Alternative binding modes of an inhibitor to two different kinases.

Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC50 1.6 micro m) (Sarno et al. (2001) FEBS Letts.496, 44-48). Three other kinases [cyclin-dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3beta] exhibit approximately 10-fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho-CDK2-cyclin A at 2.2 A resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N- and C-terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals contacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases.     

Daphnetin, one of coumarin derivatives, is a protein kinase inhibitor.

Protein kinases play key roles in the control of cell proliferation, differentiation and metabolism. In this work, we studied the effect of coumarin and its derivatives, including daphnetin, esculin, 2-OH-coumarin, 4-OH-coumarin and 7-OH-coumarin, on the activity of protein kinases. It was found that, in these compounds, only daphnetin was a protein kinase inhibitor. This compound inhibited tyrosine-specific protein kinase, EGF receptor (IC(50) = 7.67 microM), and serine/threonine-specific protein kinases, including cAMP-dependent protein kinase (PKA) (IC(50) = 9.33 microM) and protein kinase C (PKC) (IC(50) = 25.01 microM) in vitro. The inhibition of EGF receptor tyrosine kinase by daphnetin was competitive to ATP and non-competitive to the peptide substrate. The inhibition of EGF-induced tyrosine phosphorylation of EGF receptor by daphnetin was not observed in human hepatocellular carcinoma HepG2 cells. The structural comparison of daphnetin with coumarin and other coumarin derivatives suggests that the hydroxylation at C8 may be required for daphnetin acting as a protein kinase inhibitor.     

2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2

Protein kinase CK2 is a highly pleiotropic enzyme whose high constitutive activity is suspected to be instrumental to the enhancement of the tumour phenotype and to the propagation of infectious diseases. Here we describe a novel compound, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), which is superior to the commonly used specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in several respects. DMAT displays the lowest K(i) value ever reported for a CK2 inhibitor (40 nM); it is cell permeable and its efficacy on cultured cells, both in terms of endogenous CK2 inhibition and induction of apoptosis, is several fold higher than that of TBB. The selectivity of DMAT assayed on a panel of >30 protein kinases is comparable to that of TBB, with the additional advantage of being ineffective on protein kinase CK1 up to 200 microM. These properties make DMAT the first choice CK2 inhibitor for in vivo studies available to date.     

Inhibition of protein kinase CK2 by condensed polyphenolic derivatives. An in vitro and in vivo study

ATP site-directed inhibitors that can target individual kinases are powerful tools for use in signal transduction research, all the more so in the case of a pleiotropic, constitutively active protein kinase such as CK2, which is not turned on in response to specific stimuli. By screening a library of more than 200 derivatives of natural polyphenolic compounds, we have identified 16 molecules which inhibit CK2 with IC(50) values of 相似文献   

Tannic acid, a potent inhibitor of epidermal growth factor receptor tyrosine kinase

Increasing evidence supports the hypothesis that tannic acid, a plant polyphenol, exerts anticarcinogenic activity in chemically induced cancers. In the present study, tannic acid was found to strongly inhibit tyrosine kinase activity of epidermal growth factor receptor (EGFr) in vitro (IC50 = 323 nM). In contrast, the inhibition by tannic acid of p60(c-src) tyrosine kinase (IC50 = 14 microM) and insulin receptor tyrosine kinase (IC50 = 5 microM) was much weaker. The inhibition of EGFr tyrosine kinase by tannic acid was competitive with respect to ATP and non-competitive with respect to peptide substrate. In cultured cells, growth factor-induced tyrosine phosphorylation of growth factor receptors, including EGFr, platelet-derived growth factor receptor, and basic fibroblast growth factor receptor, was inhibited by tannic acid. No inhibition of insulin-induced tyrosine phosphorylation of insulin receptor and insulin-receptor substrate-1 was observed. EGF-stimulated growth of HepG2 cells was inhibited in the presence of tannic acid. The inhibition of serine/threonine-specific protein kinases, including cAMP-dependent protein kinase, protein kinase C and mitogen-activated protein kinase, by tannic acid was only detected at relatively high concentration, IC50 being 3, 325 and 142 microM respectively. The molecular modeling study suggested that tannic acid could be docked into the ATP binding pockets of either EGFr or insulin receptor. These results demonstrate that tannic acid is an in vitro potent inhibitor of EGFr tyrosine kinase.     

山奈酚抑制蛋白激酶CK2活性

研究体外以及HL-60细胞内山奈酚对蛋白激酶CK2的抑制作用及机制. 通过测定药物作用后转移到CK2底物上的[γ-32P]ATP的32P的放射性活度, 探讨山奈酚对重组人CK2全酶以及细胞内CK2活性的影响; 采用多重RT-PCR检测CK2α、α' 和 β亚基的mRNA表达水平; 通过 Lineweaver-Burk作图法,分析CK2的酶动力学机制.山奈酚能显著抑制重组人CK2活性（IC50 = 1.88 μmol/L）和HL-60细胞内的CK2活性, 对细胞内CK2的作用效果强于阳性对照四溴-2-氮杂苯并咪唑(TBB). 山奈酚作用2h,对CK2各亚基的mRNA表达水平均没有影响. 山奈酚对重组人CK2的酶动力学分析表明, 山奈酚与ATP(Ki = 1.14 μmol/L)及酪蛋白(Ki = 1.03 μmol/L)均呈非竞争性抑制作用. 结果提示, 山奈酚是一种有效的蛋白激酶CK2的抑制剂, 其作用机制可能与其阻碍CK2与ATP以及底物的结合有关.     

Inhibition of protein kinase CK2 by flavonoids and tyrphostins. A structural insight

Sixteen flavonoids and related compounds have been tested for their ability to inhibit three acidophilic Ser/Thr protein kinases: the Golgi apparatus casein kinase (G-CK) recently identified with protein FAM20C, protein kinase CK1, and protein kinase CK2. While G-CK is entirely insensitive to all compounds up to 40 μM concentration, consistent with the view that it is not a member of the kinome, and CK1 is variably inhibited in an isoform-dependent manner by fisetin and luteolin, and to a lesser extent by myricetin and quercetin, CK2 is susceptible to drastic inhibition by many flavonoids, displaying with six of them IC(50) values < 1 μM. A common denominator of these compounds (myricetin, quercetin, fisetin, kaempferol, luteolin, and apigenin) is a flavone scaffold with at least two hydroxyl groups at positions 7 and 4'. Inhibition is competitive with respect to the phospho-donor substrate ATP. The crystal structure of apigenin and luteolin in complex with the catalytic subunit of Zea mays CK2 has been solved, revealing their ability to interact with both the hinge region (Val116) and the positive area near Lys68 and the conserved water W1, the two main polar ligand anchoring points in the CK2 active site. Modeling experiments account for the observation that luteolin but not apigenin inhibits also CK1. The observation that luteolin shares its pyrocatechol moiety with tyrphostin AG99 prompted us to solve also the structure of this compound in complex with CK2. AG99 was found inside the ATP pocket, consistent with its mode of inhibition competitive with respect to ATP. As in the case of luteolin, the pyrocatechol group of AG99 is critical for binding, interacting with the positive area in the deepest part of the CK2 active site.     

Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole

Two novel crystal structures of Zea mays protein kinase CK2alpha catalytic subunit, one in complex with the specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and another in the apo-form, were solved at 2.2 A resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2alpha is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five-membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2alpha enzyme analyzed so far, with the exception of the complex with emodin. In the N-terminal lobe, the position of helix alphaC is particularly well preserved in all the structures examined; the Gly-rich loop is displaced from the intermediate position it has in the apo-form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.     

Auto-inhibition of Ca(2+)/calmodulin-dependent protein kinase II by its ATP-binding domain

Ca(2+)/calmodulin dependent protein kinase (CaMPK) II is a key enzyme in many physiological processes. The enzyme is inactive unless Ca(2+)/CaM binds to it. In this inactive form CaMPK-II does not bind ATP suggesting that the ATP-binding domain is involved in an intramolecular interaction. We show here that F12, a 12 amino acid long peptide fragment of the ATP-binding domain (CaMPK-II(23-34), GAFSVVRRCVKV) can inhibit the Ca(2+)/CaM-dependent activity (IC(50) of 3 microM) but has no effect on the Ca(2+)/CaM-independent activity of CaMPK-II. Kinetic analysis exhibited mixed inhibition with respect to autocamtide-2 and ATP. The inhibition by F12 showed specificity towards CaMPK-II, but also inhibited CaMPK-I (IC(50) = 12.5 microM), while CaMPK-IV (IC(50) = 85 microM) was inhibited poorly and cAMP-dependent protein kinase (PKA) was not inhibited. Substitution of phenylalanine at position 25 to alanine (A12), had little effect on the inhibition of different Ca(2+)/CaM-dependent protein kinases, suggesting that phenylalanine 25 does not play a crucial role in the interactions involving F12. Thus the molecular interactions involving the ATP-binding domain appears to play a role in the regulation of nonphosphorylated CaMPK-II activity.     

Discovery of cyclin-dependent kinase inhibitor, CR229, using structurebased drug screening

To generate new scaffold candidates as highly selective and potent cyclin-dependent kinase (CDK) inhibitors, structure-based drug screening was performed utilizing 3D pharmacophore conformations of known potent inhibitors. As a result, CR229 (6-bromo-2,3,4,9-tetrahydro-carbolin-1-one) was generated as the hit-compound. A computational docking study using the X-ray crystallographic structure of CDK2 in complex with CR229 was evaluated. This predicted binding mode study of CR229 with CDK2 demonstrated that CR229 interacted effectively with the Leu83 and Glu81 residues in the ATP-binding pocket of CDK2 for the possible hydrogen bond formation. Furthermore, biochemical studies on inhibitory effects of CR229 on various kinases in the human cervical cancer HeLa cells demonstrated that CR229 was a potent inhibitor of CDK2 (IC50: 3 microM), CDK1 (IC50: 4.9 microM), and CDK4 (IC50: 3 microM), yet had much less inhibitory effect (IC50: >20 microM) on other kinases, such as casein kinase 2-1 (CK2- alpha1), protein kinase A (PKA), and protein kinase C (PKC). Accordingly, these data demonstrate that CR229 is a potent CDK inhibitor with anticancer efficacy.     

The selectivity of inhibitors of protein kinase CK2: an update

CK2 (casein kinase 2) is a very pleiotropic serine/threonine protein kinase whose abnormally high constitutive activity has often been correlated to pathological conditions with special reference to neoplasia. The two most widely used cell permeable CK2 inhibitors, TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole), are marketed as quite specific CK2 blockers. In the present study we show, by using a panel of approx. 80 protein kinases, that DMAT and its parent compound TBI (or TBBz; 4,5,6,7-tetrabromo-1H-benzimidazole) are potent inhibitors of several other kinases, with special reference to PIM (provirus integration site for Moloney murine leukaemia virus)1, PIM2, PIM3, PKD1 (protein kinase D1), HIPK2 (homeodomain-interacting protein kinase 2) and DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase 1a). In contrast, TBB is significantly more selective toward CK2, although it also inhibits PIM1 and PIM3. In an attempt to improve selectivity towards CK2 a library of 68 TBB/TBI-related compounds have been tested for their ability to discriminate between CK2, PIM1, HIPK2 and DYRK1a, ending up with seven compounds whose efficacy toward CK2 is markedly higher than that toward the second most inhibited kinase. Two of these, K64 (3,4,5,6,7-pentabromo-1H-indazole) and K66 (1-carboxymethyl-2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole), display an overall selectivity much higher than TBB and DMAT when tested on a panel of 80 kinases and display similar efficacy as inducers of apoptosis.     

Tyrphostin AG213对重组人蛋白激酶CK2全酶的抑制动力学

利用基因工程克隆、表达和纯化获得重组人蛋白激酶CK2α和 β亚基 ,在体外等摩尔数混合构成有最大生物活性的重组人CK2全酶 .以重组人CK2全酶为分子靶点 ,研究tyrphostinAG2 13对该全酶的直接作用及其抑制动力学 .通过测定转移到CK2底物上的 [γ 3 2 P]GTP的 [3 2 P]放射活度 ,检测CK2活性 .结果表明 :重组人CK2是一种Ca2 + 、cAMP和cGMP等第二信使非依赖性蛋白激酶 ,与天然CK2的性质一致 .AG2 13对重组人CK2全酶具有很强的抑制作用 ,IC50 为 1 1μmol L ,抑制作用远大于已知CK2的抑制剂 5 ,6 二氯 1 β 呋喃糖苯并咪唑 (DRB)和N (2 氨乙基 ) 5 氯萘 1 硫胺 (A3) .AG2 13对重组人CK2全酶的动力学研究表明 :它与GTP呈现非竞争 竞争性混合型抑制作用 ,抑制常数Ki 和Ki′值分别为 0 6 μmol L与 1 4 μmol L ;与酪蛋白呈非竞争性抑制作用 ,Ki 值为 0 9μmol L .结果说明 ,tyrphostinAG2 13不仅是酪氨酸蛋白激酶的抑制剂 ,而且是一种十分有效的蛋白激酶CK2的抑制剂 .重组人蛋白激酶CK2可作为一种较为简便筛选和开发有效的CK2抑制剂的分子靶点 .     

Novel compounds, '1,3-selenazine derivatives' as specific inhibitors of eukaryotic elongation factor-2 kinase

The inhibitory activities of 5,6-dihydro-4H-1,3-selenazine derivatives on protein kinases were investigated. In a multiple protein kinase assay using a postnuclear fraction of v-src-transformed NIH3T3 cells, 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-6-isopropyl-4-methyl-2-p-tolyl-5,6-dihydro-4H-1, 3-selenazine (TS-4) exhibited selective inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K) over protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). In further experiments using purified kinases, TS-2 (IC(50)=0.36 microM) and TS-4 (IC(50)=0.31 microM) inhibited eEF-2K about 25-fold more effectively than calmodulin-dependent protein kinase-I (CaMK-I), and about 6-fold (TS-2) or 33-fold (TS-4) more effectively than calmodulin-dependent protein kinase-II (CaMK-II), respectively. TS-2 and TS-4 showed much weaker inhibitory activity toward PKA and PKC, while TS-4, but not TS-2, moderately inhibited immunoprecipitated v-src kinase. TS-2 (10.7-fold) and TS-4 (12.5-fold) demonstrated more potent and more specific eEF-2K inhibitory activity than rottlerin, a previously identified eEF-2K inhibitor. TS-2 inhibited ATP or eEF-2 binding to eEF-2K in a competitive or non-competitive manner, respectively. In cultured v-src-transformed NIH3T3 cells, TS-2 also decreased phospho-eEF-2 protein level (IC(50)=4.7 microM) without changing the total eEF-2 protein level. Taken together, these results suggest that TS-2 and TS-4 are the first identified selective eEF-2K inhibitors and should be useful tools for studying the function of eEF-2K.     

Selective inhibition of tyrosine protein kinase by a synthetic multisubstrate analog

Synthetic compounds were designed in an attempt to mimic the possible transition state of tyrosine protein kinases. One representative compound (RP 53801) inhibited the enzyme purified from RSV-transformed cells. A serine/threonine kinase (kinase C) was 45 fold less sensitive. The inhibition was competitive with respect to ATP and noncompetitive with respect to the phosphate acceptor poly glu4-tyr1. The degree of inhibition (IC50 = 22 microM) was however lower than that expected from a transition state analog. The compound was capable of reducing tyrosine protein kinase activity in intact cells with some selectivity at 100 microM.     

Kaempferol inhibits myosin light chain kinase

Kaempferol, 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, was found to inhibit bovine aorta myosin light chain kinase with a Ki of 0.3-0.5 microM. It was found to be competitive with ATP and non-competitive with isolated myosin light chains. The specificity of this inhibitor was studied relative to protein kinase C and cAMP dependent protein kinase (IC50 = 15 microM and 150 microM, respectively). It appears not to interact strongly with calmodulin binding proteins, such as Ca2+-calmodulin dependent phosphodiesterase (IC50 = 45 microM), and had little effect on actin-activated myosin subfragment-1 ATPase activity (IC50 greater than 100 microM) or smooth muscle phosphatase activities (IC50 greater than 100 microM).     

Propranolol, a phosphatidate phosphohydrolase inhibitor, also inhibits protein kinase C.

Propranolol, a beta-adrenergic receptor antagonist, also inhibits phosphatidate phosphohydrolase, the enzyme that converts phosphatidic acid into diacylglycerol. This latter effect has prompted recent use of propranolol in studies examining the importance of diacylglycerol and phosphatidic acid in cellular signalling events. Here, we show that propranolol is also an inhibitor of protein kinase C. At concentrations greater than or equal to 20 microM, propranolol reduced [3H]phorbol dibutyrate binding (IC50 = 200 microM) and phorbol myristate acetate-stimulated superoxide anion release (IC50 = 130 microM) in human neutrophils. Scatchard analysis showed that propranolol lowers the number of phorbol diester binding sites without significantly affecting their affinity. In vitro kinetic analysis, performed in a mixed micellar assay with protein kinase C purified from human neutrophils, suggested a competitive inhibition of propranolol with the cofactor phosphatidylserine. Complex kinetic patterns were observed with respect to diacylglycerol and ATP, approximating competitive and noncompetitive inhibition, respectively. Taken together, these results suggest that the drug interacts at the level of the regulatory domain of the enzyme. Fifty % inhibition occurred at approximately 150 microM propranolol. Similar levels of inhibition were obtained using exogenous (histone) and endogenous (p47-phox, a NADPH oxidase component) substrates. Protein kinase C-alpha and protein kinase C-beta, two protein kinase C isozymes present in human neutrophils, were inhibited by propranolol in a comparable manner. In the range of concentrations tested (30-1000 microM), neither cAMP-dependent protein kinase nor neutrophil protein tyrosine kinases were affected. The racemic form of propranolol and the (+) and the (-) stereoisomers were equally active, and other beta-adrenergic receptor antagonists (pindolol) and agonists (isoproterenol) were inactive. This suggests that the inhibitory action of propranolol on protein kinase C is related to the amphipathic nature of the drug rather than to its beta-adrenergic receptor blocking ability. Analogs of propranolol were synthesized and found to be more potent protein kinase C inhibitors, with IC50 values in the 10-20 microM range. We conclude that the ability of propranolol to inhibit both protein kinase C and PA phosphohydrolase complicates interpretation of results when this drug is used in signal transduction studies. In addition, propranolol may be a useful prototype for the synthesis of new protein kinase C inhibitors.     

Ribofuranosyl-benzimidazole derivatives as inhibitors of casein kinase-2 and casein kinase-1

5,6-Dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DiCl-RB) is a powerful inhibitor of casein kinase-2 (CK-2) [Zandomeni, R. et al. (1986) J. Biol. Chem. 261, 3414-3420]. Here a series of 17 analogues of DiCl-RB has been employed for studying the specificity and the mode of action of this family of CK-2 inhibitors. The two halogen substituents on the benzene ring are shown to play a prominent role in inhibition, the 5,6-dibromo derivative (DiBr-RB) being fivefold more effective than DiCl-RB (Ki = 2 microM, with GTP as substrate), whereas the difluoro derivative (DiF-RB) is nearly as ineffective as unsubstituted 1-(beta-D-ribofuranosyl)benzimidazole. On the other hand, although some modifications of the ribose group significantly decrease the inhibitory efficiency, the sugar moiety is not strictly required, since dichlorobenzimidazole itself (DiCl-Bz) is an inhibitor almost as good as DiCl-RB. Inhibition of CK-2 by DiCl-RB and by its analogues, DiCl-Bz included, is of the competitive type with respect to the nucleotide substrate, the Ki values being lower with GTP than with ATP. The Ki values of the most potent inhibitor, DiBr-RB, with ATP and GTP, are 6 microM and 2 microM, respectively, denoting an affinity for the enzyme higher than that of the physiological substrates, ATP and GTP. DiBr-RB has been assayed for its inhibitory capacity toward several protein kinase other than CK-2. Protein kinase-C, cAMP-dependent protein kinase, the Ser/Thr protein kinase expressed by Pseudorabies virus, and four different tyrosine protein kinases from spleen, proved insensitive to DiBr-RB concentrations capable of almost entirely suppressing the activity of rat liver and maize seedling CK-2. Casein kinase-1 however is nearly as sensitive as CK-2 to DiBr-RB. Inhibition of CK-1 is also of the competitive type with respect to ATP (Ki = 14 microM). Although the inhibitory spectrum of CK-1 by the various analogues is reminiscent of that observed with CK-2, a remarkable difference is revealed by 5'-phosphorylation of ribose which increases the Ki with CK-2 while decreasing that with CK-1.     

Inhibitory effect of mitoxantrone on activity of protein kinase C and growth of HL60 cells.

Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.     

Differential regulation of mouse equilibrative nucleoside transporter 1 (mENT1) splice variants by protein kinase CK2

Nucleosides are accumulated by cells via a family of equilibrative transport proteins (ENTs). An alternative splice variant of the most common subtype of mouse ENT (ENT1) has been identified which is missing a protein kinase CK2 (casein kinase 2) consensus site (Ser(254)) in the central intracellular loop of the protein. We hypothesized that this variant (mENT1a) would be less susceptible to modulation by CK2-mediated phosphorylation compared to the variant containing the serine at position 254 (mENT1b). Each splice variant was transfected into nucleoside transporter deficient PK15 cells, and stable transfectants assessed for their ability to bind the ENT1-selective probe [(3)H]nitrobenzylthioinosine (NBMPR) and to mediate the cellular uptake of [(3)H]2-chloroadenosine, with or without treatment with the CK2 selective inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). mENT1a had a higher affinity for NBMPR relative to mENT1b - measured both directly by the binding of [(3)H]NBMPR, and indirectly via inhibition of [(3)H]2-chloroadenosine influx by NBMPR. Furthermore, incubation of mENT1b-expressing cells with 10 microM TBB for 48 h decreased both the K(D) and B(max) of [(3)H]NBMPR binding, as well as the V(max) of 2-chloroadenosine uptake, whereas similar treatment of mENT1a-expressing cells with TBB had no effect. PK15 cells transfected with hENT1, which has Ser(254), was similar to mENT1b in its response to TBB. In conclusion, inhibition of CK2 activity, or deletion of Ser(254) from mENT1, enhances transporter affinity for the inhibitor, NBMPR, and reduces the number of ENT1 proteins functioning at the level of the plasma membrane.     

An unbiased evaluation of CK2 inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2alpha and CK2alpha' were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors.