Search

Ruxolitinib (RUX) is a kinase inhibitor used in the treatment of various medical conditions and its mechanism of action involves suppression of the immune system. While beneficial in treatment of polycythemia vera, myelofibrosis and other indications, it can also increase a patient’s susceptibility to various infections, including bacterial, viral and fungal. We present a case of a patient being treated with RUX who presented with a disseminated fungal infection. This case emphasises the need for vigilance of endemic fungal infections in individuals who are on RUX therapy.

After injury to the central nervous system (CNS), both neuron-intrinsic limitations on regenerative responses and inhibitory factors in the injured CNS environment restrict regenerative axon growth. Instances of successful axon regrowth offer opportunities to identify features that differentiate these situations from that of the normal adult CNS. One such opportunity is provided by the kinase inhibitor RO48, which dramatically enhances neurite outgrowth of neurons in vitro and substantially increased contralateral sprouting of corticospinal tract neurons when infused intraventricularly following unilateral pyramidotomy. The authors present here a transcriptomic deconvolution of RO48-associated axon growth, with the goal of identifying transcriptional regulators associated with axon growth in the CNS. Through the use of RNA sequencing (RNA-seq) and transcription factor binding site enrichment analysis, the authors identified a list of transcription factors putatively driving differential gene expression during RO48-induced neurite outgrowth of rat hippocampal neurons in vitro. The 82 transcription factor motifs identified in this way included some with known association to axon growth regulation, such as Jun, Klf4, Myc, Atf4, Stat3, and Nfatc2, and many with no known association to axon growth. A phenotypic loss-of-function screen was carried out to evaluate these transcription factors for their roles in neurite outgrowth; this screen identified several potential outgrowth regulators. Subsequent validation suggests that the Forkhead box (Fox) family transcription factor Foxp2 restricts neurite outgrowth, while FoxO subfamily members Foxo1 and Foxo3a promote neurite outgrowth. The authors' combined transcriptomic-phenotypic screening strategy therefore allowed identification of novel transcriptional regulators of neurite outgrowth downstream of a multitarget kinase inhibitor.

Synthetic lethality is a proven effective antitumor strategy that has attracted great attention. Large-scale screening has revealed many synthetic lethal genetic phenotypes, and relevant small-molecule drugs have also been implemented in clinical practice. Increasing evidence suggests that CDKs, constituting a kinase family predominantly involved in cell cycle control, are synthetic lethal factors when combined with certain oncogenes, such as MYC, TP53, and RAS, which facilitate numerous antitumor treatment options based on CDK-related synthetic lethality. In this review, we focus on the synthetic lethal phenotype and mechanism related to CDKs and summarize the preclinical and clinical discoveries of CDK inhibitors to explore the prospect of CDK inhibitors as antitumor compounds for strategic synthesis lethality in the future., Graphical abstract This review summarizes the CDKs-related synthetic lethality cases and the mechanisms, giving a guidance for drug combination antitumor strategies and tumor prediction by specific biomarkers.Image 1

A novel, specific and precise RP-HPLC method has been developed and validated for the quantification of selpercatinib in pure and its pharmaceutical dosage form. Selpercatinib is a kinase interrupter with increased selectivity for rearranged during transfection (RET) tyrosine kinase receptors (RTKs) above the additional RTK classes. It is used for the treatment of RET fusion-positive non-small cell lung carcinoma (NSCLC). The segregation was accomplished on the Zorbax C18 column (150 x 4.6 mm) with a 5 µ particle size. 0.1% orthophosphoric acid and Acetonitrile (ACN) (60:40 v/v) was used as an optimized mobile phase at a flow rate of 1ml/min. The wavelength selected was 220.0nm. The retention time for selpercatinib was 2.653 min. The linearity of selpercatinib was detected to be 5- 30μg/ml. Linearity equations obtained for selpercatinib was y = 18428x + 2196.2 with correlation coefficient 0.999. The % RSD for precision was found to be less than 2%. %Recovery was obtained as 99.74% for selpercatinib. The LOD and LOQ for selpercatinib were obtained as 0.02 µg/ml and 0.05 µg/ml.

Introduction Despite recent advances in the drug discovery field, developing selective kinase inhibitors remains a complicated issue for a number of reasons, one of which is that there are striking structural similarities in the ATP-binding pockets of kinases. Objective To address this problem, we have designed a machine learning model utilizing various structure-based and energy-based descriptors to better characterize protein-ligand interactions. Methods In this work, we use a dataset of 104 human kinases with available PDB structures and experimental activity data against 1202 small-molecule compounds from the PubChem BioAssay dataset "Navigating the Kinome". We propose structure-based interaction descriptors to build activity predicting machine learning model. Results and discussion We report a ligand-oriented computational method for accurate kinase target prioritizing. Our method shows high accuracy compared to similar structure-based activity prediction methods, and more importantly shows the same prediction accuracy when tested on the special set of structurally remote compounds, showing that it is unbiased to ligand structural similarity in the training set data. We hope that our approach will be useful for the development of novel highly selective kinase inhibitors.

Natural Products (NP), specifically from medicinal plants or herbs, have been extensively utilized to analyze the fundamental mechanisms of ultimate natural sciences as well as therapeutics. Isolation of secondary metabolites from these sources and their respective biological properties, along with their lower toxicities and cost-effectiveness, make them a significant research focus for drug discovery. In recent times, there has been a considerable focus on isolating new chemical entities from natural flora to meet the immense demand for kinase modulators, and also to overcome major unmet medical challenges in relation to signal transduction pathways. The signal transduction systems are amongst the foremost pathways involved in the maintenance of life and protein kinases play an imperative part in these signaling pathways. It is important to find a kinase inhibitor, as it can be used not only to study cell biology but can also be used as a drug candidate for cancer and metabolic disorders. A number of plant extracts and their isolated secondary metabolites such as flavonoids, phenolics, terpenoids, and alkaloids have exhibited activities against various kinases. In the current review, we have presented a brief overview of some important classes of plant secondary metabolites as kinase modulators. Moreover, a number of phytocompounds with kinase inhibition potential, isolated from different plant species, are also discussed.

The recent emergence of lyn kinase as a driver of aggressive behaviour in triple-negative breast cancer (TNBC) remains a major concern posing a burden for people living with breast cancer and drug development. The binding of UM-164 to lyn protein has been noted to impact the conformational dynamics required for drug fitness. Herein, we provide the first account of the molecular impact of an experimental drug, UM-164 binding on lyn protein using various computational approaches including molecular docking and molecular dynamics simulation. These computational modelling methods enabled us to analyse parameters, for example principal component analysis (PCA), dynamics cross-correlation matrices (DCCM) analysis, hydrogen bond occupancy, thermodynamics calculation and ligand–residue interaction. Findings from these analyses revealed that UM-164 exhibited a higher binding affinity of −9.9 kcal mol−1 with lyn protein than Dasatinib, with a binding affinity of −8.3 kcal mol−1 on docking. It was observed that the binding of UM-164 to lyn protein decreases the capacity of its loop to fluctuate, influences the ligand optimum orientation on the conformational space of lyn protein, and increases the hydrogen bond formation in the lyn-UM-164 system. Also, an increase in drug binding energy of UM-164 was recorded with increasing residue correlation in the lyn-UM-164 system. It is quite informative to note that Met85 was a key stabilising factor in the binding of UM-164 to lyn protein. These findings can provide important insights that will potentially serve as a baseline in the design of novel lyn inhibitors. It could also stimulate further research into multidimensional approaches required to curb the influence of lyn protein in TNBC.

The conformational dynamics of a kinase's activation loop have been challenging to assess due to the activation loop's intrinsic flexibility. To directly probe the conformational equilibrium of the activation loop of mitogen-activated protein kinase p38α, we present an approach based on site-directed spin labeling, electron paramagnetic resonance (EPR) distance restraints, and multilateration. We demonstrate that the activation loop of apo p38α resides in a highly flexible equilibrium state and we reveal that binding of small molecules significantly alters this equilibrium and the populated sub-states. published

Receptor-like cytoplasmic kinase (RLCK) subfamily VII members are involved in diverse biological processes, like reproduction, immunity, growth and development. Ubiquitination and proteasomal degradation of a RLCK VII member, BOTRYTIS-INDUCED KINASE1 (BIK1) play important roles in regulating immune signaling. It remains largely unknown whether most other RLCK VII members undergo ubiquitination and proteasomal degradation. Here, we select the 6-member RLCK VII-4 to examine the potential proteasomal degradation of its members. We find that three closely related RLCK VII-4 members, PBL38 (AvrPphB SUSCEPTIBLE1-LIKE38), PCRK1 (PTI-COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE1), and PCRK2 are under proteasomal control, while the other members in this group are not. Moreover, we demonstrate that PCRK2 undergoes ubiquitination and proteasomal in a kinase activity-dependent manner. However, the plasma membrane (PM) localization of PCRK2 is not required for its degradation. Our work suggests that many other RLCK VII members may undergo ubiquitination and proteasomal degradation to modulate their homeostasis and cellular functions.

While considerable success has been achieved with treating BRAF-V600E mutant tumors in several organ systems, tumors expressing non-V600E BRAF mutations remain a significant clinical challenge. Such atypical BRAF mutations are classified based on their kinase activity, RAS-dependence and their requirement for dimerization to function. We identified an unusually large in-frame deletion in a melanoma patient, encompassing codons 600-618 in the kinase domain. This patient experienced rapid disease progression when treated with dual BRAF/MEK inhibition. To determine whether and how such a large in-frame BRAF deletion might be activating, we ectopically expressed it in HEK293 cells and monitored BRAF pathway activation and response to BRAF and MEK inhibitors. We demonstrate that this mutation results in a kinase-deficient BRAF mutant which, nevertheless, activates MEK/ERK signaling in a dimerization-dependent manner, placing this mutation among the class III BRAF mutants.

KRAS is one of the most important proto-oncogenes. Its mutations occur in almost all tumor types, and KRAS mutant cancer is still lack of effective therapy. Prenyl-binding protein phosphodiesterase-δ (PDEδ) is required for the plasma membrane association and subsequent activation of KRAS oncogenic signaling. Recently, targeting PDEδ has provided new promise for KRAS mutant tumors. However, the therapeutic potential of PDEδ inhibition remains obscure. In this study, we explored how PDEδ inhibition was responded in KRAS mutant cancer cells, and identified KRAS mutant subset responsive to PDEδ inhibition. We first performed siRNA screen of KRAS growth dependency of a small panel of human cancer lines, and identified a subset of KRAS mutant cancer cells that were highly dependent on KRAS signaling. Among these cells, only a fraction of KRAS-dependent cells responded to PDEδ depletion, though KRAS plasma membrane association was effectively impaired. We revealed that the persistent RAF/MEK/ERK signaling seemed responsible for the lack of response to PDEδ depletion. A kinase array further identified that the feedback activation of EPH receptor A2 (EPHA2) accounted for the compensatory activation of RAF/MEK/ERK signaling in these cells. Simultaneous inhibition of EPHA2 and PDEδ led to the growth inhibition of KRAS mutant cancer cells. Together, this study gains a better understanding of PDEδ-targeted therapeutic strategy and suggests the combined inhibition of EPHA2 and PDEδ as a potential therapy for KRAS mutant cancer.

Introduction: Ataxia Telangiectasia (AT) is an autosomal recessive disorder due to mutations in the ATM gene (11q22.3), which encodes a kinase, which initiates and propagates the cellular response ...

In this study, the quality of frozen bull semen was evaluated with the proAKAP4 level test. Sixty straws of frozen bull semen from various batches (n = 30) belonging to six bulls were used in the current study. The frozen bull semen samples were analysed in terms of proAKAP4 levels, sperm morphology and sperm movement parameters at hour 0 and hour 3 after thawing. The semen samples were divided into three groups according to the proAKAP4 levels: low concentration (

Kinases are essential regulators of a variety of cellular signaling processes, including neurite formation – a foundational step in neurodevelopment. Aberrant axonal sprouting and failed regeneration of injured axons are associated with conditions like traumatic injury, neurodegenerative disease, and seizures. Investigating the mechanisms underlying neurite formation will allow for identification of potential therapeutics. We used a kinase inhibitor library to screen 493 kinase inhibitors and observed that 45% impacted neuritogenesis in Neuro2a (N-2a) cells. Based on the screening, we further investigated the roles of Aurora kinases A, B, and C and Nuak kinases 1 and 2. The roles of Aurora and Nuak kinases have not been thoroughly studied in the nervous system. Inhibition or overexpression of Aurora and Nuak kinases in primary cortical neurons resulted in various neuromorphological defects, with Aurora A regulating neurite initiation, Aurora B and C regulating neurite initiation and elongation, all Aurora kinases regulating arborization, and all Nuak kinases regulating neurite initiation and elongation and arborization. Our high-throughput screening and analysis of Aurora and Nuak kinases revealed their functions and may contribute to the identification of therapeutics.

Protein kinases exhibit significant structural diversity, primarily in the conformation of the activation loop and adjacent components of the active site. We previously performed a clustering of the conformation of the activation loop of all protein kinase structures in the Protein Data Bank (PDB) (Modi and Dunbrack, PNAS, 116:6818-6827, 2019) into 8 classes based on the location of the Phe side chain of the DFG motif at the N- terminus of the activation loop. This is determined with a distance metric that measures the difference in the dihedral angles that determine the placement of the Phe side chains (the φ,ψ of X, D, and F of the X-DFG motif and the χ1 of the Phe side chain). The nomenclature is based on the regions of the Ramachandran map occupied by the XDF residues and the χ1 rotamer of the Phe residue. All active structures are "BLAminus", while the most common inactive DFGin conformations are "BLBplus" and "ABAminus." Type 2 inhibitors bind almost exclusively to the DFGout "BBAminus" conformation. In this paper, we present Kincore (http://dunbrack.fccc.edu/kincore), a web resource providing access to the conformational assignments based on our clustering along with labels for ligand types (Type 1, Type 2, etc.) bound to each kinase chain in the PDB. The data are annotated with several properties including PDBid, Uniprotid, gene, protein name, phylogenetic group, spatial and dihedral labels for orientation of DFGmotif residues, C-helix disposition, ligand name and type. The user can browse and query the database using these attributes individually or perform advanced search using a combination of them like a phylogenetic group with specific conformational label and ligand type. The user can also upload a structure and determine its spatial and dihedral labels using the web server and or use a freely available standalone program. The entire database can be downloaded as text files and structure files in PyMOL sessions and mmCIF format. We believe that Kincore will help in understanding conformational dynamics of these proteins and guide development of inhibitors targeting specific states.

A moving target Abl kinase is an important signaling protein that is dysregulated in leukemia and other cancers and is the target of inhibitors such as imatinib. Like other kinases, Abl kinase is dynamic, and regulating conformational dynamics is key to regulating activity. Xie et al. used nuclear magnetic resonance to show that the Abl kinase domain interconverts between one active and two inactive states. Imatinib stabilizes an inactive conformation, and several resistance mutations act by destabilizing this conformation. In a construct that includes the regulatory domain, depending on the relative arrangement of the kinase and regulatory domains, the kinase domain is stabilized in either the active state or one of the inhibited states. Understanding the conformational dynamics of kinases can be leveraged to design selective drugs. Science , this issue p. eabc2754

[This corrects the article DOI: 10.1371/journal.pbio.3000329.].

Aims: Design of sulfonamide-triazine derivatives as JAK1 inhibitors. Background: JAK1 is a kinase involved in different autoimmune diseases. JAK1 inhibitors have shown promising results in treating autoimmune diseases. Objectives: To design new JAK1 inhibitors based on sulfonamides-triazine conjugates capable of binding interactions comparable to observed interactions anchoring potent crystallographic JAK1 inhibitors. Methods: The crystallographic structures of 4 diverse nanomolar inhibitors complexed within JAK1 were used to guide the synthesis of new diaminotriazine-sulfonamide-based JAK1 inhibitors. Results: Nineteen compounds have been prepared some of which exhibited low micromolar IC50 values against JAK1. Conclusions: Crystallographic complexes of diverse JAK1 inhibitors were successfully used to guide the synthesis of novel sulfonamide-triazine-based low micromolar JAK1 inhibitors.

DNA-encoded small molecule libraries (DELs) have enabled discovery of novel inhibitors for many distinct protein targets of therapeutic value through screening of libraries with up to billions of unique small molecules. We demonstrate a new approach applying machine learning to DEL selection data by identifying active molecules from a large commercial collection and a virtual library of easily synthesizable compounds. We train models using only DEL selection data and apply automated or automatable filters with chemist review restricted to the removal of molecules with potential for instability or reactivity. We validate this approach with a large prospective study (nearly 2000 compounds tested) across three diverse protein targets: sEH (a hydrolase), ER{\alpha} (a nuclear receptor), and c-KIT (a kinase). The approach is effective, with an overall hit rate of {\sim}30% at 30 {\textmu}M and discovery of potent compounds (IC50

The Hsp90 chaperone system interacts with a wide spectrum of client proteins, forming variable and dynamic multiprotein complexes that involve the intervention of cochaperone partners. Recent results suggest that the role of Hsp90 complexes is to establish interactions that suppress unwanted client activities, allow clients to be protected from degradation and respond to biochemical signals. Cryo-electron microscopy (cryoEM) provided the first key molecular picture of Hsp90 in complex with a kinase, Cdk4, and a cochaperone, Cdc37. Here, we use a combination of molecular dynamics (MD) simulations and advanced comparative analysis methods to elucidate key aspects of the functional dynamics of the complex, with different nucleotides bound at the N-terminal Domain of Hsp90. The results reveal that nucleotide-dependent structural modulations reverberate in a striking asymmetry of the dynamics of Hsp90 and identify specific patterns of long-range coordination between the nucleotide binding site, the client binding pocket, the cochaperone and the client. Our model establishes a direct atomic-resolution cross-talk between the ATP-binding site, the client region that is to be remodeled and the surfaces of the Cdc37-cochaperone.

The molecular chaperone 90-kDa heat-shock protein (Hsp90) assists the late-stage folding and activation of diverse types of protein substrates (called clients), including many kinases. Previous studies have established that the Hsp90 homodimer undergoes an ATP-driven cycle through open and closed conformations. Here I propose a model of client activation by Hsp90, which predicts that this cycle enables Hsp90 to use ATP energy to drive a kinase client out of thermodynamic equilibrium toward its active conformation. My model is founded on a structural feature of the closed Hsp90 dimer that allows the bound client to transition between two hypothetical intermediate conformations, one folding to the inactive conformation and the other to the active conformation. It suggests that cochaperone Cdc37 aids Hsp90 to activate kinases by differentiating between the deactivating and the activating intermediate conformations. My model makes experimentally testable predictions, such as how modulating the kinetics of different steps in the Hsp90 cycle--for example, by various cochaperones--affects the activation of its clients. It may aid the rational design of therapeutic intervention of Hsp90-mediated protein activation.

Mass spectrometry enables high-throughput screening of phosphoproteins across a broad range of biological contexts. When complemented by computational algorithms, phospho-proteomic data allows the inference of kinase activity, facilitating the identification of dysregulated kinases in various diseases including cancer, Alzheimer’s disease and Parkinson’s disease. To enhance the reliability of kinase activity inference, we present a network-based framework, RoKAI, that integrates various sources of functional information to capture coordinated changes in signaling. Through computational experiments, we show that phosphorylation of sites in the functional neighborhood of a kinase are significantly predictive of its activity. The incorporation of this knowledge in RoKAI consistently enhances the accuracy of kinase activity inference methods while making them more robust to missing annotations and quantifications. This enables the identification of understudied kinases and will likely lead to the development of novel kinase inhibitors for targeted therapy of many diseases. RoKAI is available as web-based tool at http://rokai.io., Kinases drive fundamental changes in cell state, but predicting kinase activity based on substrate-level changes can be challenging. Here the authors introduce a computational framework that utilizes similarities between substrates to robustly infer kinase activity.

Herein we report the discovery of 1-(5-(tert-butyl)isoxazol-3-yl)-3- (3-fluorophenyl)urea derivatives as new FLT3 inhibitors that are able to overcome the drug resistance mutations: the secondary D835Y and F691L mutations on the basis of the internal tandem duplications (ITD) mutation of FLT3 (FLT3-ITD/D835Y and FLT3-ITD/F691L, respectively). The most potent compound corresponds to 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3- fluorophenyl)urea (4d), which showed IC50s (half maximal inhibitory concentrations) of 0.072 nM, 5.86 nM and 3.48 nM against FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y, respectively. Compound 4d also showed good selectivity for FLT3 in a kinase profiling assay. Collectively, 4d could be a good lead compound and deserves further in-depth studies.

Cobimetinib is a kinase inhibitor indicated for use in combination with vemurafenib for treatment of unresectable/metastatic melanoma with specific BRAF mutations. Cobimetinib is extensively metabolized in liver; thus, patients with hepatic impairment (HI) might have increased cobimetinib exposure. In this study, we investigated the impact of HI on the pharmacokinetics (PK) and safety of cobimetinib. Subjects with normal hepatic function and mild to severe HI were enrolled. All subjects received a single oral dose of 10 mg cobimetinib, and serial blood samples were collected at specified times. Cobimetinib PK in subjects with mild and moderate HI was similar to that in those with normal liver function. However, subjects with severe HI, on average, showed ∼30% lower total AUC0‐∞ and ∼2‐fold higher unbound AUC0‐∞ compared with those with normal hepatic function. These exposure differences can be explained by lower albumin levels observed in subjects with severe HI, the strong correlation between albumin level and the unbound fraction and the general PK variability of cobimetinib. In addition, previous studies with cobimetinib showed a lack of an exposure‐response relationship for efficacy and safety. Therefore, collectively, our results suggest that the starting dose for patients with hepatic impairment can be the same as that for those with normal hepatic function.

Summary Protein kinases constitute a large class of signaling molecules frequently targeted in research and clinical uses. However, kinase inhibitors are notoriously non-specific, making it difficult to select an appropriate inhibitor for a given kinase. Available data from large-scale kinase inhibitor screens are often difficult to query. Here, we present KInhibition (https://kinhibition.fredhutch.org), an online portal that allows users to search publicly available datasets to find selective inhibitors for a chosen kinase or group of kinases. Compounds are sorted by a KInhibition Selectivity Score, calculated based on compounds' activity against the selected kinase(s) versus activity against all other kinases for which that compound has been profiled. The current version allows users to query four datasets, with a framework that can easily accommodate additional datasets. KInhibition represents a powerful platform through which researchers from broad areas of biology, chemistry, and pharmacology can easily interrogate large datasets to help guide their selection of kinase inhibitors., Graphical Abstract, Highlights • An easy-to-use tool that compiles datasets from kinase inhibitor screens • A KInhibition Selectivity Score quantifies each compound's selectivity • Users can select kinases, view compounds and selectivity, and download the results • KInhibition is broadly applicable across life science disciplines, Molecular Biology; Bioinformatics; Software Engineering

The pseudokinase group encompasses some 10% of protein kinases, but pseudokinases diverge from canonical kinases in key motifs. The two members of the small new kinase family 3 (NKF3) group are considered pseudokinases. These proteins, pseudopodium-enriched atypical kinase 1 (PEAK1, Sugen kinase 269, or SgK269) and pragmin (Sugen kinase 223 or SgK223), act as scaffolds in growth factor signaling pathways, and both contain a kinase fold with degraded kinase motifs at their C termini. These kinases may harbor regions that mediate oligomerization or control other aspects of signal transduction, but a lack of structural information has precluded detailed investigations into their functional roles. In this study, we determined the X-ray crystal structure of the PEAK1 pseudokinase domain to 2.3 Å resolution. The structure revealed that the PEAK1 kinase-like domain contains a closed nucleotide-binding cleft that in this conformation may deleteriously affect nucleotide binding. Moreover, we found that N- and C-terminal extensions create a highly unusual all α-helical split-dimerization region, termed here the split helical dimerization (SHED) region. Sequence conservation analysis suggested that this region facilitates a dimerization mode that is conserved between PEAK1 and pragmin. Finally, we observed structural similarities between the PEAK1 SHED region and the C-terminal extension of the Parkinson's disease-associated kinase PINK1. In summary, PEAK1's kinase cleft is occluded, and its newly identified SHED region may promote an unexpected dimerization mode. Similarities of PEAK1 with the active kinase PINK1 may reclassify the latter as a member of the new kinase family 3 group.

Kinase inhibitors are promising drugs to stabilize the endothelial barrier following inflammatory damage. However, our limited knowledge of how kinase signaling activates barrier-restorative pathways and the complexity of multi-target drugs have hindered drug discovery and repurposing efforts. Here, we apply a kinase regression approach that exploits drug polypharmacology to investigate endothelial barrier regulation. A screen of 28 kinase inhibitors identified multiple inhibitors that promote endothelial barrier integrity and revealed divergent barrier phenotypes for BCR-ABL drugs. Target deconvolution predicted 50 barrier-regulating kinases from diverse kinase families. Using gene knockdowns, we identified kinases with a role in endothelial barrier regulation and dissected different mechanisms of action of barrier-protective kinase inhibitors. These results demonstrate the importance of polypharmacology in the endothelial barrier phenotype of kinase inhibitors and provide promising new leads for barrier-strengthening therapies.

In this paper, we present the results of a ligand- and structure-based virtual screen targeting LRRK2, a kinase that has been implicated in Parkinson's disease. For the ligand-based virtual screen, the structures of 12 competitor compounds were used as queries for a variety of 2D and 3D searches. The structure-based virtual screen relied on homology models of LRRK2, as no X-ray structure is currently available in the public domain. From the virtual screening, 662 compounds were purchased, of which 35 showed IC50 values below 10μM in wild-type and/or mutant LRRK2 (a hit rate of 5.3%). Of these 35 hits, four were deemed to have potential for medicinal chemistry follow-up.

Morpholine amides are cheap and safe alternative to Weinreb amides as acylating agents of organometallic species. Herein, the in-situ lithiation/borylation of 18 ortho- meta- and para-substituted morpholine benzamides has been investigated. 10 of the 18 substrates provided the desired boronic esters as the major isomer (>90% regioselectivity) in crude isolated yields ranging from 68 to 93%. The synthetic usability of such building blocks was subsequently illustrated via the synthesis of a kinase inhibitor.

ALS-associated mutations in the kinase TBK1 with the same pathological outcome affect distinct signaling pathways.

Significance The discovery that more than 40% of the eukaryotic proteome is intrinsically disordered, and that these disordered segments are enriched in phosphorylation sites, suggests that conformational heterogeneity may be important to kinase selectivity. Indeed, phosphorylation prediction programs reliant on classic notions of conserved sequence information (i.e., “vertical information”) are only partially effective. We find that the conformational equilibrium of the phosphorylatable site, whose information is embedded in sequence-averaged energetic and structural properties of the protein (i.e., “horizontal information”), plays a major role in distinguishing phosphorylatable versus nonphosphorylatable sites. In fact, employing both horizontal and vertical information produces a state-of-the-art phosphorylation predictor, wherein the conformational equilibrium of the disordered chain is the dominant contributor., Phosphorylation sites are hyperabundant in the eukaryotic disordered proteome, suggesting that conformational fluctuations play a major role in determining to what extent a kinase interacts with a particular substrate. In biophysical terms, substrate selectivity may be determined not just by the structural–chemical complementarity between the kinase and its protein substrates but also by the free energy difference between the conformational ensembles that are, or are not, recognized by the kinase. To test this hypothesis, we developed a statistical-thermodynamics-based informatics framework, which allows us to probe for the contribution of equilibrium fluctuations to phosphorylation, as evaluated by the ability to predict Ser/Thr/Tyr phosphorylation sites in the disordered proteome. Essential to this framework is a decomposition of substrate sequence information into two types: vertical information encoding conserved kinase specificity motifs and horizontal information encoding substrate conformational equilibrium that is embedded, but often not apparent, within position-specific conservation patterns. We find not only that conformational fluctuations play a major role but also that they are the dominant contribution to substrate selectivity. In fact, the main substrate classifier distinguishing selectivity is the magnitude of change in local compaction of the disordered chain upon phosphorylation of these mostly singly phosphorylated sites. In addition to providing fundamental insights into the consequences of phosphorylation across the proteome, our approach provides a statistical-thermodynamic strategy for partitioning any sequence-based search into contributions from structural–chemical complementarity and those from changes in conformational equilibrium.

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies on their therapeutic value in the lung EMT process are lacking. Bronchial epithelial (BEAS-2B, primary HAE cells) were exposed to TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers collagen Ӏ (mRNA, protein) were analyzed. St-Ht31 disrupted AKAP-PKA interactions. TGF-β1 release was measured by ELISA. TGF-β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, forskolin were used to elevate intracellular cAMP. TGF-β1 induced morphological changes, decreased E-cadherin but increased collagen Ӏ and cell migration, a process reversed by PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95 and Yotiao. St-Ht31 decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented cell migration and reduced E-cadherin expression, a process blocked by TGF-β1 neutralizing antibody. Silencing of Ezrin, AKAP95 and Yotiao diminished TGF-β1-induced collagen Ӏ expression, as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95 and Yotiao promote TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members define the ability of fenoterol, rolipram and cilostamide to modulate the EMT process, and are potential relevant targets in the treatment of COPD.

Ryanodine Receptors are Ca2+ release channels expressed in the Endoplasmic and Sarcoplasmic Reticulum membranes. Gong et al [1] reported cryo-EM structures of the cardiac RyR2 complexed to Calmodulin, which can downregulate channel opening. Haji-Ghassemi et al [2] report crystal structures of an RyR2 domain with PKA, a kinase promoting channel opening.

The protein p62/Sequestosome 1 (p62) has been described as a selective autophagy receptor and independently as a platform for pro-inflammatory and other intracellular signaling. How these seemingly disparate functional roles of p62 are coordinated has not been resolved. Here, we show that TAK1, a kinase involved in immune signaling, negatively regulates p62 action in autophagy. TAK1 reduces p62 localization to autophagosomes, dampening the autophagic degradation of both p62 and p62-directed autophagy substrates. TAK1 also relocalizes p62 into dynamic cytoplasmic bodies, a phenomenon that accompanies the stabilization of TAK1 complex components. On the other hand, p62 facilitates the assembly and activation of TAK1 complexes, suggesting a connection between p62's signaling functions and p62 body formation. Thus, TAK1 governs p62 action, switching it from an autophagy receptor to a signaling platform. This ability of TAK1 to disable p62 as an autophagy receptor may allow certain autophagic substrates to accumulate when needed for cellular functions.

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 &le, 10 &micro, M confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition.

Biological signalling networks provide decision making and control capabilities for cells as they interact with changing physiological and environmental conditions. Studies of these signalling networks have revealed that they are greatly inter-dependent, which can make isolated analysis of their behaviour challenging. An accurate understanding of the interdependence of these systems will thus be of great utility to synthetic biologists as they attempt to engineer control systems within living cells. Cross-talk, which arises when the response of one signalling pathway can directly influence that of another, should either be considered explicitly when designing new signalling networks or controlled to ensure its effects are minimised. In this paper we consider a simple one-kinase two-phosphatase system and investigate potential control schemes that can reduce the impact of cross-talk upon its behaviour, for example by acting to selectively inhibit a kinase enzyme's affinity for one of its target substrates. We demonstrate that these schemes can be used to reduce the impact of crosstalk, and that a closed-loop control architecture provides better performance over a larger range of parameter values when compared to an open-loop equivalent.

STK38, a reader of histone H4 ufmylation, regulates ATM activation., The ATM (ataxia-telangiectasia mutated) kinase is rapidly activated following DNA damage and phosphorylates its downstream targets to launch DDR signaling. Recently, we and others showed that UFM1 signaling promotes ATM activation. We further discovered that monoufmylation of histone H4 at Lys31 by UFM1-specific ligase 1 (UFL1) is an important step in the amplification of ATM activation. However, how monoufmylated H4 enhances ATM activation is still unknown. Here, we report STK38, a kinase in the Hippo pathway, serves as a reader for histone H4 ufmylation to promote ATM activation in a kinase-independent manner. STK38 contains a potential UFM1 binding motif which recognizes ufmylated H4 and recruits the SUV39H1 to the double-strand breaks, resulting in H3K9 trimethylation and Tip60 activation to promote ATM activation. Together, STK38 is a previously unknown player in DNA damage signaling and functions as a reader of monoufmylated H4 at Lys31 to promote ATM activation.

Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a ‘hybrid’ molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity., Author summary Effective and safe treatment of multigenic diseases often involves drugs that address multiple points along disease networks, i.e., polypharmacology. Polypharmacology is increasingly appreciated as a potentially desirable property of kinase drugs. However, most known drugs that interact with multiple targets have been identified as such by chance and most polypharmacological compounds are not chemically unique, resembling structures of known kinase inhibitors. The fruit fly Drosophila provides an inexpensive, rapid, quantitative, whole animal screening platform that has the potential to complement computational approaches. We present a chemical genetics approach that efficiently combines Drosophila with structural prediction and virtual screening, creating a unique discovery platform. We demonstrate the utility of our approach by developing useful small molecules targeting a kinase network in a Drosophila model of Medullary Thyroid Cancer (MTC) driven by oncogenic dRetM955T.

International audience; No abstract available

How living systems break symmetry in an organized manner is a fundamental question in biology. In wild-type Caenorhabditis elegans zygotes, symmetry breaking during anterior-posterior axis specification is guided by centrosomes, resulting in anterior-directed cortical flows and a single posterior PAR-2 domain. We uncover that C. elegans zygotes depleted of the Aurora A kinase AIR-1 or lacking centrosomes entirely usually establish two posterior PAR-2 domains, one at each pole. We demonstrate that AIR-1 prevents symmetry breaking early in the cell cycle, whereas centrosomal AIR-1 instructs polarity initiation thereafter. Using triangular microfabricated chambers, we establish that bipolarity of air-1(RNAi) embryos occurs effectively in a cell-shape and curvature-dependent manner. Furthermore, we develop an integrated physical description of symmetry breaking, wherein local PAR-2-dependent weakening of the actin cortex, together with mutual inhibition of anterior and posterior PAR proteins, provides a mechanism for spontaneous symmetry breaking without centrosomes.

Epub ahead of print; OBJECTIVE: GCN2 is a kinase which detects amino acid deficiency and is involved in the control of protein synthesis and energy metabolism. However, the role of hepatic GCN2 in the metabolic adaptations in response to the modulation of dietary protein has been seldom studied. METHODS: Wild-type (WT) and liver GCN2-deficient (KO) mice were fed either a normo-protein diet, a low-protein diet or a high-protein diet for 3 weeks. During this period, body weight, food intake and metabolic parameters were followed. RESULTS: In mice fed normo- and high protein diets, GCN2 pathway in the liver is not activated in WT mice leading to a similar metabolic profile with the one of KO mice. On the contrary, a low protein diet activates GCN2 in WT mice inducing FGF21 secretion. In turn, FGF21 maintains a high level of lipid oxidation leading to a different postprandial oxidation profile compared with KO mice. CONCLUSIONS: Hepatic GCN2 controls FGF21 secretion under a low protein diet, and modulates a whole-body postprandial oxidation profile.

Chemical cytometry, sensitive analytical measurements of single cells, reveals inherent heterogeneity of cells within a population which is masked or averaged out when using bulk analysis techniques. A particular challenge of chemical cytometry is the development of a suitable reporter or probe for the desired measurement. These reporters must be sufficiently specific for measuring the desired process; possess a lifetime long enough to accomplish the measurement; and have the ability to be loaded into single cells. This chapter details our approach to rationally design and improve peptide substrates as reporters of enzyme activity utilizing chemical cytometry. This method details the iterative approach used to design, characterize, and identify a peptidase-resistant peptide reporter which acts as a kinase substrate within intact cells. Small-scale, rationally designed peptide libraries are generated to rapidly and economically screen candidate reporter peptides for substrate suitability and peptidase resistance. Also detailed are strategies to characterize and validate the designed reporters by determining kinetic parameters, intracellular substrate specificity, resistance to degradation by intracellular peptidases, and behavior within lysates and intact cells.

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose “open” or “closed” position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria., The bacterial stressosome is a large nanomachine and a key inducer of stress response. Here, the authors present the cryo-EM structure of the stressosome from the bacterial pathogen Listeria monocytogenes at 3.38 Å resolution and discuss its activation mechanism.

9071 Background: BRG is a kinase inhibitor approved for the treatment of patients (pts) with ALK+ metastatic NSCLC; specific details for BRG use vary by indication and country. We report long-term efficacy and safety results of the Phase 1/2 and Phase 2 (ALTA) trials of BRG. Methods: The Phase 1/2 study was a single-arm, open-label trial (NCT01449461) of BRG 30–300 mg/d in pts with advanced malignancies. ALTA (NCT02094573) randomized pts with CRZ-refractory ALK+ NSCLC to receive BRG at 90 mg qd (arm A) or 180 mg qd with 7-d lead-in at 90 mg (arm B). For the Phase 1/2 study, investigator assessments of confirmed objective response rate (cORR; RECIST v1.1), duration of response (DoR), progression-free survival (PFS), overall survival (OS), and safety in pts with ALK+ NSCLC are reported. The primary endpoint of ALTA was cORR per investigator; secondary endpoints included cORR per independent review committee (IRC), DoR, PFS, and OS. Results: In the Phase 1/2 study, 137 pts received BRG; of these, 79 pts had ALK+ NSCLC (71/79 had prior CRZ; 28/79 received 180 mg qd [7-d lead-in at 90 mg]; 14/79 received 90 mg qd). In ALTA, 222 pts with CRZ-refractory ALK+ NSCLC were randomized (n = 112/110, arm A/B). At the end of the Phase 1/2 study (Feb 18, 2020), with median 27.7 mo follow-up (̃67 mo after last pt enrolled), 4 pts remained on BRG. At the end of ALTA (Feb 27, 2020), with median 19.6/28.3 mo follow-up in arm A/B (̃53 mo after last pt enrolled), 10/17 pts in arm A/B were still on treatment. Table shows efficacy results from final analyses with long-term follow-up. In ALTA, the IRC-assessed intracranial cORR in pts with measurable baseline brain metastases was 50% (13/26) in arm A and 67% (12/18) in arm B; Kaplan-Meier (KM) estimated median intracranial DoR was 9.4 mo (95% CI, 3.7, not reached [NR]) in arm A and 16.6 mo (3.7, NR) in arm B. With long-term follow-up, no new safety signals were identified. Treatment-emergent adverse events led to dose interruption (Phase 1/2: 59%; ALTA arm A/B: 49%/61%), dose reduction (13%; 8%/33%), or discontinuation (10%; 4%/13%). Conclusions: BRG showed sustained long-term activity, PFS, and manageable safety in pts with CRZ-refractory ALK+ NSCLC. The 180 mg/d dose after 7-d lead-in at 90 mg/d led to numerically higher median PFS and OS. Final results are similar to those reported for other approved ALK tyrosine kinase inhibitors in this setting. Clinical trial information: NCT01449461, NCT02094573. [Table: see text]