did not observe CD133+ cells around the pulmonary arteries in the lungs of chronically hypoxic G6PD-deficient mice. Furthermore, knockdown of G6PD and inhibition of G6PD activity: 1) downregulated canonical and noncanonical Wnt and Fzd receptors genes; 2) upregulated Bmpr1a; 3) decreased Cxcl12, and 4) reduced HSC (CD117+ and CD133+) numbers. In all, our findings demonstrate unexpected function for bone marrow-derived HSCs in augmenting α-adrenergic receptor-mediated contraction of pulmonary arteries and remodeling of pulmonary arteries that contribute to increase pulmonary vascular resistance in PAH patients and hypoxic mice and suggest that G6PD, by regulating expression of genes in the WNT and BMPR signaling, contributed to increase and release of HSCs from the bone marrow in response to hypoxic stimuli.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Hipertensão Pulmonar/fisiopatologia , Células-Tronco Pluripotentes/metabolismo , Artéria Pulmonar/fisiopatologia , Receptores Adrenérgicos alfa/metabolismo , Animais , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Contagem de Células , Células Cultivadas , Quimiocina CXCL12/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Coração/fisiopatologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hipertensão Pulmonar/etiologia , Hipóxia/sangue , Hipóxia/complicações , Hipóxia/genética , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
The Fanconi anemia pathway orchestrates the repair of DNA interstrand cross-links and stalled replication forks. A key step in this pathway is UBE2T and FANCL-dependent monoubiquitylation of the FANCD2-FANCI complex. The Fanconi anemia pathway represents an attractive therapeutic target, because activation of this pathway has been linked to chemotherapy resistance in several cancers. However, to date, very few selective inhibitors of ubiquitin conjugation pathways are known. By using a high-throughput screen-compatible assay, we have identified a small-molecule inhibitor of UBE2T/FANCL-mediated FANCD2 monoubiquitylation that sensitizes cells to the DNA cross-linking agent, carboplatin.
Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/antagonistas & inibidores , Anemia de Fanconi/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Linhagem Celular Tumoral , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have an average survival of less than 1 year, underscoring the importance of evaluating novel targets with matched targeted agents. We recently identified that poly (ADP) ribose glycohydrolase (PARG) is a strong candidate target due to its dependence on the pro-oncogenic mRNA stability factor HuR (ELAVL1). Here, we evaluated PARG as a target in PDAC models using both genetic silencing of PARG and established small-molecule PARG inhibitors (PARGi), PDDX-01/04. Homologous repair-deficient cells compared with homologous repair-proficient cells were more sensitive to PARGi in vitro. In vivo, silencing of PARG significantly decreased tumor growth. PARGi synergized with DNA-damaging agents (i.e., oxaliplatin and 5-fluorouracil), but not with PARPi therapy. Mechanistically, combined PARGi and oxaliplatin treatment led to persistence of detrimental PARylation, increased expression of cleaved caspase-3, and increased γH2AX foci. In summary, these data validate PARG as a relevant target in PDAC and establish current therapies that synergize with PARGi. SIGNIFICANCE: PARG is a potential target in pancreatic cancer as a single-agent anticancer therapy or in combination with current standard of care.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Glicosídeo Hidrolases/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Feminino , Inativação Gênica , Glicosídeo Hidrolases/genética , Humanos , Camundongos Nus , Terapia de Alvo Molecular , Oxaliplatina/farmacologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Reparo de DNA por Recombinação , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fluorination of metabolic hotspots in a molecule is a common medicinal chemistry strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.
Assuntos
Ciclopropanos/farmacologia , Glicosídeo Hidrolases/toxicidade , Microssomos Hepáticos/efeitos dos fármacos , Administração Oral , Animais , Ciclopropanos/administração & dosagem , Ciclopropanos/química , Ciclopropanos/farmacocinética , Glicosídeo Hidrolases/administração & dosagem , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/farmacocinética , Meia-Vida , Humanos , Camundongos , Microssomos Hepáticos/metabolismoRESUMO
DNA damage repair enzymes are promising targets in the development of new therapeutic agents for a wide range of cancers and potentially other diseases. The enzyme poly(ADP-ribose) glycohydrolase (PARG) plays a pivotal role in the regulation of DNA repair mechanisms; however, the lack of potent drug-like inhibitors for use in cellular and in vivo models has limited the investigation of its potential as a novel therapeutic target. Using the crystal structure of human PARG in complex with the weakly active and cytotoxic anthraquinone 8a, novel quinazolinedione sulfonamides PARG inhibitors have been identified by means of structure-based virtual screening and library design. 1-Oxetan-3-ylmethyl derivatives 33d and 35d were selected for preliminary investigations in vivo. X-ray crystal structures help rationalize the observed structure-activity relationships of these novel inhibitors.
Assuntos
Reparo do DNA , Desenho de Fármacos , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Quinazolinonas/química , Quinazolinonas/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Domínio Catalítico , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Inibidores de Glicosídeo Hidrolases/farmacocinética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Células HeLa , Humanos , Masculino , Camundongos , Modelos Moleculares , Quinazolinonas/administração & dosagem , Quinazolinonas/farmacocinética , Relação Estrutura-AtividadeRESUMO
The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. The APTX RNA-DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. Here, we report X-ray structures of APTX engaging nicked RNA-DNA substrates that provide direct evidence for a wedge-pivot-cut strategy for 5'-AMP resolution shared with the alternate 5'-AMP processing enzymes POLß and FEN1. Our results uncover a DNA-induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Further, based on comprehensive biochemical, X-ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations.
Assuntos
Quebras de DNA de Cadeia Simples , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Doenças Neurodegenerativas/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Nucleares/genética , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , RNA/química , RNA/metabolismoRESUMO
Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the ß-nicotinamide adenine dinucleotide (ß-NAD+) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.
Assuntos
ADP-Ribosilação/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , ADP-Ribosilação/genética , Adenosina Difosfato Ribose/metabolismo , Animais , Reparo do DNA/genética , Reparo do DNA/fisiologia , Glicosilação , Humanos , Poli(ADP-Ribose) Polimerases/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/enzimologia , Regulação Alostérica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Isocitrato Desidrogenase/isolamento & purificação , Isocitrato Desidrogenase/metabolismo , Leucemia Mieloide Aguda/patologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
We present IncucyteDRC, an R package for the analysis of data from live cell imaging cell proliferation experiments carried out on the Essen Biosciences IncuCyte ZOOM instrument. The package provides a simple workflow for summarising data into a form that can be used to calculate dose response curves and EC50 values for small molecule inhibitors. Data from different cell lines, or cell lines grown under different conditions, can be normalised as to their doubling time. A simple graphical web interface, implemented using shiny, is provided for the benefit of non-R users. The software is potentially useful to any research group studying the impact of small molecule inhibitors on cell proliferation using the IncuCyte ZOOM.
RESUMO
The enzyme poly(ADP-ribose) glycohydrolase (PARG) performs a critical role in the repair of DNA single strand breaks (SSBs). However, a detailed understanding of its mechanism of action has been hampered by a lack of credible, cell-active chemical probes. Herein, we demonstrate inhibition of PARG with a small molecule, leading to poly(ADP-ribose) (PAR) chain persistence in intact cells. Moreover, we describe two advanced, and chemically distinct, cell-active tool compounds with convincing on-target pharmacology and selectivity. Using one of these tool compounds, we demonstrate pharmacology consistent with PARG inhibition. Further, while the roles of PARG and poly(ADP-ribose) polymerase (PARP) are closely intertwined, we demonstrate that the pharmacology of a PARG inhibitor differs from that observed with the more thoroughly studied PARP inhibitor olaparib. We believe that these tools will facilitate a wider understanding of this important component of DNA repair and may enable the development of novel therapeutic agents exploiting the critical dependence of tumors on the DNA damage response (DDR).
Assuntos
Reparo do DNA , Glicosídeo Hidrolases/química , Sondas Moleculares/química , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Células HeLa , Humanos , Ressonância de Plasmônio de SuperfícieRESUMO
After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.
RESUMO
RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series. Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile.