Advancements in Protein Kinase Inhibitors: From Discovery to Clinical Applications.

Attempts to understand the physiological roles of the protein kinase inhibitor (PKI) proteins have been hampered by a lack of knowledge concerning the molecular heterogeneity of the PKI family. The PKIgamma cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKIalpha and 30% identity to PKIbeta1. Residues important for the high affinity of PKIalpha and PKIbeta1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKIgamma. Northern blot analysis showed that a 1.3-kilobase PKIgamma message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKIgamma is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKIgamma is the predominant PKI message. Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKIalpha and PKIgamma identified these inhibitory activities to be PKIalpha and PKIgamma. A comparison of inhibitory potencies of PKIalpha and PKIgamma expressed in Escherichia coli revealed that PKIgamma was a potent competitive inhibitor of Calpha phosphotransferase activity in vitro (Ki = 0.44 nM) but is 6-fold less potent than PKIalpha (Ki = 0.073 nM). Like PKIalpha, PKIgamma was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKIgamma gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKIgamma is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.

To evaluate the risk of pleural disorders (PD) associated with 33 protein kinase (PK) inhibitors (PKIs) through a disproportionality analysis and to identify which PKs and pathways are involved in PKI-induced PD. To evaluate the risk of PD, reporting odds ratios (RORs) were calculated for 33 PKIs through data registered in the World Health Organization safety report database (VigiBase). We undertook a literature review to identify PKs that were possibly involved in PD caused by PKIs. Pearson correlation coefficients (r) between RORs and affinity data of 19 PKIs were calculated to identify the cellular target most likely to be involved in PKI-induced PD. A total of 235 110 individual case safety reports were extracted from the database for 33 available PKIs. Among these reports, 5001 concerned PD (2.1%). Significant and positive disproportionality for PD was found for 29 of 33 PKI included in our study with top values for dasatinib [ROR=115.3; 95% confidence interval (CI): 110.1-120.8], bosutinib (ROR=20.4; 95% CI: 15.8-26.4) and ponatinib (ROR=12; 95% CI: 9.2-15.6). Correlation analyses between the product of dissociation constant and ROR highlighted possibly Lyn involvement in PD with PKI (r=0.73, P=0.0004). Our study showed that 28 of the 33 tested PKIs were associated with PD. Besides, the study highlighted the role of Lyn in PD caused by PKIs through an immune-mediated process.

Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.

A historical overview of protein kinases and their targeted small molecule inhibitors

Heart failure (HF) is a complex clinical syndrome marked by impaired contractility, adverse remodeling, and dysregulated intracellular signaling. Protein kinases are central regulators of cardiac function, modulating calcium handling, gene transcription, hypertrophy, and apoptosis through phosphorylation of target proteins. In HF, chronic activation of kinases such as protein kinase A, protein kinase C, calcium/calmodulin-dependent kinase II, mitogen-activated protein kinases, protein kinase B, and Rho-associated protein kinase contributes to progressive cardiac dysfunction. Preclinical studies have demonstrated that selective inhibition of these kinases can reverse maladaptive remodeling, improve contractile performance, and attenuate neurohormonal activation. However, clinical translation remains limited, and most approved kinase inhibitors are oncologic agents associated with cardiotoxicity. This review explores the mechanistic roles of key kinases in HF pathogenesis, the therapeutic potential of kinase inhibitors, and the current landscape of clinical and translational research.

Effects of protein kinase inhibitors on the mitogenic activity of human hepatocyte growth factor on rat hepatocytes in primary culture

Although Fischer's extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cβ subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.

Protein kinases play crucial roles in the regulation of multiple cell signaling pathways and cellular functions. Deregulation of protein kinase function has been implicated in carcinogenesis. The inhibition of protein kinases has emerged as an important target for cancer chemoprevention and therapy. Accumulated data revealed that flavonoids exert chemopreventive effects through acting at protein kinase signaling pathways, more than as conventional hydrogen-donating antioxidants. Recent studies show that flavonoids can bind directly to some protein kinases, including Akt/protein kinase B (Akt/PKB), Fyn, Janus kinase 1 (JAK1), mitogen-activated protein kinase kinase 1 (MEK1), phosphoinositide 3-kinase (PI3K), mitogen-activated protein (MAP) kinase kinase 4 (MKK4), Raf1, and zeta chain-associated 70-kDa protein (ZAP-70) kinase, and then alter their phosphorylation state to regulate multiple cell signaling pathways in carcinogenesis processes. In this review, we report recent results on the interactions of flavonoids and protein kinases, especially their direct binding and molecular modeling. The data suggest that flavonoids act as protein kinase inhibitors for cancer chemoprevention that were thought previously as conventional hydrogen-donating antioxidant. Moreover, the molecular modeling data show some hints for creating natural compound-based protein kinase inhibitors for cancer chemoprevention and therapy.

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions, and one of the roles is as a powerful stimulator of bone resorption. LPS stimulated bone resorption via CD14 in mouse calvaria and was reported to function as a receptor for bacterial LPS complexed with serum proteins. Interleukin-6 (IL-6) is capable of stimulating the differentiation of osteoclasts from their hematopoietic precursors, and LPS elevates IL-6 synthesis in human osteoblastic cells. However, the signaling pathway of LPS-induced IL-6 synthesis in osteoblasts is unknown. In the present study, we could detect the existence of CD14 in human osteoblastic cells by RT-PCR analysis and show that LPS increased IL-6 mRNA and synthesis via CD14 in human osteoblastic cells. In human osteoblasts (SaM-1 cells) treated with 10 microg/ml LPS, increases in IL-6 mRNA and synthesis were inhibited by anti-CD14 antibody (MEM-18), PD98059 (an inhibitor of classic mitogen-activated protein kinase [MAPK]), or SB203580 (an inhibitor of p38 MAPK) but were not inhibited by H-89 (an inhibitor of protein kinase A [PKA]) and calphostin C (an inhibitor of protein kinase C [PKC]). Furthermore, LPS-induced IL-6 synthesis was inhibited by curcumin (an inhibitor of activating protein-1 [AP-1]) but not by pyrrolidine dithiocarbamate (PDTC) (an inhibitor of nuclear factor kappa B [NF-kappaB]). The findings of the present study suggest that the LPS receptor CD14, existent in human osteoblastic cells, and IL-6 synthesis in response to LPS probably occur via CD14, p38 MAPK, and MAP kinase/extracellular-regulated kinase kinase (MEK), leading to the transcriptional activation of AP-1 in human osteoblastic cells.

Forty original sequences of peptide substrates and inhibitors of protein kinases and phosphatases were aligned in a chain matrix without artificial gaps. Fifteen protein kinase peptide substrates and inhibitors (PKSI peptides) contained a common dipeptide ArgArg and also additional important tetra-, tri- and dipeptide homologies. Three further peptide substrates were significantly similar to these peptides but lacked the ArgArg dipeptide. Sequence comparison of individual PKSI peptides revealed probabilistically restricted consensus sequence--PKSI motif--comprising 8 homologous and 13 non-randomly distributed amino acids without considering mutation analysis. This template motif was compared with the consensus sequences of 12 different immunoglobulin domains. In 11 of 12 these domains, the starts of homologous segments were found at nearly the same domain related sites, beginning with serine. A single-triplet mutation of any of the first two triplet bases that encode equally localized amino acids in each of the two sequence sets (PKSI and Ig) revealed additional homologies with the other set. A primary derived motif version composed of 9 homologous and seven non-randomly distributed amino acids was consequently established by its feedback projection into the original sequence sets. This procedure yielded a second preliminary motif version (revised motif) formed by a sequence of 9 homologous amino acids and two non-randomly distributed amino acids. In addition, three shorter oligopeptide motifs called important stereotypes were derived, based on repeated homology between Ig chains and the revised motif. The most extensive similarities in terms of these stereotypes occurred in the CH2 and CH4 domains of Ig peptides, and inhibitors of cAMP dependent protein kinase and protein kinase A. Further comparisons based on a reference sequence set arranged with the aid of feedback projection revealed a lower similarity between variable Ig chains reflected in a decreased number of homologous amino acids. Two final motif versions, FMC and FMV, were found in two different subsets of constant and variable Ig chains, respectively. FMC was composed of seven homologous and one non-randomly distributed amino acids forming the dispersed structure STLR(C)LVSD, whereas 6 homologous and one questionable amino acid constituted FMV. Only CH4 and CH1 domain segments contained all five high-incidence amino acids, which represented a higher level of similarity than homologous amino acids of all preliminary and final motifs. Four such amino acids were present also in three PKSI peptides. All similarities described here occur in domain segments positionally overlapping with the CDR1 region of variable chains. The results are discussed in terms of immunoglobulin evolution, the position of Fc receptor binding sites and degeneration or mutability of the triplets of motif-constituting amino acids.

Protein kinases and phosphatases modulate c-fos expression in rat hepatocytes. Effects of angiotensin II and phorbol myristate acetate.

Involvement of tyrosine-specific protein kinase and protein kinase C in J774A.1 macrophage functions activated by Tinospora cordifolia

During random screening for chondrogenic differentiation inducers, we found that Compound-1, 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-1H-pyrazol-3-yl]benzene-1,3-diol, initiated chondrogenic differentiation of the chondroprogenitor cell line ATDC5. Compound-1 initiated chondrogenic differentiation of the mesenchymal stem cell line C3H10T1/2 in regions where cell aggregates formed and simultaneously inhibited adipogenic differentiation. In C3H10T1/2 cells, Compound-1 increased the expression of Sry-related high-mobility-group box transcription factors L-SOX5, SOX6, and SOX9 (SOX trio) more strongly than bone morphogenic protein (BMP)-2. cAMP-dependent protein kinase (PKA) inhibitors suppressed Compound-1–dependent L-SOX5 and SOX6 up-regulation. PKA inhibitors also suppressed the up-regulation of aggrecan mRNA induced by Compound-1, indicating that increases in L-SOX5 and SOX6 mRNA, in which the PKA pathway participates, are involved in the mechanisms behind the action of Compound-1. On the other hand, the SOX6 and aggrecan gene expression, which were up-regulated by BMP-2, were not affected by the PKA inhibitor. Compound-1 induced chondrogenic differentiation of bone marrow stromal cells and recovered cartilage matrix production by primary chondrocytes, which had been decreased by interleukin-1β. These results show the potential of Compound-1 to be a new cartilage repair agent for inducing chondrogenic differentiation via SOX trio up-regulation.

The effect of protein kinase inhibitors on the activation events of mouse eggs at different ages and the dependence on Ca2+ were studied. Inhibition of protein kinase(s) by staurosporine with concentration over 2 μM first induced the destruction of meiotic spindle and then the nuclear formation in metaphase II eggs, but did not stimulate the cortical granule exocytosis; staurosporine with concentration below 0.2 μM and protein kinase C inhibitor sphingosine at concentrations of 10-100 μM did not activate mouse eggs. The activation of mouse eggs by staurosporine was egg age-dependent, but calcium-independent. The number of nucleoli in the formed nucleus or naked nucleoli in the ooplasm was dose- and egg age-dependent. To further evaluate the nuclear and cytoplasmic activation by staurosporine, interphase eggs were processed for electron microscopy and it was found that most nucleoli were highly condenced like those in the pronucleus, but vacuolated or ring-shaped ones also appeared, which implied the activation of egg genome. In addition, staurosporine also induced the formation of Golgi complexes around the nucleus. The nuclear formation induced by staurosporine was completely overcome by okadaic acid. These results suggest that inhibition of staurosporine-sensitive protein kinase(s) other than protein kinase A and C is responsible for the transition from metaphase to interphase in an egg age-dependent but calcium-independent manner in mouse eggs.

To assess the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the activation of the human neutrophil respiratory burst, we have utilized an ether lipid of the type 1-O-alkyl-2-O-methylglycerol (AMG), recently shown to be an inhibitor of this kinase. AMG-C16 (with an hexadecyl chain at the sn-1 position) was found to inhibit the respiratory burst induced by sub-optimal concentrations of phorbol 12,13-dibutyrate. Respiratory burst activity was recovered by subsequent addition of a supraoptimal dose of phorbol 12-myristate 13-acetate, indicating that in the presence of the inhibitor only the activation of the NADPH:O2 oxidoreductase via protein kinase C is inhibited, but not the oxidoreductase itself. The respiratory burst induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also inhibited in the presence of AMG-C16, the extent of inhibition being dependent on the concentration of fMLP. At the concentrations applied in these studies, AMG-C16 had no effect on cell viability, did not affect the formation of inositol phosphates induced by fMLP, and did not affect the characteristics of the Ca2+ fluxes induced by the same stimulus. In a cell-free assay system, AMG-C16 had no effect on the activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinase but inhibited protein kinase C in a dose-dependent fashion. To characterize the inhibitory action of AMG-C16 on the respiratory burst activity in more detail, we studied protein phosphorylation in relation to respiratory burst activity in neutrophil cytoplasts. We focused on the phosphorylation of the 47-kDa protein, because this protein is functionally associated with the NADPH:O2 oxidoreductase. At suboptimal concentrations of phorbol 12,13-dibutyrate, AMG-C16 inhibited phosphorylation of proteins, including that of the 47-kDa protein. Recovery of protein phosphorylation in parallel to recovery of respiratory burst activity was obtained by addition of increasing doses of phorbol 12,13-dibutyrate. Recovery of respiratory burst activity at intermediate concentrations of fMLP did not result in a proportional increase in 47-kDa protein phosphorylation; phosphorylation of the 47-kDa protein was recovered only at high concentrations of fMLP. From these data we conclude that protein kinase C is involved in the activation of the respiratory burst by phorbol esters and fMLP. However, with fMLP as a stimulus, a second signal seems to be triggered, which is insensitive to AMG-C16.