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SGC-GAK-1: A Chemical Probe for Cyclin G Associated Kinase (GAK).

ABSTRACT: We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G-associated kinase (GAK), together with a structurally related negative control SGC-GAK-1N (14). 11 was highly selective in an in vitro kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent RIPK2 inhibitor lacking GAK activity. Together, this chemical probe set can be used to interrogate GAK cellular biology.

SUBMITTER: Asquith CRM

PROVIDER: S-EPMC6567991 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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SGC-GAK-1: A Chemical Probe for Cyclin G Associated Kinase (GAK).

Asquith Christopher R M CRM Berger Benedict-Tilman BT Wan Jing J Bennett James M JM Capuzzi Stephen J SJ Crona Daniel J DJ Drewry David H DH East Michael P MP Elkins Jonathan M JM Fedorov Oleg O Godoi Paulo H PH Hunter Debra M DM Knapp Stefan S Müller Susanne S Torrice Chad D CD Wells Carrow I CI Earp H Shelton HS Willson Timothy M TM Zuercher William J WJ

Journal of medicinal chemistry 20190226 5

We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G-associated kinase (GAK), together with a structurally related negative control SGC-GAK-1N (14). 11 was highly selective in an in vitro kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent RIPK2 inhibitor lacking GAK activity. Together, this chemical probe set can be used to interrogate GAK cellular biology. ...[more]

PMID: 30768268

Similar Datasets

The cyclin G-associated kinase (GAK) inhibitor SGC-GAK-1 inhibits neurite outgrowth and synapse formation.

Project description:Protein kinases are responsible for protein phosphorylation and are involved in important signal transduction pathways; however, a considerable number of poorly characterized kinases may be involved in neuronal development. Here, we considered cyclin G-associated kinase (GAK) as a candidate regulator of neurite outgrowth and synaptogenesis by examining the effects of the selective GAK inhibitor SGC-GAK-1. SGC-GAK-1 treatment of cultured neurons reduced neurite length and decreased synapse number and phosphorylation of neurofilament 200-kDa subunits relative to the control. In addition, the related kinase inhibitor erlotinib, which has distinct specificity and potency from SGC-GAK-1, had no effect on neurite growth, unlike SGC-GAK-1. These results suggest that GAK may be physiologically involved in normal neuronal development, and that decreased GAK function and the resultant impaired neurite outgrowth and synaptogenesis may be related to neurodevelopmental disorders.

| S-EPMC9327206 | biostudies-literature

Towards the Development of an In vivo Chemical Probe for Cyclin G Associated Kinase (GAK).

Project description:SGC-GAK-1 (1) is a potent, selective, cell-active chemical probe for cyclin G-associated kinase (GAK). However, 1 was rapidly metabolized in mouse liver microsomes by cytochrome P450-mediated oxidation, displaying rapid clearance in liver microsomes and in mice, which limited its utility in in vivo studies. Chemical modifications of 1 that improved metabolic stability, generally resulted in decreased GAK potency. The best analog in terms of GAK activity in cells was 6-bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) (IC50 = 1.4 μM), showing improved stability in liver microsomes while still maintaining a narrow spectrum activity across the kinome. As an alternative to scaffold modifications we also explored the use of the broad-spectrum cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) to decrease intrinsic clearance of aminoquinoline GAK inhibitors. Taken together, these approaches point towards the development of an in vivo chemical probe for the dark kinase GAK.

| S-EPMC6891286 | biostudies-literature

Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies.

Project description:GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent 'off-target' of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.

| S-EPMC3957475 | biostudies-literature

Selective Inhibitors of Cyclin G Associated Kinase (GAK) as Anti-Hepatitis C Agents.

Project description:Cyclin G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Cocrystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV life cycle (i.e., viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer, and Parkinson's disease).

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SGC-AAK1-1: A Chemical Probe Targeting AAK1 and BMP2K.

Project description:Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

| S-EPMC7073879 | biostudies-literature

Glomerular microRNA data of the kidney podocyte-specific cyclin-G associated kinase (Gak) deficient mice

Project description:Evidence for reduced expression of cyclin-G associated kinase (GAK) in glomeruli of chronic kidney disease patients was observed in the Nephroseq human database and was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak knockout mice (Gak KO) which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mutant mice to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to this process. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak KO mice. Increased podocyte calpain activity mediated proteolysis of IkB resulted in increased NF-kBp65 induced Gadd45b expression in the Gak KO mice. Our results suggest that loss of podocyte associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.

2020-12-08 | GSE158384 | GEO

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2.

Project description:Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound 9 was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound 16 was designed as a negative control to be used alongside compound 9 in experiments to interrogate CDKL2-mediated biology. A solved cocrystal structure of compound 9 bound to CDKL2 highlighted key interactions it makes within its ATP-binding site. Inhibition of downstream phosphorylation of EB2, a CDKL2 substrate, in rat primary neurons provided evidence that engagement of CDKL2 by compound 9 in cells resulted in inhibition of its activity. When used at relevant concentrations, compound 9 does not impact the viability of rat primary neurons or certain breast cancer cells nor elicit consistent changes in the expression of proteins involved in epithelial-mesenchymal transition.

| S-EPMC11318004 | biostudies-literature

Optimization of Isothiazolo[4,3- b]pyridine-Based Inhibitors of Cyclin G Associated Kinase (GAK) with Broad-Spectrum Antiviral Activity.

Project description:There is an urgent need for strategies to combat dengue and other emerging viral infections. We reported that cyclin G-associated kinase (GAK), a cellular regulator of the clathrin-associated host adaptor proteins AP-1 and AP-2, regulates intracellular trafficking of multiple unrelated RNA viruses during early and late stages of the viral lifecycle. We also reported the discovery of potent, selective GAK inhibitors based on an isothiazolo[4,3- b]pyridine scaffold, albeit with moderate antiviral activity. Here, we describe our efforts leading to the discovery of novel isothiazolo[4,3- b]pyridines that maintain high GAK affinity and selectivity. These compounds demonstrate improved in vitro activity against dengue virus, including in human primary dendritic cells, and efficacy against the unrelated Ebola and chikungunya viruses. Moreover, inhibition of GAK activity was validated as an important mechanism of antiviral action of these compounds. These findings demonstrate the potential utility of a GAK-targeted broad-spectrum approach for combating currently untreatable emerging viral infections.

| S-EPMC6335200 | biostudies-literature

Chemical Genetic Identification of Cyclin-G-associated kinase (GAK) Substrates Reveals its Role in Regulating Na+/K+-ATPase and Sipa1L1 in Neurons

Project description:Recent genetic evidence implicates the serine/threonine kinase cyclin G-associated kinase (GAK) as a Parkinson’s disease risk. However, its role in neuronal function and many downstream effectors remain unclear. Employing a chemical genetics method, we show here that the sodium potassium pump (Na+/K+-ATPase) is a GAK target in the brain. We further show that GAK modulates Na+/K+-ATPase at a novel site affecting both pump localization and function. Whole-cell patch clamp recordings from CA1 pyramidal cells in GAK conditional knockout mice show a larger change in resting membrane potential when exposed to the Na+/K+-ATPase blocker, ouabain, indicating altered Na+/K+-ATPase function in GAK knockouts. Additionally, we show that GAK-deficient neurons have enlarged dendritic spines and we identify the spine associated protein Sipa1L1 (or SPAR) as a GAK target, which may contribute to this effect. Our results reveal novel functions of GAK in neurons.

2019-01-07 | PXD011319 | Pride

Disruption of zebrafish cyclin G-associated kinase (GAK) function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development.

Project description:BackgroundThe J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.ResultsLike mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures.ConclusionIn support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

| S-EPMC2821301 | biostudies-literature

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