scholarly journals Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4898
Author(s):  
Aileen Roth ◽  
Adrian Gihring ◽  
Florian Göser ◽  
Christian Peifer ◽  
Uwe Knippschild ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Posttranslational Modifications ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Specific Inhibitor ◽  
Specific Protein ◽  
Highly Sensitive ◽  
Experimental Approaches ◽  
Inhibitory Potential

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.

scholarly journals Substrate specificities of tyrosine-specific protein kinases toward cytoskeletal proteins in vitro.

1986 ◽  
Vol 261 (31) ◽  
pp. 14797-14803 ◽  
Author(s):  
T Akiyama ◽  
T Kadowaki ◽  
E Nishida ◽  
T Kadooka ◽  
H Ogawara ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Specific Protein ◽  
Cytoskeletal Proteins ◽  
Substrate Specificities

scholarly journals Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 71-79 ◽  
Author(s):  
K Ashizawa ◽  
G J Wishart ◽  
S Katayama ◽  
D Takano ◽  
M Maeda ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Acrosome Reaction ◽  
Kinase Inhibitors ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Immunoblot Analysis ◽  
Calpain Inhibitor ◽  
Cytoplasmic Matrix ◽  
Sperm Extract

At the avian body temperature of 40 °C, intact fowl spermatozoa require Ca2+for the initiation of motility and a combination of both Ca2+and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1–100 μmol/l, neither PD 150606 (a Ca2+-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca2+-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca2+and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca2+, as well as motility initiated by calyculin A – a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 °C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.

Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 309-316 ◽  
Author(s):  
Carsten Krischek ◽  
Burkhard Meinecke
Keyword(s):  
Map Kinase ◽  
Protein Kinases ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Histone H1 ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

scholarly journals Protein kinase CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) induces apoptosis and caspase-dependent degradation of haematopoietic lineage cell-specific protein 1 (HS1) in Jurkat cells

2002 ◽  
Vol 364 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Maria RUZZENE ◽  
Daniele PENZO ◽  
Lorenzo A. PINNA
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Specific Inhibitor ◽  
Specific Protein ◽  
Jurkat Cells ◽  
Similar Data ◽  
Caspase Cleavage ◽  
Protein Substrates ◽  
Kinase Ck2

Incubation of Jurkat cells with 4,5,6,7-tetrabromobenzotriazole (TBB), a specific inhibitor of protein kinase CK2, induces dose-and time-dependent apoptosis as judged by several criteria. TBB-promoted apoptosis is preceded by inhibition of Ser/Thr phosphorylation of haematopoietic lineage cell-specific protein 1 (HS1) and is accompanied by caspase-dependent fragmentation of the same protein. Both effects are also observable if apoptosis is promoted by anti-Fas antibodies and by etoposide. Moreover, in vitro experiments show that HS1, once phosphorylated by CK2, becomes refractory to cleavage by caspase-3. These findings, in conjunction with similar data in the literature concerning two other CK2 protein substrates, Bid and Max, suggest that CK2 may play a general anti-apoptotic role through the generation of phosphorylated sites conferring resistance to caspase cleavage.

scholarly journals BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

2006 ◽  
Vol 401 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Gopal P. Sapkota ◽  
Lorna Cummings ◽  
Felicity S. Newell ◽  
Christopher Armstrong ◽  
Jennifer Bain ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Phorbol Ester ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Camp Response Element Binding ◽  
S6 Kinase ◽  
Ribosomal S6 Kinase

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC50 of 10–30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.

Secretion of Gelatinases and Activation of Gelatinase A (MMP-2) by Human Rheumatoid Synovial Fibroblasts

2001 ◽  
Vol 382 (10) ◽  
pp. 1491-1499 ◽  
Author(s):  
H. Smolian ◽  
A. Aurer ◽  
M. Sittinger ◽  
J. Zacher ◽  
J.-P. Bernimoulin ◽  
...  
Keyword(s):  
Map Kinase ◽  
Cell Line ◽  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Human Fibroblasts ◽  
Specific Inhibitor ◽  
Fibroblast Cell ◽  
Synovial Fibroblasts ◽  
P38 Map Kinase ◽  
Gelatinase A

Abstract In monolayer cultures human rheumatoid synovial fibroblasts (HRSF) secrete gelatinase A (MMP-2) and, unlike other human fibroblasts, to a minor extent also gelatinase B (MMP-9) as inactive proenzymes. In this regard HRSF resemble the fibrosarcoma cell line HT-1080. Unlike HT-1080, however, HRSF do not increase the secretion of MMP-9 in response to phorbol-12-myristate-13-acetate. This indicates that in HRSF the protein kinase C pathway for an enhanced MMP-9 secretion is inactive. None of the substances used in our study increased MMP-9 secretion, but some of them inhibited MMP-9 secretion. The secretion of MMP-2 could not be enhanced either, not even by dbcAMP, which has been reported to be effective in Sertoli and peritubular cells. Activation of MMP-2 in HRSF could be induced by treatment with concanavalin A (ConA) or cytochalasin D, as was shown for other cell types. This activation was not accompanied by a significant change in the amount of secreted TIMP-1 and TIMP-2. In contrast to reports on human skin fibroblasts, however, the activation of MMP-2 could not be induced in HRSF by treatment of the cells with monensin or sodium orthovanadate. Moreover, monensin was shown to act as an inhibitor of ConA or cytochalasin Dmediated activation. Additionally, and in contrast to a report on a rat fibroblast cell line, MMP-2 activation is not mediated via the MAP kinase pathway in HRSF: PD 98059, a specific inhibitor of MAP kinase kinase, did not inhibit the activation of MMP-2. Similarly ineffective were PD 169316, an inhibitor for p38 MAP kinase, other inhibitors for protein kinases as lavendustin A, Gö 6983, wortmannin, rapamycin, as well as the protein tyrosine kinase inhibitors herbimycin A and genistein. Only staurosporin, a broad spectrum inhibitor of protein kinases, and the ionophores monensin and A 23187 effectively inhibited MMP-2 activation in HRSF. Our results demonstrate that MMP-2 can be activated by quite different pathways, and that different cells, even when belonging to the fibroblast family, do not necessarily use the same activating pathways.

scholarly journals Site-specific phosphorylation of Huntingtin exon 1 recombinant proteins enabled by the discovery of novel kinases

2020 ◽  
Author(s):  
Anass Chiki ◽  
Jonathan Ricci ◽  
Ramanath Hegde ◽  
Luciano A. Abriata ◽  
Andreas Reif ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Proof Of Concept ◽  
Wild Type ◽  
Expression And Purification ◽  
Sumo Fusion ◽  
Exon 1 ◽  
Health And Disease ◽  
Experimental Approaches ◽  
And Function

AbstractPosttranslational modifications (PTMs) within the first 17 amino acids (Nt17) of exon1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington’s disease (HD). In this manuscript, we describe a new and simple methodology for producing milligram quantities of highly pure wild type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK); and, 2) the development of an efficient methodology for producing recombinant native Httex1 proteins using a SUMO-fusion expression and purification strategy. As proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site- specifically phosphorylated and fluorescently labeled or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 structure, aggregation, interactome and function(s) in health and disease.

scholarly journals Axl Is Essential for in-vitro Angiogenesis Induced by Vitreous From Patients With Proliferative Diabetic Retinopathy

2021 ◽  
Vol 8 ◽  
Author(s):  
Wenyi Wu ◽  
Huizuo Xu ◽  
Zhishang Meng ◽  
Jianxi Zhu ◽  
Siqi Xiong ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Diabetic Retinopathy ◽  
Proliferative Diabetic Retinopathy ◽  
Tyrosine Kinases ◽  
Vascular Endothelial Cells ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Specific Protein ◽  
Vascular Endothelial

Proliferative diabetic retinopathy (PDR), characterized mainly with abnormal epiretinal angiogenesis forming fibrovascular membranes (FVMs), threatens vision of people with diabetes; FVMs consist of extracellular matrix and a variety of cell types including vascular endothelial cells. Axl, one of receptor tyrosine kinases, can be activated indirectly by vascular endothelial growth factor-A (VEGF-A) via an intracellular route for promoting angiogenesis. In this study, we revealed that growth arrest-specific protein 6 (Gas6), a specific ligand of Axl, was elevated in vitreous from patients with PDR and that Axl was activated in FVMs from patients with PDR. In addition, we demonstrated that in cultured human retinal microvascular endothelial cells (HRECs), Axl inhibition via suppression of Axl expression with Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated protein 9 or through inactivation with its specific inhibitor R428 blocked PDR vitreous-induced Akt activation and proliferation of HRECs. Furthermore, PDR vitreous-heightened migration and tube formation of HRECs were also blunted by restraining Axl. These results indicate that in the pathogenesis of PDR, Axl can be activated by Gas6 binding directly and by VEGF-A via an intracellular route indirectly, suggesting that Axl plays a pivotal role in the development of PDR and that Axl inhibition shows a bright promise for PDR therapy.

Targeting Protein Kinase Inhibitors with Traditional Chinese Medicine

2019 ◽  
Vol 20 (15) ◽  
pp. 1505-1516
Author(s):  
Yangyang Zhang ◽  
Minghua Liu ◽  
Jun Wang ◽  
Jianlin Huang ◽  
Mingyue Guo ◽  
...  
Keyword(s):  
Chinese Medicine ◽  
Protein Kinase ◽  
Traditional Chinese Medicine ◽  
Protein Kinases ◽  
Anticancer Agents ◽  
Kinase Inhibitors ◽  
Specific Protein ◽  
Protein Kinase Inhibitors ◽  
Bioactive Substances ◽  
Phosphoinositide 3 Kinase

Protein kinases play critical roles in the control of cell growth, proliferation, migration, and angiogenesis, through their catalytic activity. Over the past years, numerous protein kinase inhibitors have been identified and are being successfully used clinically. Traditional Chinese medicine (TCM) represents a large class of bioactive substances, and some of them display anticancer activity via inhibiting protein kinases signal pathway. Some of the TCM have been used to treat tumors clinically in China for many years. The p38mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase, serine/threonine-specific protein kinases (PI3K/AKT/mTOR), and extracellular signal-regulated kinases (ERK) pathways are considered important signals in cancer cell development. In the present article, the recent progress of TCM that exhibited significant inhibitory activity towards a range of protein kinases is discussed. The clinical efficacy of TCM with inhibitory effects on protein kinases in treating a tumor is also presented. The article also discussed the prospects and problems in the development of anticancer agents with TCM.

scholarly journals SRC family kinases in hamster spermatozoa: evidence for the presence of LCK

Reproduction ◽  
2017 ◽  
Vol 153 (5) ◽  
pp. 655-669 ◽  
Author(s):  
Durgesh Kumar Singh ◽  
Rohit Kumar Deshmukh ◽  
Praveen Kumar Narayanan ◽  
Sisinthy Shivaji ◽  
Archana Bharadwaj Siva
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Specific Inhibitor ◽  
Specific Protein ◽  
Src Family Kinases ◽  
Sperm Capacitation ◽  
Protein Tyrosine

Sperm capacitation is a prerequisite for successful fertilization. Increase in tyrosine phosphorylation is considered the hallmark of capacitation and attempts to understand its regulation are ongoing. In this regard, we attempted to study the role of SRC family kinases (SFKs) in the hamster sperm functions. Interestingly, we found the presence of the lymphocyte-specific protein tyrosine kinase, LCK, in mammalian spermatozoa and further characterized it in terms of its localization and function. LCK was found in spermatozoa of several species, and its transcript was identified in the hamster testis. Autophosphorylation of LCK at the Y394 residue increased as capacitation progressed, indicating an upregulation of LCK activity during capacitation. Inhibition of LCK (and perhaps the other SFKs) with the use of a specific inhibitor showed a significant decrease in protein tyrosine phosphorylation of several proteins, implying LCK/SFKs as key tyrosine kinase(s) regulating tyrosine phosphorylation during hamster sperm capacitation. Dihydrolipoamide dehydrogenase was identified as a substrate for LCK/SFK. LCK/SFKs inhibition significantly reduced the percentage fertilization (in vitro) but had no effect on sperm motility, hyperactivation and acrosome reaction. In summary, this is the first report on the presence of LCK, an SFK of hematopoietic lineage in spermatozoa besides being the first study on the role of SFKs in the spermatozoa of Syrian hamsters.

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