Manipulation of dipeptidylpeptidase 10 in mouse and human in vivo and in vitro models indicates a protective role in asthma

We previously identified dipeptidylpeptidase 10 (DPP10) on chromosome 2 as a human asthma susceptibility gene, through positional cloning. Initial association results were confirmed in many subsequent association studies but the functional role of DPP10 in asthma remains unclear. Using the MRC Harwell N-ethyl-N-nitrosourea (ENU) DNA archive, we identified a point mutation in Dpp10 that caused an amino acid change from valine to aspartic acid in the β-propeller region of the protein. Mice carrying this point mutation were recovered and a congenic line was established (Dpp10145D). Macroscopic examination and lung histology revealed no significant differences between wild-type and Dpp10145D/145D mice. However, after house dust mite (HDM) treatment, Dpp10 mutant mice showed significantly increased airway resistance in response to 100 mg/ml methacholine. Total serum IgE levels and bronchoalveolar lavage (BAL) eosinophil counts were significantly higher in homozygotes than in control mice after HDM treatment. DPP10 protein is present in airway epithelial cells and altered expression is observed in both tissue from asthmatic patients and in mice following HDM challenge. Moreover, knockdown of DPP10 in human airway epithelial cells results in altered cytokine responses. These results show that a Dpp10 point mutation leads to increased airway responsiveness following allergen challenge and provide biological evidence to support previous findings from human genetic studies. This article has an associated First Person interview with the first author of the paper

Biomarkers of coagulation, endothelial function, and fibrinolysis in critically ill patients with COVID-19: A single-center prospective longitudinal study

Background: Immunothrombosis and coagulopathy in the lung microvasculature may lead to lung injury and disease progression in coronavirus disease 2019 (COVID-19). We aim to identify biomarkers of coagulation, endothelial function, and fibrinolysis that are associated with disease severity and may have prognostic potential. Methods: We performed a single-center prospective study of 14 adult COVID-19(+) intensive care unit patients who were age- and sex-matched to 14 COVID-19(−) intensive care unit patients, and healthy controls. Daily blood draws, clinical data, and patient characteristics were collected. Baseline values for 10 biomarkers of interest were compared between the three groups, and visualized using Fisher\u27s linear discriminant function. Linear repeated-measures mixed models were used to screen biomarkers for associations with mortality. Selected biomarkers were further explored and entered into an unsupervised longitudinal clustering machine learning algorithm to identify trends and targets that may be used for future predictive modelling efforts. Results: Elevated D-dimer was the strongest contributor in distinguishing COVID-19 status; however, D-dimer was not associated with survival. Variable selection identified clot lysis time, and antigen levels of soluble thrombomodulin (sTM), plasminogen activator inhibitor-1 (PAI-1), and plasminogen as biomarkers associated with death. Longitudinal multivariate k-means clustering on these biomarkers alone identified two clusters of COVID-19(+) patients: low (30%) and high (100%) mortality groups. Biomarker trajectories that characterized the high mortality cluster were higher clot lysis times (inhibited fibrinolysis), higher sTM and PAI-1 levels, and lower plasminogen levels. Conclusions: Longitudinal trajectories of clot lysis time, sTM, PAI-1, and plasminogen may have predictive ability for mortality in COVID-19

Privileged Structures and Polypharmacology within and between Protein Families.

Polypharmacology is often a key contributor to the efficacy of a drug, but is also a potential risk. We investigated two hits discovered via a cell-based phenotypic screen, the CDK9 inhibitor CCT250006 (1) and the pirin ligand CCT245232 (2), to establish methodology to elucidate their secondary protein targets. Using computational pocket-based analysis, we discovered intrafamily polypharmacology for our kinase inhibitor, despite little overall sequence identity. The interfamily polypharmacology of 2 with B-Raf was used to discover a novel pirin ligand from a very small but privileged compound library despite no apparent ligand or binding site similarity. Our data demonstrates that in areas of drug discovery where intrafamily polypharmacology is often an issue, ligand dissimilarity cannot necessarily be used to assume different off-target profiles and that understanding interfamily polypharmacology will be important in the future to reduce the risk of idiopathic toxicity and in the design of screening libraries

Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9.

Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 μM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM)

Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen.

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography

Functional analysis of a novel ENU-induced PHD finger 11 (Phf11) mouse mutant

Previously, human genetic studies have shown association between polymorphisms within the gene encoding plant homeodomain zinc finger protein 11 (PHF11) and asthma-related phenotypes. Initial functional studies have suggested that PHF11 may be involved in the immune response through regulation of T cell activities. In order to study further the gene’s functions, we have investigated the mouse Phf11 locus. We have established and characterised a mouse line harbouring a point mutation in the PHD domain of Phf11. Full-length mouse cDNA for Phf11 was obtained by applying rapid amplification of cDNA ends (RACE). All five exons encoding the PHD domain of Phf11 were directly sequenced in 3840 mouse DNA samples from the UK MRC Harwell ENU (N-ethyl-N-nitrosourea)-mutagenised DNA archive. Mice harbouring a valine to alanine substitution, predicted to have a significant functional impact on the PHD zinc finger domain, were re-derived. These Phf11 mutant mice were outcrossed to C3H mice and then backcrossed for ten generations in order to establish a congenic line harbouring the single point mutation in Phf11. Macroscopic examination, haematology and histological examination of lung structure revealed no significant differences between mutant and wild-type mice. After administration of lipopolysaccharide, the level of expression of Il2, NF-kB and Setdb2 were significantly increased in Phf11 mutant homozygous lungs compared to control littermates. Our results provide evidence that Phf11 can operate as a Th1 cell regulator in immune responses. Moreover, our data indicate that these mice may provide a useful model for future studies on Phf11. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00335-014-9535-x) contains supplementary material, which is available to authorised users

Helios is a key transcriptional regulator of outer hair cell maturation

The sensory cells that are responsible for hearing include the cochlear inner hair cells (IHCs) and outer hair cells (OHCs), with the OHCs being necessary for sound sensitivity and tuning1. Both cell types are thought to arise from common progenitors; however, our understanding of the factors that control the fate of IHCs and OHCs remains limited. Here we identify Ikzf2 (which encodes Helios) as an essential transcription factor in mice that is required for OHC functional maturation and hearing. Helios is expressed in postnatal mouse OHCs, and in the cello mouse model a point mutation in Ikzf2 causes early-onset sensorineural hearing loss. Ikzf2cello/cello OHCs have greatly reduced prestin-dependent electromotile activity, a hallmark of OHC functional maturation, and show reduced levels of crucial OHC-expressed genes such as Slc26a5 (which encodes prestin) and Ocm. Moreover, we show that ectopic expression of Ikzf2 in IHCs: induces the expression of OHC-specific genes; reduces the expression of canonical IHC genes; and confers electromotility to IHCs, demonstrating that Ikzf2 can partially shift the IHC transcriptome towards an OHC-like identity