Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr–Abl, fail to effectively suppress the Bcr–Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr–Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.     

Structural and spectroscopic analysis of the kinase inhibitor bosutinib and an isomer of bosutinib binding to the Abl tyrosine kinase domain

Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib, nilotinib and dasatinib are currently used to treat CML, but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML, but its binding mode is unknown. We present the 2.4 Å structure of bosutinib bound to the kinase domain of Abl, which explains the inhibitor''s activity against several imatinib-resistant mutants, and reveals that similar inhibitors that lack a nitrile moiety could be effective against the common T315I mutant. We also report that two distinct chemical compounds are currently being sold under the name “bosutinib”, and report spectroscopic and structural characterizations of both. We show that the fluorescence properties of these compounds allow inhibitor binding to be measured quantitatively, and that the infrared absorption of the nitrile group reveals a different electrostatic environment in the conserved ATP-binding sites of Abl and Src kinases. Exploiting such differences could lead to inhibitors with improved selectivity.     

Imatinib can act as an Allosteric Activator of Abl Kinase

Imatinib is an ATP-competitive inhibitor of Bcr-Abl kinase and the first drug approved for chronic myelogenous leukemia (CML) treatment. Here we show that imatinib binds to a secondary, allosteric site located in the myristoyl pocket of Abl to function as an activator of the kinase activity. Abl transitions between an assembled, inhibited state and an extended, activated state. The equilibrium is regulated by the conformation of the αΙ helix, which is located nearby the allosteric pocket. Imatinib binding to the allosteric pocket elicits an αΙ helix conformation that is not compatible with the assembled state, thereby promoting the extended state and stimulating the kinase activity. Although in wild-type Abl the catalytic pocket has a much higher affinity for imatinib than the allosteric pocket does, the two binding affinities are comparable in Abl variants carrying imatinib-resistant mutations in the catalytic site. A previously isolated imatinib-resistant mutation in patients appears to be mediating its function by increasing the affinity of imatinib for the allosteric pocket, providing a hitherto unknown mechanism of drug resistance. Our results highlight the benefit of combining imatinib with allosteric inhibitors to maximize their inhibitory effect on Bcr-Abl.     

Allosteric interactions between the myristate- and ATP-site of the Abl kinase

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors.     

Leucine-rich repeat kinase 2 mutants I2020T and G2019S exhibit altered kinase inhibitor sensitivity

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a frequent cause of late-onset autosomal dominant Parkinson’s disease (PD). Some disease-associated mutations directly affect LRRK2 kinase activity and inhibition of LRRK2 is viewed as a potential therapeutic treatment for PD. We demonstrate by both binding and enzymatic assays that alterations in the kinase activity of the PD-associated mutants I2020T and G2019S are due in part to altered ATP affinity. In binding assays, G2019S and I2020T have approximately 2-fold lower and 6-fold higher ATP affinity, respectively, than wild-type LRRK2. Furthermore, using an in vitro kinase activity assay, we demonstrate that at ATP concentrations close to cellular levels (1 mM) I2020T is approximately 10-fold more resistant to ATP-competitive kinase inhibitors than wild-type whereas G2019S is 1.6-fold more sensitive. These results predict that LRRK2 status may impact kinase inhibitor potencies in vivo or in cellular models.     

Synthesis and docking study of 2-phenylaminopyrimidine Abl tyrosine kinase inhibitors

Six analogs of imatinib, an Abl kinase inhibitor clinically used as a first-line therapeutic agent for chronic myeloid leukaemia (CML), have been synthesized and characterized. And their potency as Abl kinase inhibitors have been screened by a robust virtual screening method developed based on the crystal structure (PDB code 2hyy) of Abl-imatinib complex using Surflex-Docking. The docking results are consistent with the inhibitory potency of the compounds characterized by MS method. And the H-bonds between imatinib analogs and Thr315 and Met318 residues in Abl kinase are shown to be crucial for achieving accurate poses and high binding affinities for the ATP-competitive kinase inhibitors.     

Identification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds

Based on bioinformatics interrogation of the genome, > 500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4′-methyl-N²-3-pyridinyl-4,5′-bi-1,3-thiazole-2,2′-diamine (JNK Docking (JD) compound 123, but not the related compound (4′-methyl-N ~ 2 ~ -(6-methyl-2-pyridinyl)-4,5′-bi-1,3-thiazole-2,2′-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10 μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.     

N-Myristoylated c-Abl Tyrosine Kinase Localizes to the Endoplasmic Reticulum upon Binding to an Allosteric Inhibitor

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH₂-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH₂-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.The catalytic activity of a protein kinase can be modulated by binding of a ligand to a site distant from the active site, also referred to as the allosteric site (1). The ligand is referred to as an allosteric kinase inhibitor and induces a protein conformation that is not compatible with kinase activity. Allosteric inhibitors can potentially be exploited to elucidate kinase functions not discovered using ATP-competitive inhibitors, because they can exploit binding sites and regulatory mechanisms that are unique to a particular kinase.The c-Abl and Arg (Abl-related gene) proteins comprise the Abl family of non-receptor tyrosine kinases. Each family member has two isoforms: one that is myristoylated in the N terminus (1b or IV) and the other that is deficient in N-myristoylation due to an alternative splicing of the first exon (1a or I) (Fig. 1A). N-Myristoylation often serves as a mechanism for targeting proteins to cellular membranes. However, Abl family members localize to multiple subcellular compartments; whereas Arg is mostly found in the cytoplasm, c-Abl shuttles between the nucleus and the cytoplasm, where it localizes to the cytosol, endoplasmic reticulum, and mitochondria (2).Open in a separate windowFIGURE 1.A, domain structure of Abl family members (5). The numbers indicate amino acid residues in c-Abl 1b, and the recombinant protein constructs used in this study encompass amino acids 65–534, 83–534, and 248–531. B, ribbon representation of the c-Abl kinase NH₂-terminal half residues, including the SH3, SH2, and kinase domains (Protein Data Bank code 1OPK) (7). The NH₂-terminal cap (amino acids 2–79) is indicated by dotted lines (8). The myristate-binding site and ATP binding pocket are indicated by arrows. C, ribbon representation of an enlarged view of GNF-2 (colored gold) bound to the c-Abl myristate binding site. The location of Ala³⁵⁶ is indicated.The Abl family members share a high degree of sequence identity (∼90%) in the NH₂-terminal half residues, including the SH3,² SH2, and kinase domains (3). The kinase domain is followed by proline-rich motifs that serve as binding sites for SH3 domains. A range of proteins are reported to bind directly or indirectly to the SH3, SH2, and proline-rich domains of c-Abl and are implicated in the proper regulation of the kinase activities of Abl family members in the cytoplasm (4–6). In addition, as revealed by recent crystallographic analyses of inactive and assembled form of recombinant Abl, the kinase activity of c-Abl is modulated by the intrinsic binding of the N-myristoyl residue to a hydrophobic pocket in the C-lobe of the kinase domain, which induces conformational changes in the kinase domain and subsequently allows the SH3 and SH2 domains to pack against the kinase domain (7, 8). Altogether, these observations suggest that the kinase activities of Abl family members in normal cells are tightly regulated by both intra- and intermolecular interactions (2, 9). Disruption of these strong regulatory mechanisms results in deregulated kinase activity, as illustrated by the BCR-Abl and v-Abl oncoproteins.Recent years have seen great advances in pharmacological inhibition of deregulated c-Abl kinase activity. Among the small molecule inhibitors targeting BCR-Abl kinase are imatinib (STI-571; Gleevec), nilotinib (AMN 107), and dasatinib (BMS-354825) (10). These small molecules have been used not only for clinical intervention in patients with leukemia but also as chemical tools to further dissect BCR-Abl kinase-linked signaling pathways in tissue culture cells (11). However, efforts to analyze the effects of monospecific inhibition of BCR-Abl kinase have been complicated by cross-reactivity of ATP-competitive Abl inhibitors with other kinases. For example, in addition to inhibiting c-Abl and BCR-Abl, STI-571 and nilotinib also potently inhibit c-Kit, platelet-derived growth factor receptor, and DDR1, whereas dasatinib potently inhibits all of these kinases as well as the Src family, Tec family, and KDR kinases (12). The multitargeted nature of these ATP-competitive inhibitors makes it difficult to assign a particular biological effect to inhibition of a specific kinase target.We previously reported the discovery of the first non-ATP site-monoselective BCR-Abl inhibitor (GNF-2), which targets not only wild type BCR-Abl but also many clinically relevant STI-571-resistant mutants either alone or in combination with other BCR-Abl inhibitors (13). Molecular modeling, site-directed mutagenesis, competition assays, NMR spectroscopy, and protein crystallography were used to determine that GNF-2 binds to a myristate-binding site in the C-lobe of the c-Abl kinase domain (Fig. 1, B and C) (3). The discovery of GNF-2 was the first demonstration that c-Abl kinase activity could be pharmacologically modulated by an inhibitor that binds outside the ATP or substrate binding sites. Although it remained unclear how GNF-2 is capable of inhibiting c-Abl upon binding to the myristate-binding site, we speculated that GNF-2 probably mimics the function of the N-myristoyl residue in c-Abl. Here, we investigated the effects of GNF-2 on Abl family members with the goals of providing further insights into the mechanism of GNF-2 function and laying the foundation to utilize GNF-2 as a tool to investigate c-Abl- and Arg-linked cellular processes.     

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

The role of brain-derived neurotrophic factor in mouse oocyte maturation in vitro involves activation of protein kinase B

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation, but the signal transduction pathways are not clear. In this study, we investigated (using western blots) the effects of BDNF on the phosphorylation of protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) in mouse oocytes and cumulus cells cultured in vitro. Treatment with BDNF enhanced phosphorylation of PKB in oocytes at 2 h (P = 0.0006) and 3 h (P < 0.0001) of in vitro maturation, compared with control oocytes. However, the pan-specific tyrosine kinase (Trk) inhibitor K252a together with BDNF completely inhibited phosphorylation of PKB in the oocytes. Furthermore, BDNF increased phosphorylation of MAPK in oocytes at 16 h of in vitro maturation (P = 0.0041), but K252a together with BDNF did not reduce phosphorylation of MAPK in the oocytes. For cumulus cells, BDNF significantly prolonged the phosphorylation of PKB and MAPK and increased the total amounts of PKB and MAPK proteins after 16 h of in vitro maturation. However, BDNF did not affect apoptosis of the cumulus cells during oocyte maturation in vitro. In conclusion, the PKB pathway is likely to be one signaling cascade activated by BDNF in combination with the TrkB receptor, whereas the MAPK pathway is not involved. These findings may have relevance for BDNF-induced promotion of developmental capacity of in vitro-matured oocytes.     

The tyrosine phosphatase TC48 interacts with and inactivates the oncogenic fusion protein BCR-Abl but not cellular Abl

The chimeric oncoprotein BCR-Abl exhibits deregulated protein tyrosine kinase activity and is responsible for the pathogenesis of certain human leukemias, such as chronic myelogenous leukemia. The activities of cellular Abl (c-Abl) and BCR-Abl are stringently regulated and the cellular mechanisms involved in their inactivation are poorly understood. Protein tyrosine phosphatases can negatively regulate Abl mediated signaling by dephosphorylating the kinase and/or its substrates. This study investigated the ability of the intracellular T cell protein tyrosine phosphatase (TCPTP/PTPN2) to dephosphorylate and regulate the functions of BCR-Abl and c-Abl. TCPTP is expressed as two alternately spliced isoforms — TC48 and TC45, which differ in their C-termini and localize to the cytoplasm and nucleus respectively. We show that TC48 dephosphorylates BCR-Abl but not c-Abl and inhibits its activity towards its substrate, CrkII. Y1127 and Y1294 residues whose phosphorylation corresponds with BCR-Abl activation status were the primary sites targeted by TC48. Co-localization and immunoprecipitation experiments showed that TC48 interacted with BCR-Abl but not with c-Abl, and BCR domain was sufficient for interaction. TC48 expression resulted in the stabilization of Bcr-Abl protein dependent on its phosphatase activity. Inactivation of cellular TC48 in K562 cells by stable expression of a dominant negative catalytically inactive mutant TC48, enhanced proliferation. TC48 expressing K562 clones showed reduced proliferation and enhanced sensitivity to STI571 compared to control clones suggesting that TC48 can repress the growth of CML cells. This study identifies a novel cellular regulator that specifically inhibits the activity of oncogenic BCR-Abl but not that of the cellular Abl kinase.     

Biomolecular response of oxanine in DNA strands to T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO₂-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5′-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa > Gua > adenine (Ade) ∼ thymine (Thy) > cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.     

Synthesis and SAR of novel quinazolines as potent and brain-penetrant c-jun N-terminal kinase (JNK) inhibitors

Quinazoline 3 was discovered as a novel c-jun N-terminal kinase (JNK) inhibitor with good brain penetration and pharmacokinetic (PK) properties. A number of analogs which were potent both in the biochemical and cellular assays were discovered. Quinazoline 13a was found to be a potent JNK3 inhibitor (IC₅₀ = 40 nM), with >500-fold selectivity over p38, and had good PK and brain penetration properties. With these properties, 13a is considered a potential candidate for in vivo evaluation.     

P2X7 receptor mediated phosphorylation of p38MAP kinase in the hippocampus

This study was designed to explore the effect of P2X7 receptor (P2X7R) activation on the expression of p38 MAP kinase (p38 MAPK) enzyme in hippocampal slices of wild-type (WT) and P2X7R^−/− mice using the Western blot technique and to clarify its role in P2X7 receptor mediated [³H]glutamate release. ATP (1 mM) and the P2X7R agonist BzATP (100 μM) significantly increased p38 MAPK phosphorylation in WT mice, and these effects were absent in the hippocampal slices of P2X7R^−/− mice. Both ATP- and BzATP-induced p38 MAPK phosphorylations were sensitive to the p38 MAP kinase inhibitor, SB203580 (1 μM). ATP elicited [³H]glutamate release from hippocampal slices, which was significantly attenuated by SB203580 (1 μM) but not by the extracellular signal-regulated kinase (ERK1/2) inhibitor, PD098095 (10 μM). Consequently, we suggest that P2X7Rs and p38 MAPK are involved in the stimulatory effect of ATP on glutamate release in the hippocampal slices of WT mice.     

Abl tyrosine kinase promotes dorsal ruffles but restrains lamellipodia extension during cell spreading on fibronectin

The nonreceptor Abl tyrosine kinase stimulates F-actin microspikes and membrane ruffles in response to adhesion and growth factor signals. We show here that induced dimerization of Abl-FKBP, but not the kinase-defective AblKD-FKBP, inhibits cell spreading on fibronectin. Conversely, knockdown of cellular Abl by shRNA stimulates cell spreading. The Abl kinase inhibitor, imatinib, also stimulates cell spreading and its effect is overridden by the imatinib-resistant AblT315I. Expression of Abl but not AbkKD in Abl/Arg-deficient cells again inhibits spreading. Furthermore, Abl inhibits spreading of cells that express the activated Rac, RacV12, correlating with RacV12 localization to dorsal membrane protrusions. Ectopic expression of CrkII, a Rac activator that is inactivated by Abl-mediated tyrosine phosphorylation, antagonizes Abl-mediated dorsal membrane localization of RacV12. Ectopic expression of a dynamin-2 mutant, previously shown to induce Rac-GTP localization to the dorsal membrane, abolishes the stimulatory effect of imatinib on cell spreading. These results suggest that Abl tyrosine kinase, through CrkII phosphorylation and in collaboration with dynamin-2 can regulate the partitioning of Rac-GTP to favor dorsal ruffles during cell spreading. The Abl-dependent dorsal membrane localization of activated Rac explains its positive role in ruffling and negative role in cell spreading and migration.     

STI-571: an anticancer protein-tyrosine kinase inhibitor

STI-571 (imatinib, Gleevec, Glivec, CGP 57148) is an inhibitor of the Abl group of protein-tyrosine kinases. One of these enzymes, the Bcr-Abl oncoprotein, results from the fusion of the BCR and ABL genes that result from the reciprocal chromosomal translocation that forms the Philadelphia chromosome. The Philadelphia chromosome occurs in 95% of people with chronic myeloid leukemia. ABL is the cellular homologue of the oncogene found in murine Abelson leukemia virus, and BCR refers to breakpoint cluster region. The Bcr-Abl oncoprotein exhibits elevated protein-tyrosine kinase activity, which is strongly implicated in the mechanism of development of chronic myeloid leukemia. STI-571 is effective in the treatment of the stable phase of chronic myeloid leukemia. The c-Abl protein kinase domain exists in an active and inactive conformation. STI-571 binds only to the inactive state of the enzyme as shown by X-ray crystallography. The drug binds to a portion of the ATP-binding site and extends from there into adjacent hydrophobic regions. STI-571 is a competitive inhibitor of Abl kinase with respect to ATP. Resistance to STI-571 is often the result of mutations in residues of the Bcr-Abl kinase that ordinarily bind to the drug. Inhibition of target protein kinases represents an emerging therapeutic strategy for the treatment of cancer.     

Hybrid compounds as new Bcr/Abl inhibitors

A series of 2,4-disubstituted thiazole derivatives were designed and synthesized as new Bcr/Abl inhibitors by hybriding the structural moieties from FDA approved imatinib, nilotinib and dasatinib. The new inhibitors strongly suppressed the activity of Bcr/Abl kinase and potently inhibited the proliferation of K562 and KU812 leukemia cancer cells. Compound 4i displayed comparable potency with that of nilotinib in both biochemical kinase assay and cancer cell growth inhibition assay. These inhibitors might serve as lead compounds for further developing new anticancer drugs.     

Focal adhesion kinase,RhoA, and p38 mitogen-activated protein kinase modulates apoptosis mediated by angiotensin II AT2 receptors

Apoptosis plays an important role in cellular processes such as development, differentiation, and homeostasis. Although the participation of angiotensin II (Ang II) AT₂ receptors (AT ₂R) in cellular apoptosis is well accepted, the signaling pathway involved in this process is not well established. We evaluated the participation of signaling proteins focal adhesion kinase (FAK), RhoA, and p38 mitogen-activated protein kinase (p38MAPK) in apoptosis induced by Ang II via AT ₂R overexpressed in HeLa cells. Following a short stimulation time (120 to 240 minutes) with Ang II, HeLa-AT ₂ cells showed nuclear condensation, stress fibers disassembly and membrane blebbing. FAK, classically involved in cytoskeleton reorganization, has been postulated as an early marker of cellular apoptosis. Thus, we evaluated FAK cleavage, detected at early stimulation times (15 to 30 minutes). Apoptosis was confirmed by increased caspase-3 cleavage and enzymatic activity of caspase-3/7. Participation of RhoA was evaluated. HeLa-AT ₂ cells overexpressing RhoA wild-type (WT) or their mutants, RhoA V14 (constitutively active form) or RhoA N19 (dominant-negative form) were used to explore RhoA participation. HeLa-AT ₂ cells expressing the constitutively active variant RhoA V14 showed enhanced apoptotic features at earlier times as compared with cells expressing the WT variant. RhoA N19 expression prevented nuclear condensation/caspase activation. Inhibition of p38MAPK caused an increase in nuclear condensation and caspase-3/7 activation, suggesting a protective role of p38MAPK. Our results clearly demonstrated that stimulation of AT ₂R induce apoptosis with participation of FAK and RhoA while p38MAPK seems to play a prosurvival role.     

Mixed lineage kinase ZAK utilizing MKK7 and not MKK4 to activate the c-Jun N-terminal kinase and playing a role in the cell arrest

The leucine-zipper (LZ) and sterile-alpha motif (SAM) kinase (ZAK) belongs to the MAP kinase kinase kinase (MAP3K) when upon over-expression in mammalian cells activates the JNK/SAPK pathway. The mechanisms by which ZAK activity is regulated are not well understood. Co-expression of dominant-negative MKK7 but not MKK4 and ZAK significantly attenuates JNK/SAPK activation. This result suggests that ZAK activates JNK/SAPK mediated by downstream target, MKK7. Expression of ZAK but not kinase-dead ZAK in 10T1/2 cells results in the disruption of actin stress fibers and morphological changes. Therefore, ZAK activity may be involved in actin organization regulation. Expression of wild-type ZAK increases the cell population in the G(2)/M phase of the cell cycle, which may indicate G(2) arrest. Western blot analysis shows that the decreased cyclin E level correlated strongly with the low proliferative capacity of ZAK-expressed cells.     

Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.