scholarly journals Structural Analysis of Inhibitor Binding to CAMKK1 Identifies Features Necessary for Design of Specific Inhibitors

2018 ◽  
Author(s):  
André da Silva Santiago ◽  
Rafael M. Couñago ◽  
Priscila Zonzini Ramos ◽  
Paulo H. C. Godoi ◽  
Katlin B. Massirer ◽  
...  
Keyword(s):  
Neural Development ◽  
Kinase Inhibitor ◽  
Binding Energies ◽  
Titration Calorimetry ◽  
Inhibitor Binding ◽  
High Sequence Identity ◽  
Calcium Calmodulin Dependent ◽  
Competitive Inhibitors ◽  
Dependent Protein ◽  
Specific Inhibitors

The calcium/calmodulin-dependent protein kinases (CAMKKs) are upstream activators of CAMK1 and CAMK4 signalling and have important functions in neural development, maintenance and signalling, as well as in other aspects of biology such as Ca2+ signalling in the cardiovascular system. To support the development of specific inhibitors of CAMKKs we have determined the crystal structure of CAMKK1 with two ATP-competitive inhibitors. The structures reveal small but exploitable differences between CAMKK1 and CAMKK2, despite the high sequence identity, which could be used in the generation of specific inhibitors. Screening of a kinase inhibitor library revealed molecules that bind potently to CAMKK1. Isothermal titration calorimetry revealed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy. Together, the data provide a foundation for future inhibitor development activities.

scholarly journals Binding and structural analyses of potent inhibitors of the human Ca2+/calmodulin dependent protein kinase kinase 2 (CAMKK2) identified from a collection of commercially-available kinase inhibitors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gerson S. Profeta ◽  
Caio V. dos Reis ◽  
André da S. Santiago ◽  
Paulo H. C. Godoi ◽  
Angela M. Fala ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Titration Calorimetry ◽  
Dependent Protein Kinase ◽  
Regulatory Pathways ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
Protein Kinase Kinase

Abstract Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.

Are tyrosine kinases involved in mediating contraction-stimulated muscle glucose transport?

2006 ◽  
Vol 290 (1) ◽  
pp. E123-E128 ◽  
Author(s):  
David C. Wright ◽  
Paige C. Geiger ◽  
Dong-Ho Han ◽  
John O. Holloszy
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Tyrosine Kinase ◽  
Glucose Transport ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Muscle Contractions ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Muscle contractions and insulin stimulate glucose transport into muscle by separate pathways. The contraction-mediated increase in glucose transport is mediated by two mechanisms, one involves the activation of 5′-AMP-activated protein kinase (AMPK) and the other involves the activation of calcium/calmodulin-dependent protein kinase II (CAMKII). The steps leading from the activation of AMPK and CAMKII to the translocation of GLUT4 to the cell surface have not been identified. Studies with the use of the tyrosine kinase inhibitor genistein suggest that one or more tyrosine kinases could be involved in contraction-stimulated glucose transport. The purpose of the present study was to determine the involvement of tyrosine kinases in contraction-stimulated glucose transport in rat soleus and epitrochlearis muscles. Contraction-stimulated glucose transport was completely prevented by pretreatment with genistein (100 μM) and the related compound butein (100 μM). However, the structurally distinct tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4-d]pyridine and herbimycin did not reduce contraction-stimulated glucose transport. Furthermore, genistein and butein inhibited glucose transport even when muscles were exposed to these compounds after being stimulated to contract. Muscle contractions did not result in increases in tyrosine phosphorylation of proteins such as proline-rich tyrosine kinase and SRC. These results provide evidence that tyrosine kinases do not mediate contraction-stimulated glucose transport and that the inhibitory effects of genistein on glucose transport result from direct inhibition of the glucose transporters at the cell surface.

scholarly journals Effects of Specific Inhibitors for CaMK1D on a Primary Neuron Model for Alzheimer’s Disease

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7669
Author(s):  
Paige Grant ◽  
Jitendra Kumar ◽  
Satyabrata Kar ◽  
Michael Overduin
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Death ◽  
Cortical Neurons ◽  
Kinase Inhibitors ◽  
Tau Hyperphosphorylation ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Molecular Components ◽  
Specific Inhibitors

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. Despite extensive research and targeting of the main molecular components of the disease, beta-amyloid (Aβ) and tau, there are currently no treatments that alter the progression of the disease. Here, we examine the effects of two specific kinase inhibitors for calcium/calmodulin-dependent protein kinase type 1D (CaMK1D) on Aβ-mediated toxicity, using mouse primary cortical neurons. Tau hyperphosphorylation and cell death were used as AD indicators. These specific inhibitors were found to prevent Aβ induced tau hyperphosphorylation in culture, but were not able to protect cells from Aβ induced toxicity. While inhibitors were able to alter AD pathology in cell culture, they were insufficient to prevent cell death. With further research and development, these inhibitors could contribute to a multi-drug strategy to combat AD.

Exploring the Stability of Inhibitor Binding to SIK2 Using Molecular Dynamics Simulation and Binding Free Energy Calculation

2021 ◽  
Author(s):  
Mingsong Shi ◽  
Min Zhao ◽  
Lun Wang ◽  
Kongjun Liu ◽  
Penghui Li ◽  
...  
Keyword(s):  
Binding Free Energy ◽  
Dynamics Simulation ◽  
Free Energy Calculation ◽  
Energy Calculation ◽  
Inhibitor Binding ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Biological Phenomena ◽  
Dependent Protein ◽  
The Stability

Salt inducible kinase 2 (SIK2) is a calcium/calmodulin-dependent protein kinase-like kinase that is implicated in a variety of biological phenomena, including cellular metabolism, growth, and apoptosis. SIK2 is the key...

scholarly journals A Purine Analog Kinase Inhibitor, Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor 59, Reveals a Role for Calcium/Calmodulin-Dependent Protein Kinase II in Insulin-Stimulated Glucose Transport

Endocrinology ◽  
2007 ◽  
Vol 148 (1) ◽  
pp. 374-385 ◽  
Author(s):  
Nicky Konstantopoulos ◽  
Seb Marcuccio ◽  
Stella Kyi ◽  
Violet Stoichevska ◽  
Laura A. Castelli ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Kinase Inhibitor ◽  
Glut4 Translocation ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein

Olomoucine is known as a cyclin-dependent kinase inhibitor. We found that olomoucine blocked insulin’s ability to stimulate glucose transport. It did so without affecting the activity of known insulin signaling proteins. To identify the olomoucine-sensitive kinase(s), we prepared analogs that could be immobilized to an affinity resin to isolate binding proteins. One of the generated analogs inhibited insulin-stimulated glucose uptake with increased sensitivity compared with olomoucine. The IC50 for inhibition of insulin-stimulated glucose uptake occurred at analog concentrations as low as 0.1 μm. To identify proteins binding to the analog, [35S]-labeled cell lysates prepared from 3T3-L1 adipocytes were incubated with analog chemically cross-linked to a resin support and binding proteins analyzed by SDS-PAGE. The major binding species was a doublet at 50–60 kDa, which was identified as calcium/calmodulin-dependent protein kinase II (CaMKII) by N-terminal peptide analysis and confirmed by matrix-assisted laser desorption ionization-mass spectrometry as the δ- and β-like isoforms. To investigate CaMKII involvement in insulin-stimulated glucose uptake, 3T3-L1 adipocytes were infected with retrovirus encoding green fluorescent protein (GFP)-hemagluttinin tag (HA)-tagged CaMKII wild-type or the ATP binding mutant, K42M. GFP-HA-CaMKII K42M cells had less kinase activity than cells expressing wild-type GFP-HA-CaMKII. Insulin-stimulated glucose transport was significantly decreased (∼80%) in GFP-HA-CaMKII K42M cells, compared with nontransfected cells, and cells expressing either GFP-HA-CaMKII or GFP-HA. There was not a concomitant decrease in insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII K42M cells when compared with GFP-HA alone. However, insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII cells was significantly higher, compared with either GFP-HA or GFP-HA-CaMKII K42M cells. Our results implicate the involvement of CaMKII in glucose transport in a permissive role.

scholarly journals Structural and binding studies of human CAMKK2 kinase domain bound to small molecule ligands

2019 ◽  
Author(s):  
Gerson S. Profeta ◽  
Caio V. dos Reis ◽  
Paulo H. C. Godoi ◽  
Angela M. Fala ◽  
Roger Sartori ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Titration Calorimetry ◽  
Binding Studies ◽  
Regulatory Pathways ◽  
Calcium Calmodulin Dependent ◽  
Small Molecule Ligands ◽  
Dependent Protein

AbstractCalcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no specific small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homologue, CAMKK1, are thought to have overlapping biological roles. Here we present six novel co-structures of CAMKK2 bound to potent ligands identified from a library of ATP-competitive kinase inhibitors. Isothermal titration calorimetry (ITC) revealed that binding to some of these molecules is enthalpy driven. We expect our results to further advance current efforts to discover small molecule kinase inhibitors specific to each human CAMKK.

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