scholarly journals Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes

2020 ◽  
Author(s):  
benjamin eduful ◽  
Sean O'Byrne ◽  
Louisa Temme ◽  
Christopher R. M. Asquith ◽  
Yi Liang ◽  
...  
Keyword(s):  
Scaffold Hopping ◽  
Chemical Probes ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Calcium Calmodulin Dependent ◽  
Cellular Assays ◽  
Dependent Protein ◽  
Binding Residues ◽  
Protein Kinase Kinase

<div>CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934 (CAMKK2 IC50 = 3 nM), a total of 32 compounds, composed of single ring, 5,6-, and 6,6-fused heteroaromatic cores were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. These compounds were evaluated in vitro in biochemical and cellular assays for CAMKK2 inhibition. Compared to GSK650394 and STO-609, thirteen of our compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had greatly improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs aimed at the identification of CAMKK2 chemical probes and clinical candidates<br></div>

scholarly journals Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes

2020 ◽  
Author(s):  
benjamin eduful ◽  
Sean O'Byrne ◽  
Louisa Temme ◽  
Christopher R. M. Asquith ◽  
Yi Liang ◽  
...  
Keyword(s):  
Scaffold Hopping ◽  
Chemical Probes ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Calcium Calmodulin Dependent ◽  
Cellular Assays ◽  
Dependent Protein ◽  
Binding Residues ◽  
Protein Kinase Kinase

<div>CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934 (CAMKK2 IC50 = 3 nM), a total of 32 compounds, composed of single ring, 5,6-, and 6,6-fused heteroaromatic cores were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. These compounds were evaluated in vitro in biochemical and cellular assays for CAMKK2 inhibition. Compared to GSK650394 and STO-609, thirteen of our compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had greatly improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs aimed at the identification of CAMKK2 chemical probes and clinical candidates<br></div>

scholarly journals TAOK2 is an ER-localized Kinase that Catalyzes the Dynamic Tethering of ER to Microtubules

2021 ◽  
Author(s):  
Kimya Nourbakhsh ◽  
Amy A. Ferreccio ◽  
Matthew J. Bernard ◽  
Smita Yadav
Keyword(s):  
Endoplasmic Reticulum ◽  
Catalytic Activity ◽  
Membrane Dynamics ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
High Affinity ◽  
Amphipathic Helices ◽  
Cellular Assays ◽  
Membrane Spanning

SummaryThe endoplasmic reticulum (ER) depends on extensive association with the microtubule cytoskeleton for its structure, function and mitotic inheritance. The identity of molecular tethers that mediate ER-microtubule coupling, and mechanisms through which dynamic tethering is regulated are poorly understood. Here, we identify, Thousand And One amino acid Kinase 2 (TAOK2) as a pleiotropic protein kinase that mediates tethering of ER to microtubules. We show that TAOK2 is a unique multipass membrane spanning serine/threonine kinase localized in distinct ER domains via four transmembrane and amphipathic helices. Using in vitro and cellular assays, we find that TAOK2 directly binds microtubules with high affinity. We define the minimal TAOK2 determinants that induce ER-microtubule tethering, and delineate the mechanism for its autoregulation. While ER membrane dynamics are increased in TAOK2 knockout cells, the movement of ER along growing microtubule plus-ends is disrupted. We show that ER-microtubule tethering is tightly regulated by catalytic activity of TAOK2 in both interphase and mitotic cells, perturbation of which leads to profound defects in ER morphology and cell division. Our study identifies TAOK2 as an ER-microtubule tether, and reveals a kinase-regulated mechanism for control of ER dynamics critical for cell growth and division.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

scholarly journals Regulation of the tumour suppressor Fbw7α by PKC-dependent phosphorylation and cancer-associated mutations

2010 ◽  
Vol 432 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Joanne Durgan ◽  
Peter J. Parker
Keyword(s):  
Mammalian Cells ◽  
Tumour Suppressor ◽  
Dependent Manner ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Pkc Protein Kinase C ◽  
Specific Regulation ◽  
The Relationship ◽  
Substrate Binding Domain

Fbw7 (F-box WD40 protein 7) is a major tumour suppressor, which mediates the degradation of several potent oncogenes. PKC (protein kinase C) comprises a serine/threonine kinase family that can promote transformation when dysregulated. In the present study, we investigated the relationship between Fbw7 and PKC. Multiple members of the PKC superfamily interact with the substrate-binding domain of Fbw7. However, we find no evidence for Fbw7-mediated degradation of PKC. Instead, we demonstrate that Fbw7 is a novel substrate for PKC. Two residues within the isoform-specific N-terminus of Fbw7α are phosphorylated in a PKC-dependent manner, both in vitro and in mammalian cells (Ser10 and Ser18). Mutational analyses reveal that phosphorylation of Fbw7α at Ser10 can regulate its nuclear localization. Cancer-associated mutations in nearby residues (K11R and the addition of a proline residue at position 16) influence Fbw7α localization in a comparable manner, suggesting that mislocalization of this protein may be of pathological significance. Together these results provide evidence for both physical and functional interactions between the PKC and Fbw7 families, and yield insights into the isoform-specific regulation of Fbw7α.

scholarly journals Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Echinococcus Granulosus ◽  
Calcium Content ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

scholarly journals 1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors

Molecules ◽  
2018 ◽  
Vol 23 (5) ◽  
pp. 1221 ◽  
Author(s):  
Christopher Asquith ◽  
Paulo Godoi ◽  
Rafael Couñago ◽  
Tuomo Laitinen ◽  
John Scott ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Narrow Spectrum ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Protein Kinase Kinase

Increased amount of a 25-kilodalton phosphoprotein after v-mos transfection of CHO cells

1988 ◽  
Vol 8 (11) ◽  
pp. 4685-4691
Author(s):  
J K Mayo ◽  
K E Sampson ◽  
L D Adams ◽  
E R Crumm ◽  
S L Kelly ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cho Cells ◽  
Kinase Activity ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Serine Threonine Kinase ◽  
Dependent Protein Kinase ◽  
Threonine Kinase ◽  
Dependent Protein ◽  
A Cell

We transfected Chinese hamster ovary (CHO) cells with a cloned v-mos gene (pHT25). The mos family of oncogenes has previously been shown to have serine-threonine kinase activity. This kinase activity may be required for oncogenic transformation, although its exact biological role is unknown. We found that the transfected cells had an altered morphology, a slower doubling time, and an apparent increase in the amount of a 25-kilodalton (kDa) phosphoprotein that appeared to be of low abundance. Transfection of CHO cells with a cloned temperature-sensitive mos gene (ts159) led to isolation of a cell line that showed the presence of the 25-kDa phosphoprotein at the permissive but not at the nonpermissive temperature, suggesting a direct relationship between mos activity and the presence of this phosphoprotein. The characteristics of altered morphology and depressed growth rate were reminiscent of changes seen after the activation of the cyclic AMP-dependent protein kinase (PKA) in CHO cells. However, PKA activation did not stimulate phosphorylation of this 25-kDa protein, nor was there a change in total PKA activity in these cells. We suggest that the increased presence of the 25-kDa phosphoprotein is a consequence of the v-mos transfection and that it may be involved in the change of morphology and growth rate seen in the CHO cells. Phosphorylation of this protein may be a useful marker of mos and have some functional importance in the transformation of cells by the v-mos oncogene.

scholarly journals WNK4 Kinase: from structure to physiology

2021 ◽  
Author(s):  
Adrian Rafael Murillo-de-Ozores ◽  
Alejandro Rodriguez-Gama ◽  
Hector Carbajal-Contreras ◽  
Gerardo Gamba ◽  
Maria Castaneda-Bueno
Keyword(s):  
Oxidative Stress ◽  
Mouse Models ◽  
Serine Threonine Kinase ◽  
Gene Encoding ◽  
Threonine Kinase ◽  
Physiological Conditions ◽  
Different Levels ◽  
Familial Hyperkalemic Hypertension

With No Lysine (K) kinase 4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that cause Familial Hyperkalemic Hypertension (FHHt). This disease is mainly driven by increased downstream activation of the Ste20-related Proline Alanine Rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1)-NCC pathway, which increases salt reabsorption in the DCT and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

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