RESUMO
For the discovery of new candidate molecules in the pharmaceutical industry, library synthesis is a critical step, in which library size, diversity, and time to synthesise are fundamental. In this work we propose stopped-flow synthesis as an intermediate alternative to traditional batch and flow chemistry approaches, suited for small molecule pharmaceutical discovery. This method exploits the advantages of both techniques enabling automated experimentation with access to high pressures and temperatures; flexibility of reaction times, with minimal use of reagents (µmol scale per reaction). In this study, we integrate a stopped-flow reactor into a high-throughput continuous platform designed for the synthesis of combinatory libraries with at-line reaction analysis. This approach allowed â¼900 reactions to be conducted in an accelerated timeframe (192 hours). The stopped flow approach used â¼10% of the reactants and solvents compared to a fully continuous approach. This methodology demonstrates a significantly improved synthesis success rate of smaller libraries by simplifying the implementation of cross-reaction optimisation strategies. The experimental datasets were used to train a feed-forward neural network (FFNN) model providing a framework to guide further experiments, which showed good model predictability and success when tested against an external set with fewer experiments. As a result, this work demonstrates that combining experimental automation with machine learning strategies can deliver optimised analyses and enhanced predictions, enabling more efficient drug discovery investigations across the design, make, test and analysis (DMTA) cycle.
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
Faced with ageing infrastructure and ever-increasing demands from hit discovery and lead optimisation functions, AstraZeneca has chosen to develop innovative technologies and process solutions to support the future of drug discovery. These include the miniaturisation of compound storage tubes for high-density storage and rapid access to the corporate collection for feeding samples to the predicted tripling number of high throughput screening (HTS) campaigns. The acoustically- compatible tubes also enable the first fully-acoustic plate production process for faster sample supply to screening with less waste and continued high quality. Operating at a smaller scale reduces compound synthesis, storage, and consumption, prompting miniaturisation of upstream chemistry and downstream biological assays, while offering a transformative and sustainable solution to many drug discovery issues applicable across the industry.
Assuntos
Descoberta de Drogas/tendências , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas/análise , Automação/métodos , Química Farmacêutica/tendências , Técnicas de Química Combinatória/instrumentação , Técnicas de Química Combinatória/métodos , Indústria Farmacêutica/tendências , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Miniaturização/métodos , Melhoria de Qualidade , Tecnologia Farmacêutica/tendências , Fluxo de TrabalhoRESUMO
Compound management (CM) is a critical discipline enabling hit discovery through the production of assay-ready compound plates for screening. CM in pharma requires significant investments in manpower, capital equipment, repairs and maintenance, and information technology. These investments are at risk from external factors, for example, new technology rendering existing equipment obsolete and strategic site closures. At AstraZeneca, we faced the challenge of evaluating the number of CM sites required to support hit discovery in response to site closures and pressure on our operating budget. We reasoned that overall equipment effectiveness, a tool used extensively in the manufacturing sector, could determine the equipment capacity and appropriate number of sites. We identified automation downtime as the critical component governing capacity, and a connection between automation downtime and the availability of skilled staff. We demonstrated that sufficient production capacity existed in two sites to meet hit discovery demand without the requirement for an additional investment of $7 million in new facilities. In addition, we developed an automated capacity model that incorporated an extended working-day pattern as a solution for reducing automation downtime. The application of this solution enabled the transition to a single site, with an annual cost saving of $2.1 million.
Assuntos
Automação Laboratorial/economia , Automação Laboratorial/métodos , Avaliação Pré-Clínica de Medicamentos/economia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Custos e Análise de CustoRESUMO
Ghrelin plays a major physiological role in the control of food intake, and inverse agonists of the ghrelin receptor (GHS-R1a) are widely considered to offer utility as antiobesity agents by lowering the set-point for hunger between meals. We identified an acylurea series of ghrelin modulators from high throughput screening and optimized binding affinity through structure-activity relationship studies. Furthermore, we identified specific substructural changes, which switched partial agonist activity to inverse agonist activity, and optimized physicochemical and DMPK properties to afford the non-CNS penetrant inverse agonist 22 (AZ-GHS-22) and the CNS penetrant inverse agonist 38 (AZ-GHS-38). Free feeding efficacy experiments showed that CNS exposure was necessary to obtain reduced food intake in mice, and it was demonstrated using GHS-R1a null and wild-type mice that this effect operates through a mechanism involving GHS-R1a.
Assuntos
Agonismo Inverso de Drogas , Receptores de Grelina/agonistas , Receptores de Grelina/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologia , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Receptores de Grelina/metabolismo , Relação Estrutura-Atividade , Ureia/químicaRESUMO
A novel series of DGAT-1 inhibitors was discovered from an oxadiazole amide high throughput screening (HTS) hit. Optimisation of potency and ligand lipophilicity efficiency (LLE) resulted in a carboxylic acid containing clinical candidate 53 (AZD3988), which demonstrated excellent DGAT-1 potency (0.6 nM), good pharmacokinetics and pre-clinical in vivo efficacy that could be rationalised through a PK/PD relationship.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Hipoglicemiantes/síntese química , Oxidiazóis/síntese química , Animais , Diabetes Mellitus/tratamento farmacológico , Diacilglicerol O-Aciltransferase/metabolismo , Cães , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Meia-Vida , Ensaios de Triagem em Larga Escala , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Ligantes , Camundongos , Obesidade/tratamento farmacológico , Oxidiazóis/farmacocinética , Relação Quantitativa Estrutura-Atividade , RatosRESUMO
Obesity represents a rapidly emerging epidemic amongst pregnant women. Our study looks at the impact of morbid obesity on pregnant singleton nulliparous women in comparison with normal body mass index women. We conclude that morbid obesity is associated with a significantly higher risk of pre-existing medical conditions, developing antenatal complications, induction of labour, caesarean section and greater birth weight. However, there was no significant difference in caesarean section rates when adjusted for induction of labour. We also found no significant difference in length of hospital stay, postnatal complications and neonatal morbidity.
Assuntos
Parto Obstétrico/métodos , Obesidade Mórbida/complicações , Paridade , Complicações na Gravidez , Resultado da Gravidez , Serviços de Saúde Rural , Adolescente , Adulto , Peso ao Nascer , Índice de Massa Corporal , Estudos de Coortes , Feminino , Humanos , Trabalho de Parto Induzido , Serviços de Saúde Materna , Gravidez , Estudos Retrospectivos , Adulto JovemRESUMO
Deregulation of the cell cycle has long been recognized as an essential driver of tumorigenesis, and agents that selectively target key cell cycle components continue to hold promise as potential therapeutics. We have developed AZD5438, a 4-(1-isopropyl-2-methylimidazol-5-yl)-2-(4-methylsulphonylanilino) pyrimidine, as a potent inhibitor of cyclin-dependent kinase (cdk) 1, 2, and 9 (IC(50), 16, 6, and 20 nmol/L, respectively). In vitro, AZD5438 showed significant antiproliferative activity in human tumor cell lines (IC(50) range, 0.2-1.7 micromol/L), causing inhibition of the phosphorylation of cdk substrates pRb, nucleolin, protein phosphatase 1a, and RNA polymerase II COOH-terminal domain and blocking cell cycling at G(2)-M, S, and G(1) phases. In vivo, when orally administered at either 50 mg/kg twice daily or 75 mg/kg once daily, AZD5438 inhibited human tumor xenograft growth (maximum percentage tumor growth inhibition, range, 38-153; P < 0.05). In vivo, AZD5438 reduced the proportion of actively cycling cells. Further pharmacodynamic analysis of AZD5438-treated SW620 xenografts showed that efficacious doses of AZD5438 (>40% tumor growth inhibition) maintained suppression of biomarkers, such as phospho-pRbSer(249)/Thr(252), for up to 16 hours following a single oral dose. A comparison of different schedules indicated that chronic daily oral dosing provided optimal cover to ensure antitumor efficacy. These data indicate that broad cdk inhibition may provide an effective method to impair the dysregulated cell cycle that drives tumorigenesis and AZD5438 has the pharmacologic profile that provides an ideal probe to test this premise.
Assuntos
Antineoplásicos/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Imidazóis/farmacologia , Neoplasias/tratamento farmacológico , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Western Blotting , Proteína Quinase CDC2/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Ratos , Ratos Nus , Células Tumorais CultivadasRESUMO
The development of a novel series of imidazole pyrimidine amides as cyclin-dependent kinase (CDK) inhibitors is described. The series was found to have much improved CDK2 inhibition and potent in vitro anti-proliferative effects against cancer cell lines. Control of overall lipophilicity was important to achieve good in vitro potency along with acceptable physiochemical properties and margins against inhibition of both CYP isoforms and the hERG potassium ion channel. A compound with an attractive overall balance of properties was profiled in vivo and possessed suitable physiochemical and pharmacokinetic profiles for oral dosing.
Assuntos
Proteínas Inibidoras de Quinase Dependente de Ciclina/síntese química , Imidazóis/química , Pirimidinas/química , Administração Oral , Animais , Linhagem Celular Tumoral , Proteínas Inibidoras de Quinase Dependente de Ciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Nus , Ratos , Ratos WistarRESUMO
An imidazole series of cyclin-dependent kinase (CDK) inhibitors has been developed. Protein inhibitor structure determination has provided an understanding of the emerging structure activity trends for the imidazole series. The introduction of a methyl sulfone at the aniline terminus led to a more orally bioavailable CDK inhibitor that was progressed into clinical development.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Imidazóis/química , Compostos de Anilina/química , Animais , Proteínas de Ciclo Celular/química , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
A piperazine series of cyclin-dependent kinase (CDK) inhibitors have been identified. The compounds exhibit excellent physiochemical properties and a novel binding mode, whereby a bridging interaction via a water molecule with Asp 86 of CDK2, leads to selectivity for the CDK family of enzymes over other kinases. Piperazines 2e and 2i were subsequently shown to inhibit tumour growth when dosed orally in a nude mouse xenograft study. Additional chemical series that exploit this unexpected interaction with Asp 86 are also described.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Imidazóis/síntese química , Imidazóis/farmacologia , Piperazinas/síntese química , Piperazinas/farmacologia , Animais , Antineoplásicos/química , Ácido Aspártico/química , Ácido Aspártico/genética , Sítios de Ligação , Técnicas de Química Combinatória , Quinase 2 Dependente de Ciclina/metabolismo , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Imidazóis/química , Camundongos , Camundongos Nus , Estrutura Molecular , Piperazinas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In model studies towards the quaternary centre at the heart of diazonamide A (early structure 2; revised structure 1), cyclisations of the alkene-substituted iodoaryls 4, 13, 18 and 23, under Heck reaction conditions, were shown to lead to the corresponding benzodihydrofuran 5, benzofuranone 14 and the oxindoles 19 and 24 respectively, in 50-80% yield. Further manipulation of the benzodihydrofuran 5 then led to the intermediates 30, 33 and 39, which make up parts of the oxazole-indole heterocyclic core in diazonamide A. Attempts to perform a corresponding 13-exo-trig Heck cyclisation from the precursor 46a, prepared from 44 and 45, leading to 47 were not successful. A similar outcome was obtained during attempts to effect Heck cyclisations from the ester 57 and the related ether 59. Treatment of the chromene-substituted iodoaryl 62 with Pd(OAc)2, PPh3 and Ag2CO3 led to the spirocycle 64 as a crystalline solid. X-Ray crystal structure analysis established that the quaternary centre in 64 had the same configuration as that present in diazonamide A (1).