Calmodulin, activated cyclic nucleotide phosphodiesterase, microtubules, and vinca alkaloids

Calmodulin 1.8 and 10.6 microM, inhibited the polymerization of bovine brain microtubules by 30 and 50%, respectively. Two 55,000- to 68,000-dalton calmodulin-binding protein as well as calmodulin-dependent and -independent phosphodiesterases (PDE) were found associated with microtubule proteins. Among the antimicrotubule drugs, such as colchicine, podophyllotoxin, griseofulvin, and vinca alkaloids (vinblastine, desacetylvinblastine amide, and vincristine), the vinca alkaloids were selective inhibitors of calmodulin-activated PDE activity. This action of vinca alkaloids resides in the catharanthine moiety of vinblastine molecule. An alpha 2 inhibitor, yohimbine, affects the microtubules, and a series of alpha-adrenoceptor blocking agents were examined for their effects on calmodulin-dependent PDE. The relative order of the potency is phenoxybenzamine = dibenamine greater than phentolamine greater than yohimbine greater than prazosin greater than tolazoline, and the first four drugs in this series were selective inhibitors of calmodulin action. Inasmuch as phenoxybenzamine and dibenamine are alkylating agents, the effects of antineoplastic alkylating agents on the calmodulin action were also examined. Busulphan, melphalan, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and streptozotocin (up to 4 mM) were not selective inhibitors of calmodulin action. Maytansine, a vinca alkaloid-type antimicrotubule agents as well as an alkylating agent, selectively inhibited the calmodulin with a potency similar to vincristine. In addition, phenoxybenzamine affected Ca2+-dependent fluorescence induced by the interaction between calmodulin and hydrophobic fluorescent probes, whereas vinblastine was ineffective. However, the binding of vinblastine to calmodulin is calcium dependent. Studies such as these suggest the importance of physical and structural considerations in drugs binding to calmodulin as well as at least two different binding sites for drugs on calmodulin.     

Inhibition of Calmodulin-Stimulated Phosphodiesterase Activity by Vasoactive Intestinal Peptide

The effects of certain peptides of the glucagon family on calmodulin activity were determined from their capacity to inhibit a calmodulin-dependent form of phosphodiesterase. Vasoactive intestinal peptide and secretin were potent inhibitors of calmodulin activity, having IC50 values of 0.5 microM and 2 microM, respectively. By contrast, glucagon failed to inhibit calmodulin activity even at concentrations of 100 microM. None of these compounds significantly inhibited the basal activity of phosphodiesterase at concentrations up to 100 microM. These findings support the suggestion that important structural features of peptides for anticalmodulin activity include a net positive charge and a hydrophobic surface.     

Isolation of NPY-25 (neuropeptide Y[12-36]), a potent inhibitor of calmodulin, from porcine brain

In the present purification of low molecular weight fractions (Mr: 2000-4000) containing basic peptides, twenty nmol of novel calmodulin binding peptide, possessing a potent affinity for calmodulin, was isolated from 18 kg of porcine brain. By analysis with gas phase sequencer, the sequence was determined to be APAEDLARYYSALRHYINLITRQRY. Carboxy terminus of the peptide was determined to be Tyr-NH2. The peptide was a carboxy terminal pentacosanepeptide of neuropeptide Y and was termed NPY-25. NPY-25 competitively inhibited the activation of cAMP-phosphodiesterase through CaM binding in a Ca++ dependent fashion, but did not inhibit the basal activity of cAMP phosphodiesterase. NPY-25 elicited a more potent activity than did neuropeptide Y. IC50 values of NPY-25 and Neuropeptide Y were 0.06 microM and 0.54 microM respectively.     

Effects of ruthenium red on activation of Ca2(+)-dependent cyclic nucleotide phosphodiesterase.

Ruthenium red inhibited Ca2(+)-dependent phosphodiesterase (Ca2(+)-PDE) selectively with an IC50 value of 15 microM. Increasing calmodulin concentration in the presence of both 100 microM and 4000 microM Ca2+ completely reversed the inhibition of Ca2(+)-PDE activity by ruthenium red. Ruthenium red-induced inhibition of Ca2(+)-PDE activity was also overcome by increasing the concentration of Ca2+ in the presence of both 200 ng and 2000 ng calmodulin, in sharp contrast to fluphenazine-induced inhibition of Ca2(+)-PDE. These results indicate that ruthenium red has distinct inhibitory mechanism which differs from that of calmodulin antagonists previously reported.     

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-949: mechanism of inhibitory activity

CI-949 [5-methyl-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H- indole-2- carboxamide, L-arginine salt] inhibits human neutrophil activation in response to stimuli which promote calcium mobilization or calcium influx. This report further examines the effect of CI-949 on phosphoinositide-dependent stimulus-response coupling. At 100 microM, CI-949 had no inhibitory effect on human neutrophil phospholipase C or protein kinase C. In contrast, CI-949 inhibited FMLP-stimulated intracellular calcium mobilization with an IC50 of 8.4 microM. The compound was also a potent calmodulin antagonist, inhibiting calmodulin-dependent phosphodiesterase activity with an IC50 of 31.0 microM. The calmodulin antagonist activity of CI-949 was confirmed by fluorescence spectroscopy. These results demonstrate that CI-949 may function through inhibition of calcium- and calmodulin-dependent signal transduction processes.     

Benzylisoquinoline compounds inhibit the ability of calmodulin to activate cyclic nucleotide phosphodiesterase

Benzylisoquinoline compounds antagonised the ability of calmodulin (CaM) to stimulate the activity of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE). This 'anti-CaM' activity was related to the hydrophobicity of the non-polar terminal region of the antagonist molecule. Antagonistic potency increased with the increase of hydrophobicity; the anti-CaM activity did not change when the polar terminus was a tertiary amine or quarternary amine. The anti-CaM potency was greater for bisbenzylisoquinoline compounds than for monobenzylisoquinoline compounds. Among the bisbenzylisoquinoline compounds anti-CaM pathway was: D3 greater than D2 berbamine greater than daurisoline greater than dauricine. Compound D3, which exhibited an IC50 value of 2.8 microM, was one of the most potent calmodulin antagonists, among benzylisoquinoline compounds, so far reported.     

Inhibition of the type 1 inositol 1,4,5-trisphosphate-sensitive Ca2+ channel by calmodulin antagonists

This study describes the effects of a number of calmodulin antagonists on the cerebellar type 1 inositol 1,4,5-trisphosphate (InsP3) receptor. All the antagonists tested (trifluoperazine, fluphenazine, chlorpromazine and calmidazolium) inhibited the extent of InsP3-induced Ca2+ release (IICR) with similar IC(50) values (between 60 and 85 microM). They did not affect the efficacy of InsP3 to release Ca2+, since the concentrations of InsP3 required to cause half-maximal release was little affected in the presence of these agents. In addition, these agents did not affect InsP3 binding to its receptor. Stopped-flow studies to determine the rate constants of IICR showed this process to be biphasic with a fast and slow component. All the calmodulin antagonists appeared to reduce the rate constants for Ca2+ release in a phase-specific manner, preferentially reducing the fast phase component. Chlorpromazine (75 microM) appeared to have the most potent effect on the fast phase rate constant, reducing it from 1.0 to 0.08 s(-1), while only reducing the rate constant for the slow phase about twofold (0.2-0.08 s(-1)). The fact that calmodulin itself inhibits both IICR and InsP3 binding, while these calmodulin antagonists also reduce Ca2+ release and do not affect InsP3 binding, suggests that the mechanism of action of these agents is unlikely to be due to the reversal of the modulatory action of calmodulin on this receptor.     

Inhibition of the beta-adrenergic receptor kinase by polyanions

The beta-adrenergic receptor kinase, which specifically phosphorylates the agonist-occupied beta-adrenergic receptor, is strongly inhibited by polyanions. Heparin and dextran sulfate inhibit the enzyme with an IC50 of approximately 0.15 microM. De-N-sulfated heparin is approximately 8-fold less potent. Other acid mucopolysaccharides such as heparan sulfate and chondroitin sulfates B and C are also less effective. Polyaspartic and polyglutamic acid also inhibit with IC50 values of 1.3-2 microM. Inositol hexasulfate, with an IC50 of 13 microM is approximately 270-fold more potent than inositol hexaphosphate implicating the sulfate group as a major determinant of the inhibition. The inhibition by heparin is competitive with substrate and of mixed type with respect to ATP. Polycations also inhibit receptor phosphorylation by beta-adrenergic receptor kinase. Polylysine is more effective with an IC50 of 69 microM, while spermine (990 microM) and spermidine (2570 microM) are less potent. Polylysine, spermine, and spermidine are also able to block effectively the inhibition by heparin. The identification of compounds which specifically inhibit beta-adrenergic receptor kinase should prove useful in further defining the biological role of this enzyme.     

Selective antimitochondrial agents inhibit calmodulin

Certain cationic-lipophilic compounds are known to selectively accumulate in tumor mitochondria and inhibit energy production. Since these substances bear a structural resemblance to known inhibitors of calmodulin, we studied whether rhodamine-123 or a bis-4-aminoquinaldinium could antagonize the action of calmodulin. Rhodamine-123 (IC50 = 58 microM) and dequalinium (IC50 = 1 microM) inhibited the activity of a calmodulin-stimulated cyclic nucleotide phosphodiesterase. Propylinium, a compound similar to dequalinium except for having a 3 rather than 10 carbon alkyl bridge connecting two non-substituted quinoline rings, had no inhibitory effect. Kinetic analysis showed that dequalinium competitively inhibited calmodulin's activation of phosphodiesterase. We also studied the antiproliferative effects of the compounds on the C6 astrocytoma cell line. Rhodamine-123 and dequalinium inhibited the proliferation of this cell line while propylinium had no effect. These studies demonstrate that rhodamine-123 and dequalinium are calmodulin-antagonists and inhibit cellular proliferation.     

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.     

Indolicidin,a 13-residue basic antimicrobial peptide rich in tryptophan and proline,interacts with Ca(2+)-calmodulin

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.     

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).     

Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2.     

Affinity labelling of alpha-adrenoceptors in intact liver cells by [3H]phenoxybenzamine.

[³H]phenoxybenzamine of high specific activity (5.3 Ci/mmol) was synthesized and its binding to isolated, viable rat liver cells was studied. Phentolamine suppressible binding of [³H]phenoxybenzamine was irreversible and saturable (EC50: 10 nM, b_max: 200 fmol/mg wet cell weight). Competition-inhibition studies showed structural and stereoselectivity compatible with α-receptors. The IC50 of unlabelled phenoxybenzamine to reduce specific binding (9 nM) or to block adrenaline-induced phosphorylase activation in the same cells (2 nM) was similar, whereas the IC50 of agonists to suppress binding was higher than their EC50's for phosphorylase activation. The results represent the first example of labelling α-adrenoceptors in intact liver cells. The sites labelled by [³H]phenoxybenzamine mediate the block of phosphorylase activation by α-adrenoceptor antagonists. However, the relationship of these sites to receptors that mediate responses to physiological, low concentrations of catecholamines remains to be clarified.     

Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.     

Synthesis and biological evaluation of imidazo[1,2-a]pyridine derivatives as novel PI3 kinase p110alpha inhibitors

3-{1-[(4-Fluorophenyl)sulfonyl]-1H-pyrazol-3-yl}-2-methylimidazo[1,2-a]pyridine, 2a, was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 0.67microM, through screening in a scintillation proximity assay. Optimization of the substituents of 2a increased the p110alpha inhibitory activity by more than 300-fold (2g: IC(50)=0.0018microM). Further structural modification of 2g afforded thiazole derivative 12, which has potent p110alpha inhibitory activity (IC(50) of 0.0028microM) and is highly selective for p110alpha over other PI3K isoforms. Compound 12 also inhibited serum-induced cell proliferation of A375 and HeLa cells in vitro with IC(50) values of 0.14microM and 0.21microM, respectively, and suppressed tumor growth by 37% in a mouse HeLa xenograft model when dosed intraperitoneally at 25mg/kg. These results suggest that selective p110alpha inhibitors may have potential as cancer therapeutic agents.     

Differentiation of the drug-binding sites of calmodulin

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various 'calmodulin antagonists' for these sites was investigated. The Ki values for the inhibition of calmodulin-activated cyclic-nucleotide phosphodiesterase and myosin light-chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and II compounds inhibited competitively the activation of the phosphodiesterase and myosin light-chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light-chain kinase at identical concentrations. In contrast, group II compounds inhibited the activation of the phosphodiesterase at 5-10-fold lower concentrations than that of myosin light-chain kinase. Group III compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 microM and did not affect the activation of myosin light-chain kinase. Binding of [3H]bepridil to calmodulin under equilibrium conditions yielded one high-affinity site (apparent Kd 0.4 microM) and four low affinity sites (apparent Kd 44 microM). Group I compounds interfered with the binding of bepridil to the high and low-affinity sites in a competitive manner. Group II compounds interfered in a non-competitive manner with the high-affinity site and apparently competed only with one of the low-affinity sites. Group III compounds did not compete with any of the bepridil-binding sites. Nimodipine, a group III compound, bound to one site on calmodulin with a Kd value of 1.1 microM. Other dihydropyridines competed with [3H]nimodipine for this site. The group I and II compounds, trifluoperazine and prenylamine, did not affect the binding of [3H]nimodipine. These data show that 'calmodulin antagonists' can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.     

A new peptide (1150Da) selectively activates the calcium-calmodulin sensitive isoform of cyclic nucleotide phosphodiesterase from human myometrium.

The cyclic nucleotide phosphodiesterase enzymatic system is examined in extracts of human myometrium and four individual phosphodiesterase isoforms have been isolated and characterized. A new thermostable peptide, recently purified in rat and calf myometrium, is able to stimulate up to 55-fold, the calcium-calmodulin dependent phosphodiesterase isoform. Activation of cAMP hydrolysis is by far the most marked with a 55-fold maximal stimulation at a concentration of 0.1 microM peptide and a IC50 value estimated at 30nM. For cGMP hydrolysis, the maximal effect (x25) obtained at 40nM peptide is lesser and the IC50 value is in the 10nM range. Furthermore, we verified that classical calmodulin antagonists such as calmidazolium or trifluoroperazine did not change stimulation of the calcium-calmodulin phosphodiesterase by the peptide, indicating that the myometrial peptide is different from calmodulin. To our knowledge, this is the first evidence for such a strong and selective stimulation of one isoform of the phosphodiesterase enzymatic system by a natural peptide.     

Inhibition of calcium and calmodulin-dependent phosphodiesterase activity in rats by capsaicin

Capsaicin, reported to elevate hormone sensitive lipase (HSL), is also found to inhibit the Ca++ and calmodulin-dependent cAMP phosphodiesterase (PDE) activity in adipose tissue of rats, fed high fat diet. The dependence of the enzyme activity on Ca++ and calmodulin in vitro, in control rats, is shown by its substantial lowering in the presence of EGTA and inhibition by trifluoperazine (TFP) (IC50 between 10-20 microM). This enzyme activity is also inhibited by both red pepper extract (80% inhibition with 50 microliter) and capsaicin (IC50 between 0.3-1 microM) in a dose dependent manner. Capsaicin has been found to inhibit Ca++-dependent PDE activity by 60% in the test rats. Enzyme inhibition in vivo, due to capsaicin, was overcome by addition of calmodulin to the assay system. Inclusion of fluphenazine or capsaicin in assay inhibited not only the calmodulin-restored enzyme activity from test rats but also that of control rats. These results suggest a possible mechanism for the stimulation of lipolytic activity by capsaicin in vivo.     

In vivo regulation of cyclic AMP phosphodiesterase in Xenopus oocytes. Stimulation by insulin and insulin-like growth factor 1

Cyclic AMP phosphodiesterase activity was measured in vivo after microinjection of [3H]cAMP into intact Xenopus oocytes. This activity was inhibited by extracellular application of methylxanthines, and the dose-dependent inhibition of phosphodiesterase activity correlated with the abilities of isobutylmethylxanthine and theophylline to inhibit oocyte maturation induced by progesterone, with IC50 values of approximately 0.3 and 1.5 mM, respectively. Insulin stimulated in vivo phosphodiesterase activity measured after microinjection of 200 microM [3H]cAMP in a time- and dose-dependent fashion without affecting phosphodiesterase activity measured after microinjection of 2 microM [3H]cAMP. Although progesterone alone had no effect on in vivo phosphodiesterase activity, low concentrations of progesterone (0.01 microM) accelerated the time course of insulin stimulation of both phosphodiesterase activity and oocyte maturation. The EC50 for stimulation of in vivo phosphodiesterase activity by insulin correlated with the IC50 for inhibition of oocyte membrane adenylate cyclase activity measured in vitro (2 and 4 nM, respectively). Twenty-fold higher concentrations of insulin were required to stimulate oocyte maturation. In contrast, insulin-like growth factor 1 stimulated in vivo phosphodiesterase, inhibited in vitro adenylate cyclase, and induced oocyte maturation at concentrations of 0.3-1.0 nM. These results demonstrate a dual regulation of oocyte phosphodiesterase and adenylate cyclase by insulin and insulin-like growth factor 1.