Despite decades of intensive search for compounds that modulate the activity of particular protein targets, a large proportion of the human kinome remains as yet undrugged. Effective approaches are ...therefore required to map the massive space of unexplored compound-kinase interactions for novel and potent activities. Here, we carry out a crowdsourced benchmarking of predictive algorithms for kinase inhibitor potencies across multiple kinase families tested on unpublished bioactivity data. We find the top-performing predictions are based on various models, including kernel learning, gradient boosting and deep learning, and their ensemble leads to a predictive accuracy exceeding that of single-dose kinase activity assays. We design experiments based on the model predictions and identify unexpected activities even for under-studied kinases, thereby accelerating experimental mapping efforts. The open-source prediction algorithms together with the bioactivities between 95 compounds and 295 kinases provide a resource for benchmarking prediction algorithms and for extending the druggable kinome.
Abstract only
Phosphoinositides are key regulators in many signaling cascades and several cellular processes. These lipids are located in cellular membranes containing different forms of these ...lipids. The plasma membrane (PM) is enriched in two phosphorylated variants, phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate (PIP2).
To discover the role of these lipids, several applications have been developed. One of them is the rapamycin inducible heterodimerization system, based on the rapid and irreversible interaction of the FRB and FKBP12 proteins. In our system FRB was targeted to the PM using the localization sequence of the Fyn tyrosine kinase, whereas the FKBP was fused to an enzymatically active cytoplasmic 5‐phosphatase. After rapamycin treatment, the enzyme recruited to the PM, where rapid depletion of PIP2 could be achieved. Our goal was to generate a genetically modified mouse line containing this lipid depletion system, which can be highly capable to investigate the physiological role of PIP2 in primary prepared cells or in intact tissues and organs.
Our plasmid used for the transgenic application contained both protein coding sequences of the depletion system in the same vector. To reach the stochiometric expression of the two distinct fragments, a viral T2A sequence was inserted between the protein coding sequences. For further detection of the transgene the PM targeted protein was fluorescently tagged. The transgenic construct was driven by a CMV promoter to reach a general expression in various animal tissues. The transgenic modification was carried out by applying the Sleeping Beauty transposase in FVB/Ant mouse strain. Based on the genotyping data of the founder generation, two distinct lines with low and high copy number were selected for the further investigations. Interestingly, transgene showed detectable expressions only in pancreatic acinar cells and in keratinocytes.
To investigate the functional effect of the PIP2 depletion we carried out cytoplasmic calcium ion measurements. Using freshly prepared pancreatic acinar cells of the F2 generation rapamycin treatment did not resulted a reduced cytoplasmic calcium signal upon stimulation the cells with carbachol, indicating the low expression level of the PIP2 depletion system. A mathematical model was created, which predicts of the increase of protein expression, so we decided to select the animals with the highest copy numbers in each generation and use them as parents of the further generations. The efficiency of lipid depletion is monitored by measuring the translocation of PLCdelta1‐PH‐domain based PIP2 sensors, as well as by measuring other PIP2‐dependent cellular responses.
Support or Funding Information
National Research, Development and Innovation Fund (NKFI K105006)
The Columbia Cancer Target Discovery and Development (CTD2) Center is developing PANACEA, a resource comprising dose-responses and RNA sequencing (RNA-seq) profiles of 25 cell lines perturbed with ...∼400 clinical oncology drugs, to study a tumor-specific drug mechanism of action. Here, this resource serves as the basis for a DREAM Challenge assessing the accuracy and sensitivity of computational algorithms for de novo drug polypharmacology predictions. Dose-response and perturbational profiles for 32 kinase inhibitors are provided to 21 teams who are blind to the identity of the compounds. The teams are asked to predict high-affinity binding targets of each compound among ∼1,300 targets cataloged in DrugBank. The best performing methods leverage gene expression profile similarity analysis as well as deep-learning methodologies trained on individual datasets. This study lays the foundation for future integrative analyses of pharmacogenomic data, reconciliation of polypharmacology effects in different tumor contexts, and insights into network-based assessments of drug mechanisms of action.
Display omitted
•Drug-perturbed RNA sequencing data can be used to identify drug targets•Technology-based drug-target definitions often subsume literature definitions•Literature and screening datasets provide complementary information on drug mechanisms
Douglass et al. report the results of a crowdsourced challenge to develop machine-learning algorithms that use drug-perturbed transcriptome data to rapidly predict drug targets on a proteomic scale. Winning methods effectively predicted off-target binding of clinical kinase inhibitors and clarified disparate literature on these drugs’ mechanisms of action.
In this study we investigated the functional consequences of intradimeric interactions within type I angiotensin receptor (AT1R) homodimer. To achieve this, we stimulated selectively one protomer of ...the AT1R dimer, and followed the activation process of the other protomer. For the selective stimulation, we used the S109Y mutant AT1R which is resistant to the non peptide AT1R antagonists like candesartan. Expressing this mutant receptor together with the wild type in CHO cells, in the presence of candesartan we could examine the interaction between them by stimulating the S109Y mutant receptor, and following the activation of the wild type receptor. The activation of the wild type receptor was monitored either by measuring the interaction between the receptor and β‐arrestin2 or by following the conformational changes of the receptor with an intramolecular biosensor. Under these conditions we could detect conformational changes and β‐arrestin2 binding of the nonstimulated protomer. Introduction of the DRY/AYY mutation into the directly activated protomer completely abolished the described effects, suggesting the crucial role of this motif in the intradimeric interactions.
Heterodimerization between angiotensin type 1A receptor (AT
R) and β
-adrenergic receptor (β
AR) has been shown to modulate G protein-mediated effects of these receptors. Activation of G ...protein-coupled receptors (GPCRs) leads to β-arrestin binding, desensitization, internalization and G protein-independent signaling of GPCRs. Our aim was to study the effect of heterodimerization on β-arrestin coupling. We found that β-arrestin binding of β
AR is affected by activation of AT
Rs. Costimulation with angiotensin II and isoproterenol markedly enhanced the interaction between β
AR and β-arrestins, by prolonging the lifespan of β
AR-induced β-arrestin2 clusters at the plasma membrane. While candesartan, a conventional AT
R antagonist, had no effect on the β-arrestin2 binding to β
AR, TRV120023, a β-arrestin biased agonist, enhanced the interaction. These findings reveal a new crosstalk mechanism between AT
R and β
AR, and suggest that enhanced β-arrestin2 binding to β
AR can contribute to the pharmacological effects of biased AT
R agonists.
In adrenal zona glomerulosa cells angiotensin II (Ang II) is a key regulator of steroidogenesis. Our purpose was to compare the mechanisms of Ang II-induced changes in the expression level of early ...transcription factors NR4A1 (NGFIB) and NR4A2 (Nurr1) genes, and the CYP11B2 gene encoding aldosterone synthase in H295R human adrenocortical tumor cells and in primary rat adrenal glomerulosa cells. Real-time PCR studies have demonstrated that Ang II increased the expression levels of NR4A1 and NR4A2 in H295R cells within one hour after stimulation, which persisted up to 6 hrs; whereas in rat adrenal glomerulosa cells the kinetics of the expression of these genes were more rapid and transient. Ang II also induced prolonged nuclear translocation of Nurr1 and NGFIB proteins in both cell types. Studies using MEK inhibitor (PD98059, 20μM), protein kinase C inhibitor (BIM1, 3μM) and calmodulin kinase (CAMK) inhibitor (KN93, 10μM) revealed that in rat adrenal glomerulosa cells CAMK-mediated mechanisms play a predominant role in the regulation of CYP11B2. In accordance with earlier findings, in H295R cells MEK inhibition increased the expression of NR4A1, NR4A2 and CYP11B2 genes, however, it decreased the Ang II-induced gene expression levels, suggesting that ERK activation has a role in control of expression of these genes. No such mechanism was detected in rat glomerulosa cells. Sar-Ile-Ile-AngII, which can cause G protein-independent ERK activation, also stimulated the expression of CYP11B2 in H295R cells. These data suggest that the previously reported CAMK-mediated stimulation of early transcription factors NGFIB and Nurr1 has a predominant role in Ang II-induced CYP11B2 activation in rat adrenal glomerulosa cells, whereas in H295R cells ERK activation and G protein-independent mechanisms also contribute to this process.
PDF
