Acquisition of inhibitor-sensitive protein kinases through protein design.

Protein phosphorylation is the major post-translational modification used by eukaryotic cells to control cellular signaling. Protein kinases have emerged as attractive drug targets because heightened protein kinase activity has been associated with several proliferative diseases, most notably cancer and restenosis. Until now, it has been very difficult to confirm the utility of protein kinases as inhibitor targets because very few small molecules that selectively inhibit one particular kinase are known. Discovery of highly specific kinase inhibitors has been slow because the protein family contains approximately 2000 members, all of which share a conserved active site fold. Recent work in several laboratories has sought to circumvent the problem of kinase structural degeneracy by engineering drug sensitivity into Src family tyrosine kinases and mitogen-activated protein kinases through site-directed mutagenesis. By introducing a unique non-naturally occurring amino acid into a conserved region of the enzyme's binding site, a target protein kinase can be rapidly sensitized to a small molecule. Introduction of the engineered kinase into a cell line or animal model should greatly expedite the investigation of protein kinase inhibition as a viable drug treatment. The purpose of this review is to summarize these recent advances in protein kinase drug sensitization.     

Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)⁺IL-23R⁺IL-17⁺ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4⁺ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4⁺ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.Nitric oxide (NO; a product of nitrite reduction or the NO synthases NOS1, NOS2, and NOS3; Culotta and Koshland, 1992), is a pleiotropic regulator of neurotransmission, inflammation, and autoimmunity (Culotta and Koshland, 1992; Bogdan, 1998, 2001; Kolb and Kolb-Bachofen, 1998) implicated both in cancer progression and its immune-mediated elimination (Culotta and Koshland, 1992; Coussens and Werb, 2002; Hussain et al., 2003; Mantovani et al., 2008). In different mouse models, NO has been paradoxically shown to both promote inflammation (Farrell et al., 1992; Boughton-Smith et al., 1993; McCartney-Francis et al., 1993; Weinberg et al., 1994; Hooper et al., 1997) and to suppress autoimmune tissue damage through nonselective suppression of immune cell activation (Bogdan, 2001; Bogdan, 2011), especially at high concentrations (Mahidhara et al., 2003; Thomas et al., 2004; Niedbala et al., 2011). Although previous studies demonstrated a positive impact of NO on the induction of Th1 cells (Niedbala et al., 2002) and forkhead box P3–positive (FoxP3⁺) regulatory T (T reg) cells (Feng et al., 2008) in murine models, the regulation and function of the NO synthase (NOS)–NO system have shown profound differences between mice and humans (Schneemann and Schoedon, 2002, Schneemann and Schoedon, 2007; Fang, 2004), complicating the translation of these findings from mouse models to human disease.In cancer, NOS2-derived NO plays both cytotoxic and immunoregulatory functions (Bogdan, 2001). It can exert distinct effects on different subsets of tumor-infiltrating T cells (TILs), capable of blocking the development of cytotoxic T lymphocytes (CTLs; Bronte et al., 2003), suppressing Th1 and Th2 cytokine production, and modulating the development of FoxP3⁺ T reg cells (Brahmachari and Pahan, 2010; Lee et al., 2011). NOS2-driven NO production is a prominent feature of cancer-associated myeloid-derived suppressor cells (MDSCs; Mazzoni et al., 2002; Kusmartsev et al., 2004; Vuk-Pavlović et al., 2010; Bronte and Zanovello, 2005), which in the human system are characterized by a CD11b⁺CD33⁺HLA-DR^low/neg phenotype consisting of CD14⁺ monocytic (Serafini et al., 2006; Filipazzi et al., 2007; Hoechst et al., 2008; Obermajer et al., 2011) and CD15⁺ granulocytic (Zea et al., 2005; Mandruzzato et al., 2009; Rodriguez et al., 2009) subsets (Dolcetti et al., 2010; Nagaraj and Gabrilovich, 2010).Production of NO in chronic inflammation is supported by IFN-γ and IL-17 (Mazzoni et al., 2002; Miljkovic and Trajkovic, 2004), the cytokines produced by human Th17 cells (Veldhoen et al., 2006; Acosta-Rodriguez et al., 2007a,b; van Beelen et al., 2007; Wilson et al., 2007). Human Th17 cells secrete varying levels of IFN-γ (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; Kryczek et al., 2009; Miyahara et al., 2008; van Beelen et al., 2007; Wilson et al., 2007) and have been implicated both in tumor surveillance and tumor progression (Miyahara et al., 2008; Kryczek et al., 2009; Martin-Orozco and Dong, 2009). Induction of Th17 cells typically involves IL-1β, IL-6, and IL-23 (Bettelli et al., 2006; Acosta-Rodriguez et al., 2007a,b; Ivanov et al., 2006; van Beelen et al., 2007; Veldhoen et al., 2006; Wilson et al., 2007; Zhou et al., 2007), with the additional involvement of TGF-β in most mouse models (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007; Ghoreschi et al., 2010), but not in the human system (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007). IL-1β₁, IL-6, and IL-23 production by monocytes and DCs, and the resulting development of human Th17 cells, can be induced by bacterial products, such as LPS or peptidoglycan (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; van Beelen et al., 2007). However, the mechanisms driving Th17 responses in noninfectious settings, such as autoimmunity or cancer, remain unclear.Here, we report that the development of human Th17 cells from naive, effector, and memory CD4⁺ T cell precursors induced by the previously identified Th17-driving cytokines (IL-1β, IL-6, and IL-23) or by IL-1β/IL-6/IL-23-producing MDSCs, is promoted by exogenous NO (or NO produced by human MDSCs) and critically depends on the induction of endogenous NOS2 in differentiating CD4⁺ T cells.     

The soluble guanylate cyclase stimulator BAY 41-2272 inhibits vascular smooth muscle growth through the cAMP-dependent protein kinase and cGMP-dependent protein kinase pathways

Vascular smooth muscle (VSM) proliferation and migration are key components in vessel remodeling. Cyclic nucleotide signaling is protective and has long-served as a therapeutic target against undesired VSM growth. The present work analyzed the effects of the soluble guanylate cyclase (sGC) stimulator 3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine [BAY 41-2272 (BAY)] on VSM growth, and we hypothesize that BAY has the capacity to reduce proliferation and migration via cyclic nucleotide-driven kinase signaling. Perivascular BAY postballoon injury reduced neointimal growth by ～ 40% compared with vehicle controls after 2 weeks. In VSM cells, BAY (10 μM) reduced proliferation by ～ 40% after 72 h and migration by ～ 40% after 6 h and ～ 60% after 18 h without deleterious effects on cell viability. cGMP content peaked (248 ×) 20 min after BAY treatment and remained elevated (140 ×) through 60 min; however, BAY did not affect cAMP levels compared with controls. Conventional and In-Cell Western analyses showed increases in vasodilator-stimulated phosphoprotein (VASP) phosphorylation (pVASP) at serines 239 (3 ×) and 157 (2 ×), respective markers of cGMP- and cAMP-directed protein kinases (PKG and PKA, respectively). The PKG inhibitor YGRKKRRQRRRPPLRKKKKKH peptide (DT-2) completely reversed BAY-mediated increases in pVASPSer(239) and BAY-mediated inhibition of migration. In comparison, the PKA inhibitor peptide PKI further potentiated BAY-stimulated pVASPSer(157) and pVASPSer(239) and partially reversed the antiproliferative effects of BAY. This is the first report demonstrating the effectiveness of BAY in reducing neointimal growth with direct evidence for PKG-specific antimigratory and PKA-specific antiproliferative mechanisms. Conclusively, the sGC stimulator BAY reduces VSM growth through cGMP-dependent PKG and PKA processes, providing support for continued evaluation of its clinical utility.     

Cyclic nucleotide-dependent protein kinases.

The actions of several hormones and neurotransmitters evoke signal transduction pathways which rapidly elevate the cytosolic concentrations of the intracellular messengers, cAMP and cGMP. The cyclic-nucleotide dependent protein kinases, cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), are the major intracellular receptors of cAMP and cGMP. These enzymes become active upon binding respective cyclic nucleotides and modulate a diverse array of biochemical events through the phosphorylation of specific substrate proteins. The focus of this review is to describe the progress made in understanding the structure and function of both PKA and PKG.     

Designing bisubstrate analog inhibitors for protein kinases

Protein kinases play critical roles in signal transduction pathways by transmitting extracellular signals across the cell membrane to distant locations in the cytoplasm and the nucleus. The development of protein kinase inhibitors has been hindered by the broad overlapping substrate specificities exhibited by these enzymes. The design of bisubstrate analog inhibitors could provide for the enhancement of specificity and potency in protein kinase inhibition. Bisubstrate analog inhibitors form a special group of protein kinase inhibitors that mimic two natural substrates/ligands and that simultaneously associate with two regions of given kinases. Most bisubstrate analogs have been designed to mimic the phosphate donor (ATP) and the acceptor components (Ser-, Thr-, or Tyr-containing peptides). Recent studies have emphasized the importance of maintaining a specific distance between these two components to achieve potent inhibition. In this review, we present a discussion of the methods for designing protein kinase inhibitors by mechanism-based approaches. Emphasis is given to bivalent approaches, with an interpretation of what has been learned from more and less successful examples. Future challenges in this area are also highlighted.     

ATP site-directed competitive and irreversible inhibitors of protein kinases

Several tyrosine and serine/threonine protein kinases have emerged in the last few years as attractive targets in the search for new therapeutic agents being applicable in many different disease indications. Initially, inhibition of these protein kinases by ATP site-directed inhibitors was considered less prone to success, but medicinal chemists from both academia and industry have been able to impart potency and selectivity to a limited number of scaffolds by modulating and fine-tuning the interactions of the modified template with the ATP binding site of the selected kinase. The chemical templates that have been used in the synthesis of ATP site-directed protein kinase inhibitors are reviewed with emphasis on the kinase inhibitors that have entered or are about to enter clinical trials. Examples have been selected to illustrate how structure-based design approaches and new methods to increase compound diversity have had an impact on this area of research.     

Mitogen‐activated protein kinases in hemostasis and thrombosis

Summary. The mitogen‐activated protein (MAP) kinases ERK2, p38 and JNK1 are present in platelets and are activated by various stimuli, such as thrombin, collagen, von Willebrand factor (VWF) and ADP. Until recently, MAP kinases were only studied in the conventional model of agonist‐induced platelet aggregation mediated by fibrinogen and integrin α_IIbβ₃. However, this approach is likely to be too limited for a physiological understanding of platelet MAP kinases and their signaling pathways. Recent studies with varying blood‐flow conditions and animal models of thrombosis have provided deeper insight into the role of MAP kinases in thrombus formation and the dependence of these kinases on shear conditions. This review summarizes and discusses the physiological functions of these kinases in hemostasis and thrombosis as revealed by various technical approaches.     

The role of protein kinases in pancreatic carcinogenesis

BACKGROUND: Pancreatic cancer is a devastating disease with a very poor prognosis. METHODS: Protein kinases are aberrantly expressed in pancreatic ductal adenocarcinoma as analyzed by microarray-based expression analysis and have an impact for pancreatic cancer. Many regulatory proteins have an impact on cancer progression similar to the kinases. The list contains several regulators of kinases derived from the cell cycle control or the mitogen-activated protein (MAP)-kinase pathway. CONCLUSION: Both signalling pathways are essential for tumor progression and pancreatic ductal adenocarcinoma (PDAC) malignancy.     

Viral and host-cell protein kinases: enticing antiviral targets and relevance of nucleoside, and viral thymidine, kinases.

Numerous targets are known for development of antiviral agents, and some significant successes have been achieved with nucleoside analogues. These are "activated" by phosphorylation by viral and/or host-cell nucleoside kinases, the final target being principally the viral polymerase. With latency of herpes viruses, the viral thymidine kinase may be the ultimate target. Less attention has been devoted to viral protein kinases as antiviral targets, largely because 5 years ago, these the study of such enzymes was considered "still in its infancy." In the interim, identification of viral and host-cell protein kinases involved in viral gene expression, and viral replication, has made impressive advances. In conjunction with current progress in development of specific inhibitors of cellular protein kinases, and the differences in sequence motifs between these and the viral enzymes, the latter are indeed attractive targets, as are also some host-cell protein kinases. Examples include, amongst others, the essential protein kinases of vaccinia virus; the nonsegmented negative-strand RNA viruses, all essentially dependent on host-cell kinases, e.g., protein kinase CK-II (casein kinase-II), for which good inhibitors, such as halogenated benzimidazoles and benzotriazoles, are known; herpes viruses, with emphasis on human cytomegalovirus, the UL97 gene of which codes for a protein kinase that, like viral thymidine kinases, "activates," by phosphorylation, a nonpeptide antiviral acyclonucleoside ganciclovir, an analogue of the antiherpes aciclovir. The latter, in turn, is active against animal cytomegaloviruses following phosphorylation by the products of their UL97 gene homologues. Attention is also directed to the antiviral activity of the cyclic phosphate of ganciclovir, a structural analogue of the second messenger cyclic GMP.     

Altered expression and activity of G-proteins, mitogen activated protein kinases, and tyrosine kinases in aging kidney cortex.

BACKGROUND: Renal function declines with age and this may be related to changes in the expression or activity of various signal transduction proteins in the kidney. METHODS: The present study compared the expression and activity of G alpha i(1-3) and G alpha s phosphorylation of mitogen activated protein kinases (MAP-K) (44 and 42 kd) and the activity of tyrosine kinase in renal cortical homogenates of young (4-month-old) and aging (14-month-old) rats. RESULTS: The GTP/(GTP + GDP) binding ratio of G alpha s was significantly decreased in the kidney cortex of aging rats compared to young rats, while the GTP/(GTP + GDP) binding ratio of G alpha i(1-3) increased significantly in kidney cortex of aging rats. Tyrosine kinase activity and phosphorylation of MAP-K (44 and 42 kd) were also reduced in the kidney cortex of aging rats compared to young rats. CONCLUSIONS: These results suggest that diminished phosphorylation of MAP-K and tyrosine kinase activity as well as changes in the binding of GTP/(GTP + GDP) to G alpha i(1-3) and G alpha s may contribute to the age-related decline in renal tubular and vascular function seen in aging animals.     

内脂素对巨噬细胞凋亡的影响及其机制研究

目的：探讨内脂素对巨噬细胞凋亡的影响及其机制。方法：血清饥饿法诱导THP-1细胞凋亡，加入不同浓度的内脂素（O～100ng／mL5个浓度组），每组分别孵育24～48h。同时设立2-羟基萘甲基磷酸三乙酸甲基酯（HNMPA）组。用Annex—inV-碘化丙啶流式细胞仪检测及末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）检测细胞凋亡情况，并检测caspase-3活性及细胞色素C的释放。结果：内脂素增加THP-1细胞凋亡百分比，呈浓度依赖（P〈0．05）；增加caspase-3活性及细胞色素C的释放（P〈O．05）。HNMPA能完全抵消内脂素的促进凋亡作用。结论：内脂素促进巨噬细胞凋亡，凋亡过程涉及增加线粒体内细胞色素C的释放；其机制可能通过胰岛素受体信号途径发挥作用。     

Conformational diversity of catalytic cores of protein kinases.

X-ray crystallography of the protein kinase family has provided an impressive array of crystal structures, setting the stage for rational design of specific inhibitors of these vitally important regulators of the signaling pathways of the cell. Initial work on the first crystal structure of a protein kinase, cyclic AMP-dependent protein kinase, has provided evidence of conformational changes suggested to be critical for the common catalytic event of transferring the gamma phosphate from ATP onto the targeted protein. This review updates the current status of the extent of conformational diversity of the protein kinase family and suggests that both the nature and the extent of those changes can provide a rationale for the increased occurrence of specific protein kinase inhibitors targeted at the ATP-binding site. It focuses on the fact that in addition to the sequence diversities in ATP binding clefts reported recently, there is conformational diversity in the beta sheets of the upper domains of the catalytic cores. This difference is directly related to the regulation of kinases by multiple mechanisms.     

Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.     

Regulation of cardiac hypertrophy in vivo by the stress-activated protein kinases/c-Jun NH2-terminal kinases

Cardiac hypertrophy often presages the development of heart failure. Numerous cytosolic signaling pathways have been implicated in the hypertrophic response in cardiomyocytes in culture, but their roles in the hypertrophic response to physiologically relevant stimuli in vivo is unclear. We previously reported that adenovirus-mediated gene transfer of SEK-1(KR), a dominant inhibitory mutant of the immediate upstream activator of the stress-activated protein kinases (SAPKs), abrogates the hypertrophic response of neonatal rat cardiomyocytes to endothelin-1 in culture. We now report that gene transfer of SEK-1(KR) to the adult rat heart blocks SAPK activation by pressure overload, demonstrating that the activity of cytosolic signaling pathways can be inhibited by gene transfer of loss-of-function mutants in vivo. Furthermore, gene transfer of SEK-1(KR) inhibited pressure overload-induced cardiac hypertrophy, as determined by echocardiography and several postmortem measures including left ventricular (LV) wall thickness, the ratio of LV weight to body weight, cardiomyocyte diameter, and inhibition of atrial natriuretic factor expression. Our data suggest that the SAPKs are critical regulators of cardiac hypertrophy in vivo, and therefore may serve as novel drug targets in the treatment of hypertrophy and heart failure.     

Effects of dioxins on protein kinases and disruption of signal transduction pathways

Dioxins(polychlorinated dibenzo-p-dioxins and related compounds, e.g. polychlorinated dibenzofurans and coplanar polychlorinated biphenyls) has been recognized as endocrine disruptors. Typical endocrine disrupting effects on human of dioxins were shown in 'Yusho' disease(mass PCB poisoning in Japan and Taiwan) patients. A major symptom was chloracne-like lesion on skin, indicating that growth on keratinocytes was stimulated by dioxins. In case of in utero exposure, the body weights of children were lowered, and in some case children had poorer cognitive development in Taiwan episode. Dioxins stimulate the expression of genes(e.g. cytochrome P450 1A1 and related drug metabolizing enzymes) via a ligand dependent transactivating factor, arylhydrocarbon receptor(Ah-R). Besides gene expression of drug metabolizing enzyme, down-regulation of epidermal growth factor receptor has been observed in tissue and cultured cells by exposure to TCDD. Alternative pathways for gene expression such as cross-talk between epidermal growth factor/MAP kinase pathway and Ah-R pathway are reviewed with respect to understanding the endocrine disrupting activity of dioxins.     

Phosphorylation of GSK-3beta by cGMP-dependent protein kinase II promotes hypertrophic differentiation of murine chondrocytes

cGMP-dependent protein kinase II (cGKII; encoded by PRKG2) is a serine/threonine kinase that is critical for skeletal growth in mammals; in mice, cGKII deficiency results in dwarfism. Using radiographic analysis, we determined that this growth defect was a consequence of an elongated growth plate and impaired chondrocyte hypertrophy. To investigate the mechanism of cGKII-mediated chondrocyte hypertrophy, we performed a kinase substrate array and identified glycogen synthase kinase-3beta (GSK-3beta; encoded by Gsk3b) as a principal phosphorylation target of cGKII. In cultured mouse chondrocytes, phosphorylation-mediated inhibition of GSK-3beta was associated with enhanced hypertrophic differentiation. Furthermore, cGKII induction of chondrocyte hypertrophy was suppressed by cotransfection with a phosphorylation-deficient mutant of GSK-3beta. Analyses of mice with compound deficiencies in both protein kinases (Prkg2(-/-)Gsk3b(+/-)) demonstrated that the growth retardation and elongated growth plate associated with cGKII deficiency were partially rescued by haploinsufficiency of Gsk3b. We found that beta-catenin levels decreased in Prkg2(-/-) mice, while overexpression of cGKII increased the accumulation and transactivation function of beta-catenin in mouse chondroprogenitor ATDC5 cells. This effect was blocked by coexpression of phosphorylation-deficient GSK-3beta. These data indicate that hypertrophic differentiation of growth plate chondrocytes during skeletal growth is promoted by phosphorylation and inactivation of GSK-3beta by cGKII.