Phenocopy – A Strategy to Qualify Chemical Compounds during Hit-to-Lead and/or Lead Optimization

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype-determined phenotype of another individual. The phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of biological systems with new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor β Receptor I (TGF-βR1) were qualified by high-throughput RNA expression profiling. This chemical genomics approach resulted in a precise time-dependent insight to the TGF-β biology and allowed furthermore a comprehensive analysis of each NCE's off-target effects. The evaluation of off-target effects by the phenocopy approach allows a more accurate and integrated view on optimized compounds, supplementing classical biological evaluation parameters such as potency and selectivity. It has therefore the potential to become a novel method for ranking compounds during various drug discovery phases

New Alzheimer Amyloid β Responsive Genes Identified in Human Neuroblastoma Cells by Hierarchical Clustering

Alzheimer's disease (AD) is characterized by neuronal degeneration and cell loss. Aβ42, in contrast to Aβ40, is thought to be the pathogenic form triggering the pathological cascade in AD. In order to unravel overall gene regulation we monitored the transcriptomic responses to increased or decreased Aβ40 and Aβ42 levels, generated and derived from its precursor C99 (C-terminal fragment of APP comprising 99 amino acids) in human neuroblastoma cells. We identified fourteen differentially expressed transcripts by hierarchical clustering and discussed their involvement in AD. These fourteen transcripts were grouped into two main clusters each showing distinct differential expression patterns depending on Aβ40 and Aβ42 levels. Among these transcripts we discovered an unexpected inverse and strong differential expression of neurogenin 2 (NEUROG2) and KIAA0125 in all examined cell clones. C99-overexpression had a similar effect on NEUROG2 and KIAA0125 expression as a decreased Aβ42/Aβ40 ratio. Importantly however, an increased Aβ42/Aβ40 ratio, which is typical of AD, had an inverse expression pattern of NEUROG2 and KIAA0125: An increased Aβ42/Aβ40 ratio up-regulated NEUROG2, but down-regulated KIAA0125, whereas the opposite regulation pattern was observed for a decreased Aβ42/Aβ40 ratio. We discuss the possibilities that the so far uncharacterized KIAA0125 might be a counter player of NEUROG2 and that KIAA0125 could be involved in neurogenesis, due to the involvement of NEUROG2 in developmental neural processes

RNAi screen for NRF2 inducers identifies targets that rescue primary lung epithelial cells from cigarette smoke induced radical stress

Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism

Anticipated responses to a hypothetical minimum price for cigarettes and roll-your-own tobacco: an online cross-sectional survey with cigarette smokers and ex-smokers in the UK

Objectives As tobacco companies can circumvent tax increases, a minimum retail price per-cigarette/per-gram of roll-your-own tobacco presents an additional mechanism for governments to reduce smoking. We examined (1) anticipated responses to a hypothetical minimum price-per-cigarette/per-gram among smokers in the UK; (2) what demographic and smoker characteristics are associated with anticipated responses; and (3) whether minimum pricing may help ex-smokers stay quit.Design Cross-sectional survey (May–July 2019).Setting UK.Participants Adult cigarette smokers (n=2412) and ex-smokers (n=700).Main outcome measurements Anticipated responses to a hypothetical minimum price of £10.00 for 20 cigarettes (£0.50 per-cigarette) and £13.50 for 30 grams of roll-your-own tobacco (£0.45 per-gram); approximately £0.10 per-cigarette/per-gram increases on the cheapest prices in leading UK supermarkets (January 2019). Smokers were presented with ten options (eg, ‘Try to quit’) and asked which they would do (Yes/No) and then which they would most likely do. Ex-smokers were asked to what extent the minimum prices would help them stay quit (A lot vs Lesser agreement).Results Among smokers, 55.6% said they would most likely smoke the same amount, 10.7% they would smoke less, 9.5% they would try to quit and 5.8% they would use e-cigarettes more often. Anticipated reactions were associated with demography and smoker characteristics, for example, C2DE (lower social grade) smokers were less likely than ABC1 (higher social grade) smokers to say they would smoke the same as they do now (ORAdj=0.74, 95% CI 0.62 to 0.88). Among ex-smokers, 38.5% said the minimum prices would help them stay quit ‘A lot’, more so among C2DE than ABC1 participants (ORAdj=1.80, 95% CI 1.30 to 2.49).Conclusions In response to a hypothetical minimum price for cigarettes and roll-your-own tobacco, approximately a fifth of smokers in the UK indicated they would smoke less or quit and almost two-fifths of ex-smokers indicated the prices would help them stay quit

Influence of TCF7L2 gene variants on the therapeutic response to the dipeptidylpeptidase-4 inhibitor linagliptin

Aims/hypothesis Individuals carrying variants of the transcription factor 7-like 2 gene (TCF7L2) are at increased risk for type 2 diabetes. These metabolic genetic risk factors have been linked to diminished pancreatic islet-cell responsiveness to incretins, thus pharmacological interventions aimed at amplifying endogenous incretin biology may be affected. However, clinical evidence from randomised controlled trials so far is lacking. We investigated the influence of TCF7L2 risk alleles on the response to treatment with the dipeptidylpeptidase-4 (DPP-4) inhibitor linagliptin from four 24 week, phase III, placebo-controlled trials. Methods Pharmacogenomic samples and clinical data were available from 961 patients with type 2 diabetes. Whole-blood DNA samples were genotyped for TCF7L2 single-nucleotide polymorphisms in conjunction with assessments of 24 week changes in HbA1c. Results Linagliptin lowered HbA1c meaningfully in all three genotypes of rs7903146 (non-risk variant carriers CC [n = 356]: −0.82% [−9.0 mmol/mol], p < 0.0001; heterozygous CT [n = 264]: −0.77% [−8.4 mmol/mol], p < 0.0001; homozygous risk variant carriers TT [n = 73]: −0.57% [−6.2 mmol/mol], p < 0.0006). No significant treatment differences were seen between CC and CT patients, although HbA1c response was reduced in TT compared with CC patients (~0.26% [~2.8 mmol/mol], p = 0.0182). Conclusions/interpretation Linagliptin significantly improved hyperglycaemia in patients with type 2 diabetes both with and without the TCF7L2 gene diabetes risk alleles. However, differences in treatment response were observed, indicating that diabetes susceptibility genes may be an important contributor to the inter-individual variability of treatment response.Accepted versio

FOOTPRINTS study protocol: rationale and methodology of a 3-year longitudinal observational study to phenotype patients with COPD

Introduction A better understanding is needed of the different phenotypes that exist for patients with chronic obstructive pulmonary disease (COPD), their relationship with the pathogenesis of COPD and how they may affect disease progression. Biomarkers, including those associated with emphysema, may assist in characterising patients and in predicting and monitoring the course of disease. The FOOTPRINTS study (study 352.2069) aims to identify biomarkers associated with emphysema, over a 3-year period.Methods and analysis The FOOTPRINTS study is a prospective, longitudinal, multinational (12 countries), multicentre (51 sites) biomarker study, which has enrolled a total of 463 ex-smokers, including subjects without airflow limitation (as defined by the 2015 Global Initiative for Chronic Obstructive Lung Disease (GOLD) strategy report), patients with COPD across the GOLD stages 1–3 and patients with COPD and alpha1-antitrypsin deficiency. The study has an observational period lasting 156 weeks that includes seven site visits and additional phone interviews. Biomarkers in blood and sputum, imaging data (CT and magnetic resonance), clinical parameters, medical events of special interest and safety are being assessed at regular visits. Disease progression based on biomarker values and COPD phenotypes are being assessed using multivariate statistical prediction models.Ethics and dissemination The study protocol was approved by the authorities and ethics committees/institutional review boards of the respective institutions where applicable, which included study sites in Belgium, Canada, Denmark, Finland, Germany, Japan, Korea, Poland, Spain, Sweden, UK and USA; written informed consent has been obtained from all study participants. Ethics committee approval was obtained for all participating sites prior to enrolment of the study participants. The study results will be reported in peer-reviewed publications.Trial registration number NCT02719184