scholarly journals Small molecule ERK5 kinase inhibitors paradoxically activate ERK5 signalling: be careful what you wish for…

2020 ◽  
Vol 48 (5) ◽  
pp. 1859-1875
Author(s):  
Simon J. Cook ◽  
Julie A. Tucker ◽  
Pamela A. Lochhead
Keyword(s):  
Protein Kinase ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Kinase Domain ◽  
Transactivation Domain ◽  
Nuclear Localisation Signal ◽  
Nuclear Localisation ◽  
Cancer And Inflammation

ERK5 is a protein kinase that also contains a nuclear localisation signal and a transcriptional transactivation domain. Inhibition of ERK5 has therapeutic potential in cancer and inflammation and this has prompted the development of ERK5 kinase inhibitors (ERK5i). However, few ERK5i programmes have taken account of the ERK5 transactivation domain. We have recently shown that the binding of small molecule ERK5i to the ERK5 kinase domain stimulates nuclear localisation and paradoxical activation of its transactivation domain. Other kinase inhibitors paradoxically activate their intended kinase target, in some cases leading to severe physiological consequences highlighting the importance of mitigating these effects. Here, we review the assays used to monitor ERK5 activities (kinase and transcriptional) in cells, the challenges faced in development of small molecule inhibitors to the ERK5 pathway, and classify the molecular mechanisms of paradoxical activation of protein kinases by kinase inhibitors.

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.

scholarly journals New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors

2018 ◽  
Vol 475 (15) ◽  
pp. 2417-2433 ◽  
Author(s):  
Dominic P. Byrne ◽  
Yong Li ◽  
Krithika Ramakrishnan ◽  
Igor L. Barsukov ◽  
Edwin A. Yates ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Heparan Sulfate ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitors ◽  
Fluorescent Substrate ◽  
Raf Kinase ◽  
Shift Analysis

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

scholarly journals Current translational potential and underlying molecular mechanisms of necroptosis

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Tamás Molnár ◽  
Anett Mázló ◽  
Vera Tslaf ◽  
Attila Gábor Szöllősi ◽  
Gabriella Emri ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Immune Surveillance ◽  
Kinase Domain ◽  
Tumor Formation

Abstract Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.

scholarly journals A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency

2020 ◽  
Vol 117 (40) ◽  
pp. 24802-24812 ◽  
Author(s):  
Salima Daou ◽  
Manisha Talukdar ◽  
Jinle Tang ◽  
Beihua Dong ◽  
Shuvojit Banerjee ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Therapeutic Potential ◽  
Protein Kinase Inhibitors ◽  
Response To Treatment ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
In Cells

The oligoadenylate synthetase (OAS)–RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.

scholarly journals Small-Molecule Protein Kinases Inhibitors and the Risk of Fungal Infections

2019 ◽  
Vol 13 (4) ◽  
pp. 229-243 ◽  
Author(s):  
Katie Bechman ◽  
James B Galloway ◽  
Kevin L Winthrop
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Fungal Infections ◽  
Break Point ◽  
Pharmacokinetic Profile ◽  
Protein Kinase Inhibitors ◽  
Janus Kinase ◽  
The Impact

Abstract Purpose of Review This review discusses fungal infections associated with licenced small-molecule protein kinase inhibitors. For each major drug class, the mechanism of action and targeted pathways and the impact on host defence against fungi are described. Recent Findings Protein kinase inhibitors are successfully used in the treatment of malignancies and immune-mediated diseases, targeting signalling pathways for a broad spectrum of cytokines and growth-stimuli. These agents predispose to fungal infections by the suppression of integral components of the adaptive and innate immune response. Summary The greatest risk of fungal infections is seen with bruton tyrosine kinase inhibitors, e.g. ibrutinib. Infections are also reported with agents that target mTOR, Janus kinase and break point cluster (Bcr) gene–Abelson (Abl) tyrosine kinase (BCR-ABL). The type of fungal infection fits mechanistically with the specific pathway targeted. Infections are often disseminated and present soon after the initiation of therapy. The pharmacokinetic profile, possibility of off-target kinase inhibition, and underlying disease pathology contribute to infection risk.

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.

scholarly journals New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

2018 ◽  
Author(s):  
Dominic P Byrne ◽  
Yong Li ◽  
Krithika Ramakrishnan ◽  
Igor L Barsukov ◽  
Edwin A Yates ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Heparan Sulphate ◽  
Protein Kinase Inhibitors ◽  
Fluorescent Substrate ◽  
Shift Analysis ◽  
In Cells

ABSTRACTSulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3’-phosphoadenosine 5’-phosphosulphate) to the 2-Oposition of α-L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compoundsin vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.SUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.

Mutation Mediated Resistance to the Tyrosine Kinase Inhibitors Imatinib, Dasatinib & Nilotinib in Philadelphia Positive Leukaemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4245-4245 ◽  
Author(s):  
Lisa M O’ Connor ◽  
Stephen Langabeer ◽  
Shaun R. McCann ◽  
Eibhlin Conneally
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Molecular Mechanisms ◽  
Second Generation ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Patient Specific ◽  
Abl Kinase ◽  
Clinically Significant

Abstract The Philadelphia chromosome is formed as a result of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the hybrid oncoprotein BCR-ABL. It is observed in over 95% of Chronic Myeloid Leukaemia (CML) and approximately 30% of adult Acute Lymphoblastic Leukaemia (ALL) cases. Imatinib Mesylate (IM), a tyrosine kinase inhibitor that specifically binds BCR-ABL in its inactive conformation has revolutionized therapy for CML and Ph+ ALL. However, resistance develops in a significant proportion of patients and is predominantly mediated by single base-pair substitutions within the BCR-ABL kinase domain leading to changes in the amino acid composition that inhibit IM binding whilst retaining BCR-ABL phosphorylation capacity. Second generation tyrosine kinase inhibitors such as Dasatinib and Nilotinib retain activity in IM-resistant patients due to less stringent binding requirements and represent viable alternatives for IM-resistant patients with a suitable molecular profile. In this study, we undertook to examine the molecular mechanisms underlying IM resistance. A cohort of 40 patients with either primary or acquired resistance or intolerance to IM was identified by persistent high or increasing levels of BCR-ABL transcripts determined by real-time quantitative PCR. An allele-specific PCR screen was used to sensitively detect the clinically significant T315I mutation, which renders patients insensitive to currently available tyrosine kinase inhibitors: five (12.5%) IM resistant/intolerant patients were T315I positive. To further elucidate the molecular mechanisms of mutation induced resistance, the BCR-ABL kinase domain was screened for the presence of a mutation using a sensitive denaturing high performance liquid chromatography (dHPLC) approach. Samples showing evidence of mutation were examined by direct sequencing to identify the mutation(s) present. Kinase domain mutations have been identified in 20 of the 40 (50%) patients examined to date and these include p-loop mutations (M244V, G250E, Q252H), IM-binding domain mutations (T315I), catalytic domain mutations (M351T), an activation-loop mutation (L387M). Three previously unreported mutations were identified in patients with indications of IM resistance (T267A, E275Q) and Nilotinib resistance (L273M). The L273 residue lies adjacent to a region of the BCR-ABL kinase domain bound by Nilotinib. Three patients were found to harbour mutations at two distinct kinase domain residues while one patient harboured mutations at three distinct residues, supporting the theory that patients who develop mutation-mediated resistance to one kinase inhibitor may become resistant to subsequent inhibitors by a similar mechanism. The identification of clinically significant mutations facilitates selection of alternative approaches to therapy such as dose escalation of IM, second generation tyrosine kinase inhibitors or allogeneic stem cell transplant, if eligible, at an early stage in a patient’s disease, tailoring patient specific approaches to therapy.

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