RESUMO
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
The biphenyl amides (BPAs) are a series of p38alpha MAP kinase inhibitors. Compounds are able to bind to the kinase in either the DFG-in or DFG-out conformation, depending on substituents. X-ray, binding, kinetic and cellular data are shown, providing the most detailed comparison to date between potent compounds from the same chemical series that bind to different p38alpha conformations. DFG-out-binding compounds could be made more potent than DFG-in-binding compounds by increasing their size. Unexpectedly, compounds that bound to the DGF-out conformation showed diminished selectivity. The kinetics of binding to the isolated enzyme and the effects of compounds on cells were largely unaffected by the kinase conformation bound.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Amidas/sangue , Amidas/química , Aminoácidos/genética , Aminoácidos/metabolismo , Sítios de Ligação , Compostos de Bifenilo/sangue , Compostos de Bifenilo/química , Técnicas de Química Combinatória , Cristalografia por Raios X , Desenho de Fármacos , Lipopolissacarídeos/farmacologia , Conformação Molecular , Estrutura Molecular , Naftalenos/farmacologia , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
The biphenyl amides (BPAs) are a novel series of p38alpha MAP kinase inhibitor. The optimisation of the series to give compounds that are potent in an in vivo disease model is discussed. SAR is presented and rationalised with reference to the crystallographic binding mode.
Assuntos
Benzamidas/síntese química , Benzamidas/farmacologia , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Benzamidas/sangue , Benzamidas/química , Compostos de Bifenilo/sangue , Compostos de Bifenilo/química , Técnicas de Química Combinatória , Cristalografia por Raios X , Modelos Animais de Doenças , Conformação Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/química , RatosRESUMO
The biphenyl amides are a novel series of p38 MAP kinase inhibitors. Structure-activity relationships of the series against p38alpha are discussed with reference to the X-ray crystal structure of an example. The series was optimised rapidly to a compound showing oral activity in an in vivo disease model.
Assuntos
Amidas/farmacologia , Compostos de Bifenilo/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Administração Oral , Amidas/química , Amidas/farmacocinética , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacocinética , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Oxidiazóis/química , Oxidiazóis/farmacocinética , Oxidiazóis/farmacologia , Piperazinas/química , Piperazinas/farmacocinética , Piperazinas/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.
Assuntos
Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Especificidade de Anticorpos , Linhagem Celular , Histonas/imunologia , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Permeabilidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas PequenasRESUMO
p38alpha MAP kinase is a key anti-inflammatory target for rheumatoid arthritis, influencing biosynthesis of pro-inflammatory cytokines TNFalpha and IL-1beta at a translational and transcriptional level. In this paper, we describe how we have optimized a series of novel p38alpha/beta inhibitors using crystal structures of our inhibitors bound to p38alpha, classical medicinal chemistry, and modeling of virtual libraries to derive a molecule suitable for progression into clinical development.