Inhibition of phospholipid/Ca2+-dependent protein kinase and phosphorylation of leukemic cell proteins by CP-46,665-1, a novel antineoplastic lipoidal amine

CP-46,665-1, an antineoplastic lipoidal amine, was found to inhibit phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C), with an IC50 (concentration causing a 50% inhibition) of 10 microM. Its inhibition of the enzyme was reversed by phosphatidylserine, but not by Ca2+. The agent also inhibited the enzyme activity which was further augmented by 12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein or diolein. Phosphorylation of endogenous proteins from HL-60 cells by the enzyme, with or without being further augmented by TPA, was inhibited by CP-46,665-1 as well as by alkyllysophospholipid (an antineoplastic agent). CP-46,665-1, while without effect on cyclic AMP-dependent protein kinase, also inhibited myosin light chain kinase (a calmodulin/Ca2+-dependent protein kinase). The present findings suggest that inhibition of the Ca2+-effector enzymes may be related in part to the antimetastatic activity of the lipoidal amine.     

Calphobindins (placental annexins) inhibit protein kinase C.

Calphobindins (CPBs, placental annexins) are intracellular Ca(2+)- and phospholipid-dependent proteins like protein kinase C [EC 2.7.1.37]. We investigated the inhibitory effects of calphobindins on the protein kinase C activity in vitro. CPB I inhibited the protein kinase C activity for both histone phosphorylation and lipocortin phosphorylation, but CPB II and CPB III inhibited only the protein kinase C activity for histone phosphorylation. In the case of histone phosphorylation, all CPBs inhibited the protein kinase C activity in a concentration-dependent manner, and the IC50 (concentration required for 50% inhibition) value of CPB I was 70 nM. The inhibition of protein kinase C by CPB I was Ca(2+)-dependent, and did not disappear upon increasing the concentration of phosphatidyl-serine. Kinetic analysis by double-reciprocal plots indicated that CPB I interacted not only with phosphatidylserine but also with protein kinase C. Although CPB I partially interacts with phospholipid, it is conceivable that the inhibitory action of CPB I on protein kinase C results from direct interaction of CPB I with protein kinase C. Since CPBs are mainly present under the plasma membrane, it is presumed that CPB I is an endogenous inhibitor of protein kinase C, and according to intracellular circumstances, CPB II and CPB III may also be endogenous inhibitors.     

Membrane interactions of amphiphilic polypeptides mastoparan, melittin, polymyxin B, and cardiotoxin. Differential inhibition of protein kinase C, Ca2+/calmodulin-dependent protein kinase II and synaptosomal membrane Na,K-ATPase, and Na+ pump and differentiation of HL60 cells.

Interactions of certain naturally occurring, amphiphilic polypeptides with membranes were investigated. Mastoparan (wasp venom toxin), melittin (bee venom toxin), cardiotoxin (cobra venom toxin), and polymyxin B (antibacterial antibiotic) inhibited protein kinase C stimulated by phosphatidylserine bilayer or arachidonate monomer and blocked binding of [3H] phorbol 12,13-dibutyrate to protein kinase C in the presence of phosphatidylserine bilayer, with IC50 values (concentrations causing 50% inhibition) of 1-8 microM. Mastoparan and polymyxin B were much less inhibitory (IC50, 10-20 microM), whereas melittin and cardiotoxin were similarly inhibitory (IC50, 1-4 microM), when protein kinase C was activated instead by synaptosomal membrane. Kinetic analysis indicate that mastoparan inhibited protein kinase C, assayed using phosphatidylserine or synaptosomal membrane as the phospholipid cofactor, competitively with the phospholipid cofactor, in a mixed manner with CaCl2 or diacylglycerol, noncompetitively with histone, and uncompetitively with ATP, with apparent Ki values of 1.6-18.7 microM. Inhibition of Na,K-ATPase in the membrane by these polypeptides had relative potencies different from those for their inhibition of protein kinase C activated by the same membrane preparation; mastoparan and melittin inhibited the two activities with comparable potencies, but polymyxin B and cardiotoxin were far less effective in inhibiting Na,K-ATPase. The same relative inhibitory potencies of the polypeptides (melittin greater than mastoparan greater than polymyxin B) for inhibition of Na,K-ATPase were also noted for their inhibition of Ca2+/calmodulin-dependent protein kinase II, 86Rb uptake (Na+ pump) by HL60 cells and the phorbol ester-induced differentiation of the leukemia cells. These findings were consistent with discrete interactions of the polypeptides with functionally distinct sites on the membrane, leading to differential inhibition of biological activities associated with the membrane. Actions of certain polypeptides appeared to be more specific compared to those of lipid second messengers such as lyso-phosphatidylcholine and sphingosine, and the antineoplastic ether lipid analogs such as 1-O-octadecyl-2-methyl-rac-glycero-3-ophosphocholine.     

N-alpha-Tosyl-L-lysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C

TLCK (N-alpha-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca2+ and phospholipid. TLCK (IC50 = 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) (IC50 = 8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.     

Terbium as a luminescent probe of metal-binding sites in protein kinase C

In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.     

Second messengers in platelet aggregation evoked by serotonin and A23187, a calcium ionophore.

We investigated the combined effect of 5-hydroxytryptamine (5-HT, serotonin) and calcium ionophore (A23187) on human platelet aggregation. Aggregation, monitored at 37 degrees C using a Dual-channel Lumi-aggregometer, was recorded for 5 min after challenge by a change in light transmission as a function of time. 5-HT (2-200 microM) alone did not cause platelet aggregation, but markedly potentiated A23187 (low dose) induced aggregation. Inhibitory concentration (IC50) values for a number of compounds were calculated as means +/- SEM from dose-response determinations. Synergism between 5-HT (2-5 microM) and A23187 (0.5-2 microM) was inhibited by 5-HT receptor blockers, methysergide (IC50 = 18 microM) and cyproheptadine (IC50 = 20 microM), and calcium channel blockers (verapamil and diltiazem, IC50 = 20 microM and 40 microM respectively). Interpretation of the effects of these blockers is complicated by their lack of specificity. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect at an IC50 value of 9.2 microM. Wortmannin, a phosphatidylinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC50 = 2.6 microM). However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C inhibitor, affected aggregation at concentrations up to 10 microM. We conclude that the synergistic interaction between 5-HT and ionophore may be mediated by activation of PLC/Ca2+ and PI 3-kinase signalling pathways, but definitive proof will require other enzyme inhibitors with greater specificity.     

Effects of inhibitors on aldolase breakdown after its microinjection into HeLa cells.

The tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces insulin secretion from isolated pancreatic islets, and this suggests a potential role for protein kinase C in the regulation of stimulus-secretion coupling in islets. In the present study, the hypothesis that the insulinotropic effect of TPA is mediated by activation of protein kinase C in pancreatic islets has been examined. TPA induced a gradual translocation of protein kinase C from the cytosol to a membrane-associated state which correlated with the gradual onset of insulin secretion. The pharmacologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not mimic this effect. TPA also induced a rapid time-dependent decline of total protein kinase C activity in islets and the appearance of a Ca2+- and phospholipid-independent protein kinase activity. Insulin secretion induced by TPA was completely suppressed (IC50 approximately 10 nM) by staurosporine, a potent protein kinase C inhibitor. Staurosporine also inhibited islet cytosolic protein kinase C activity at similar concentrations (IC50 approximately 2 nM). In addition, staurosporine partially (approximately 60%) inhibited glucose-induced insulin secretion at concentrations (IC50 approximately 10 nM) similar to those required to inhibit TPA-induced insulin secretion, suggesting that staurosporine may act at a step common to both mechanisms, possibly the activation of protein kinase C. However, stimulatory concentrations of glucose did not induce down-regulation of translocation of protein kinase C, and the inhibition of glucose-induced insulin release by staurosporine was incomplete. Significant questions therefore remain unresolved as to the possible involvement of protein kinase C in glucose-induced insulin secretion.     

Monoclonal antibody recognition of two subtype forms of protein kinase C in human platelets

Using a hydroxylapatite column chromatographic technique, we obtained the evidence for two subtype forms of protein kinase C in human platelets. These subtypes had a similar chromatographic property to Type II, Type III protein kinase C from the rabbit brain. In addition, in monoclonal antibodies (MC-1a, 2a, 3a) (1) which reacted with specifically Type I, II, III rabbit brain protein kinase C, respectively, only MC-2a and MC-3a reacted with human platelet protein kinase C. All these brain and platelet subtypes have a similar Km value for ATP, the range being from 8.0 to 20.0 microM and a similar IC50 value with regard to the effect of the protein kinase C inhibitor, H-7. Thus, the possibility that specific functions of platelet may be derived from a deficiency of Type I protein kinase C warrants attention.     

Effect of spermine on the activity of synaptosomal plasma membrane Ca(2+)-ATPase reconstituted in neutral or acidic phospholipids

The activity of purified plasma membrane Ca(2+)-ATPase (PMCA) from pig brain was inhibited by spermine (a naturally occurring and highly abundant polycation in brain). The level of inhibition was dependent on the phospholipid used for reconstitution as well as on the intact or truncated state of the enzyme. An IC(50) value of 12.5 mM spermine was obtained for both, the intact protein plus calmodulin and the trypsin-digested protein, reconstituted in phosphatidylcholine (PC). In the absence of calmodulin the intact Ca(2+)-ATPase gave an IC(50) of 27 mM. This form was more sensitive to spermine inhibition when it was reconstituted with phosphatidylserine (PS), showing an IC(50) value of 2.5 mM spermine. However, the truncated form was less responsive to spermine inhibition, having an IC(50) value of 12.5 mM. Spermine has no effect on the affinity of the PMCA for Ca(2+) or ATP, but its effect on the protein is pH-dependent. It is suggested that spermine could bind to negatively charged residues on the ATPase with different accessibility, depending on the structural rearrangement of the protein. Further, when the protein is reconstituted in PS, spermine also binds to the lipid.     

Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C

Sphingosine inhibited [3H]methylhistidine-thyrotropin-releasing hormone (MeTRH) binding to intact GH3 cells and to GH3 membranes. This inhibition was dependent on the concentration of sphingosine and on the ratio of sphingosine to cell number (or membrane protein) and was partly reversed by washing. In intact cells, the IC50 was 63 microM (1.8 X 10(6) cells/ml; 2 nM MeTRH), and 100 microM sphingosine was found, by Scatchard analysis, to increase the apparent dissociation constant (Kd) from 1.1 +/- 0.3 to 6.5 +/- 2.3 nM and to decrease the maximal binding capacity (Bmax) to 41 +/- 9.5% of control. Kinetic analysis showed that the major effect of sphingosine on Kd was due to a marked decrease in the apparent association rate constant for MeTRH from 2.5 +/- 0.4 X 10(5) M-1 s-1 to 0.10 +/- 0.015 X 10(5) M-1 s-1. At 100 microM, sterylamine was as effective as sphingosine in inhibiting MeTRH binding, whereas sphinganine was less effective, and psychosine and steroylsphingosine were without effect. The following observations show that sphingosine inhibition of MeTRH binding did not involve protein kinase C. The IC50 for sphingosine inhibition of MeTRH binding was the same in GH3 cells that had been incubated with 1 microM phorbol 12-myristate 13-acetate for 16 h, to "down-regulate" protein kinase C, as in control cells. Sphingosine inhibited MeTRH binding to membranes isolated from GH3 cells that contain very little protein kinase C activity. In GH3 membranes, 100 microM sphingosine increased the Kd for MeTRH from 3.4 +/- 0.1 to 13 +/- 3.1 nM but did not significantly decrease Bmax (12 +/- 5.0% of control, p greater than 0.05). And, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C, failed to decrease MeTRH binding to intact GH3 cells or to membranes, and did not interfere with the effects of sphingosine. These data show that sphingosine and its analogs have complex actions to inhibit MeTRH binding to GH3 cells, at least some of which are independent of protein kinase C, and thereby demonstrate that sphingolipids cannot be used as specific inhibitors of protein kinase C.     

Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C

Calphostin C (UCN-1028C), a newly isolated compound from Cladosporium cladosporioides, is a potent and specific inhibitor of protein kinase C, because it was 1000 times more inhibitory to protein kinase C (IC50, 0.05 microM) than other protein kinases such as cAMP-dependent protein kinase and tyrosine-specific protein kinase (IC50, greater than 50 microM). Calphostin C did not inhibit calcium activated neutral protease (calpain)-digested protein kinase C, indicating that it interacts with the regulatory domain of protein kinase C. In addition this compound showed inhibitory effects on the binding of [3H]PDBu to protein kinase C. The potent cytotoxic activity and antitumor activity of calphostin C might be due to the inhibition of protein kinase C, and thus it may be potentially useful for the therapeutic application.     

A continuous fluorescence assay for protein kinase C

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.     

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.     

Ca2+/calmodulin mediated pathways regulate the uptake of L-DOPA in mouse neuroblastoma neuro 2A cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca2+/calmodulin mediated pathways on the uptake of L-DOPA through the L-type amino acid transporter in Neuro 2A cells, an in vitro model of neuronal cells. Non-linear analysis of the saturation curve for L-DOPA revealed a Km value (in microM) of 54+/-2 and a Vmax value (in nmol mg protein/6 min) of 34+/-1. L-DOPA uptake was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 mM), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC50=82 microM). The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC50's of 33 and 105 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutirate, phorbol 12-myristate 13-acetate and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in Neuro 2A cells is promoted through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin mediated pathways.     

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.     

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.     

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).     

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.     

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.     

Plasma from uninephrectomized rats stimulates phospholipid methylation and arachidonic acid release in renal-cortical slices.

Phospholipid methylation and arachidonic acid release in renal-cortical slices was investigated in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats ('uni-plasma') stimulated phospholipid methylation when obtained within the first 3 h after uninephrectomy. With different amounts of added plasma a graded response in phospholipid methylation was obtained. Addition of 50 nM-12-O-tetradecanoylphorbol 13-acetate for 10 min to intact slices also stimulated phospholipid methylation, whereas incubation of slices before addition of 'uni-plasma' with 100 microM-1-(5-isoquinolinylsulphonyl)-2-methylpiperazine prevented it, suggesting that protein kinase C stimulates phospholipid methylation in renal-cortical slices. Plasma from uninephrectomized rats also stimulates [3H]arachidonic acid release from phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) via activation of phospholipase A2. Two mechanisms of phospholipase A2 activation are proposed: first, in which it is activated by protein kinase C and releases 3H radioactivity from PtdCho, and second, in which phospholipase A2 is stimulated by Ca2+ ions and releases 3H radioactivity from PtdEtn.