CGS 9343B, a novel, potent, and selective inhibitor of calmodulin activity

1,3-Dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]- benzoxazepin-4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-o ne (1:1) maleate was synthesized in six steps from methyl anthranilate and designated CGS 9343B. CGS 9343B inhibited calmodulin-stimulated cAMP phosphodiesterase activity with an IC50 value of 3.3 microM. CGS 9343B was 3.8 times more potent than trifluoperazine (IC50 = 12.7 microM) as an inhibitor of calmodulin activity. CGS 9343B did not inhibit protein kinase C activity at concentrations up to 100 microM, whereas trifluoperazine inhibited protein kinase C activity with an IC50 value of 43.9 microM. CGS 9343B weakly displaced [3H]spiperone from postsynaptic dopamine receptors with an IC50 value of 4.8 microM while the value for trifluoperazine, a potent antipsychotic agent, was 0.018 microM. It is concluded that CGS 9343B is a novel, potent, and selective inhibitor of calmodulin activity. Unlike trifluoperazine, CGS 9343B does not inhibit protein kinase C activity and does not possess potential antidopaminergic activity.     

Inhibition of membrane currents and rises of intracellular Ca2+ in PC12 cells by CGS 9343B, a calmodulin inhibitor.

The calmodulin inhibitor 1,3-dihydro-1-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin - 4-yl)methyl)-4-piperidinyl]-2H-benzimidazol-2-one maleate (CGS 9343B) caused a reversible block of voltage-activated Ca2+, Na+, and K+ currents in differentiated rat pheochromocytoma (PC12) cells. The drug also inhibited nicotinic acetylcholine receptor (nAChR) channel currents but not inward currents evoked by extracellular ATP. Depolarization-induced intracellular Ca2+ transients were almost completely inhibited in growth cones and cell bodies by CGS 9343B. Our results suggest actions of CGS 9343B on ion fluxes unrelated to calmodulin inhibition.     

Effect of the calmodulin inhibitor trifluoperazine on phosphorylation of P-glycoprotein and topoisomerase II: relationship to modulation of subcellular distribution, DNA damage and cytotoxicity of doxorubicin in multidrug resistant L1210 mouse leukemia cells.

The results from the present study using the sensitive and progressively DOX resistant L1210 model system demonstrated that the effects of TFP are not due to redistribution of DOX to the nucleus, and modulation of cytotoxicity is related to effects on DOX-induced DNA strand breaks. Although TFP affects phosphorylation of PGP and TOPO II (R2 greater than R1), the comparable DNA strand breaks at lower DOX levels with TFP in the resistant sublines suggest that modulation of TOPO II function related to drug-induced DNA damage by calmodulin-mediated events may be an important mode of action.     

Differential effect of the calmodulin inhibitor trifluoperazine in modulating cellular accumulation, retention and cytotoxicity of doxorubicin in progressively doxorubicin-resistant L1210 mouse leukemia cells. Lack of correlation between cellular doxorubicin levels and expression of resistance

Calmodulin inhibitors are effective in enhancing cytotoxic effects of doxorubicin (DOX) in DOX-resistant cells, possibly by enhancing cellular levels of drug. In the present study, L1210 mouse leukemia cells adapted to grow in vitro, in the presence of 0.025 to 0.25 microgram/ml DOX, and identified as L1210/DOX0.025, L1210/DOX0.05, L1210/DOX0.1, and L1210/DOX0.25 were approximately 5-, 10-, 20-, and 40-fold DOX resistant, respectively, compared to parent-sensitive cells (L1210/S). Using a soft agar colony assay and 3-hr drug exposure, the IC50 concentration of DOX in the progressively DOX-resistant (5- to 40-fold) L1210 cells ranged from 0.25 to 2.0 micrograms/ml and from 0.08 to 0.25 microgram/ml in the absence and presence of a non-cytotoxic concentration of 5 microM trifluoperazine (TFP) respectively. Further, based on the observed in vitro cytotoxic response, the IC50 concentration of DOX in the presence of 5 microM TFP was 2.5-, 4-, 6.7- and 8-fold lower than DOX without 5 microM TFP in the L1210/DOX0.025, L1210/DOX0.05, L1210/DOX0.1, and L1210/DOX0.25 resistant sublines respectively. In contrast, the IC50 of DOX in L1210/S cells was approximately 0.05 microgram/ml with or without 5 microM TFP. Cellular accumulation of DOX was 15-50% lower in the progressively resistant L1210 sublines compared to similarly treated L1210/S cells. However, in the presence of 5 microM TFP, cellular accumulation of DOX in the L1210/DOX0.05 and L1210/DOX0.1 but not L1210/DOX0.25 was comparable to the L1210/S cells. Cellular retention of DOX in the absence or presence of 5 microM TFP was comparable in similarly treated L1210/S, L1210/DOX0.05 and L1210/DOX0.1 cells, and a 2-fold reduction in the retention of DOX in the absence versus the presence of 5 microM TFP was apparent only in L1210/DOX0.25 cells. At the IC50 of DOX in the presence of 5 microM TFP, although cellular accumulation of DOX was concentration dependent over the range of 1-20 microM TFP, enhancement in cytotoxicity of DOX was dose dependent at 1-5 microM TFP but not 5-20 microM TFP. In cells treated for 3 hr at the IC50 concentration of DOX alone or DOX plus 5 microM TFP, cellular accumulation of DOX was 7- to 14-fold and 2.5- to 3.5-fold higher, respectively, in resistant than in sensitive cells. Additionally, following treatment for 3 hr at the IC50 dose of DOX in the absence or presence of 5 microM TFP, drug retention at 3 hr was 4- to 6-fold and 1.5-fold higher, respectively, in the resistant versus sensitive cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of CGS 9343B, an inhibitor of calmodulin activity, on histamine release from isolated rat mast cells.

Investigations of calmodulin involvement in cell responses has been complicated by the lack of selective calmodulin antagonists. A novel inhibitor, CGS 9343B, reportedly without influence on protein kinase C, is used in the present study of mast cell responses. The histamine release induced by antigen and compound 48/80 in the presence of calcium was enhanced by 10-20 microM CGS 9343B and inhibited by higher concentrations. Only inhibitory effects on the response to compound 48/80 in the absence of calcium and to the ionophore A23187 were observed, the latter being inhibited by 20 microM CGS 9343B. The influence on responses to combinations of the phorbol ester TPA and the ionophore A23187 was more complex, giving rise to enhancement at lower and inhibition at higher concentrations of CGS 9343B in a manner which depended on the experimental conditions. Unlike previously used calmodulin antagonists, CGS 9343B is devoid of detergent effects and without serious metabolic interference. The inhibitor seems useful to reveal differences in the mechanisms involved in responses to various histamine liberators. Our results conform with an inhibition of calmodulin by CGS 9343B but are at present inconclusive.     

Relationship between expression of P-glycoprotein and efficacy of trifluoperazine in multidrug-resistant cells.

Tumor cell resistance due to enhanced efflux of drugs with diverse structures and/or mechanisms of action is termed multidrug resistance (MDR), and modulation of the MDR phenotype by calcium blockers or calmodulin inhibitors is suggested to involve P-glycoprotein. In drug-sensitive (S) and 5-fold doxorubicin (DOX)-resistant (R0) L1210 mouse leukemia cells, no obvious differences in mdr mRNA or P-glycoprotein expression or alterations in cellular uptake, retention, or cytotoxicity of vincristine (VCR) were observed. However, in the 10-fold (R1) and 40-fold (R2) DOX-resistant sublines, expression of P-glycoprotein was correlated with the level of resistance (R2 greater than R1). An RNase protection assay revealed that elevated levels of mdr1 and mdr2 mRNA were detected in R1 and R2 cells, with an additional increase in mdr3 mRNA in the R2 subline. Further, in the R1 and R2 sublines, no VCR dose-dependent cytotoxicity was apparent, and cell kill of greater than 40% was not achievable following a 3-hr drug exposure. Cellular uptake and retention of VCR were 2- to 4-fold lower in the R1 and R2 sublines, compared with similarly treated S or R0 cells. Potentiation of VCR cytotoxicity by a noncytotoxic concentration of 5 microM trifluoperazine (TFP) was greater than 2-fold in S and R0 cells and less than 1.3-fold in the R1 and R2 sublines. Modulation of VCR uptake by 5 microM TFP in the S and R0 cells was 2-fold and it was 4- to 7-fold in the R1 and R2 sublines. The presence of 5 microM TFP, by competing for efflux, enhanced VCR retention 1.5-fold in S and R0 cells and 2- to 4-fold in the R1 and R2 sublines. In contrast to these results with VCR, dose-dependent cytotoxicity of DOX was apparent in all the resistant sublines, and modulation of DOX cytotoxicity by 5 microM TFP was dependent on the level of resistance. Cellular accumulation of DOX was 20 and 50% lower in the R1 and R2 sublines, respectively, compared with similarly treated S or R0 cells. Marked increases (greater than 1.5-fold) in cellular accumulation of DOX by TFP were apparent only in the R2 subline. Results suggest that a relationship between overexpression of P-glycoprotein isoforms and their role in affecting cellular drug levels and consequent cytotoxicity in MDR L1210 cells determines resistance to VCR but not DOX.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of the calmodulin antagonist CGS 9343B on skin burns

1. CGS 9343B is a novel, potent and selective inhibitor of calmodulin activity, which unlike other known calmodulin antagonists, does not inhibit protein kinase C activity and does not possess potential antidopaminergic activity. Here we show that CGS 9343B, like other calmodulin antagonists reported previously, is most effective in treatment of burns. 2. The effectiveness of CGS 9343B on skin burns was evaluated by electron microscopic studies, as well as by measurements of hemoglobin, ATP and enzymes which are markedly changed in the burned skin. 3. As CGS 9343B is a more selective probe for calmodulin function than other inhibitors, the similarity of its effects on burns to that of other calmodulin antagonists, strongly suggest that their action on burns is mediated through calmodulin inhibition.     

Differential regulation by Ca(2+) of calmodulin- and PKC-dependent contractile pathways in cat lower oesophageal sphincter

1. In the present investigation we examined the regulation of calmodulin (CaM)- and protein kinase C (PKC)-dependent pathways by cytosolic Ca(2+) in the contraction of cat lower oesophageal sphincter (LES). 2. Force developed in response to increasing doses of acetylcholine (ACh) was directly related to the increase of the [Ca(2+)](i) measured by fura-2. Thapsigargin, which depletes Ca(2+) stores, reduced the contraction and the [Ca(2+)](i). In addition, contraction in response to maximal ACh was reduced by the CaM inhibitor CGS9343B but not by the PKC inhibitor chelerythrine. The contraction in response to submaximal ACh was reduced by chelerythrine but not by CGS9343B. 3. In permeabilized cells, the contraction in response to low Ca(2+) (0.54 microm) was also reduced by CGS9343B. 4. The response to high Ca(2+) (1.0 microm) was reduced by CGS9343B. ACh also inhibited PKC activation induced by diacylglycerol, which activation is inhibited by the N-myristoylated peptide inhibitor derived from pseudosubstrate sequences of PKCalphabetagamma (myr-PKC-alphabetagamma), but not of myr-PKC-alpha. 5. These data are consistent with the view that activated CaM-dependent pathways inhibit PKC-dependent pathways, this switch mechanism might be regulated by Ca(2+) in the LES.     

CGS 9343B and W7 (calmodulin antagonists) inhibit KCl-induced increase in cytosolic free calcium and insulin secretion of RINm5F cells

Summary CGS 9343B:1,3-Dihydro-l-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin-4-yl)methyl)-4-piperidinyl]-2 H-benzimidazol-2-one maleate and W7: N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide) are calmodulin antagonists with different specificities. The effects of CGS 9343B and W7 on cytosolic free calcium concentration ([Ca²⁺]_i) and insulin release were investigated in rat insulinoma cells (RINm5F). As measured with the Quin-2 technique, preincubation with CGS 9343B (0.3–10 M) and W7 (5–50 M) concentration dependently decreased KCl (25 mM)-mediated accumulation of cytosolic calcium. Both, CGS 9343B (10 M) and W7 (50–100 M) almost abolished the alanine- and KCl-induced increase in [Ca²⁺]_i and significantly inhibited KCl (25 mM)- and alanine (10 mM)-mediated insulin release. W5 (100 LM), the chlorine-deficient analogue of W7 with decreased affinity for calmodulin, did not inhibit the KCl-induced increase in [Ca²⁺]_i and enhanced basal and KCl-mediated insulin release by 56% and 189%, respectively. Our data suggest that CGS 9343B and W7 inhibit the depolarization-induced calcium uptake and subsequent increase in [Ca²⁺]_i. Send offprint requests to H. P. T. Ammon at the above address     

Factors governing the modulation of vinca-alkaloid resistance in doxorubicin-resistant cells by the calmodulin inhibitor trifluoperazine

Calmodulin inhibitors enhance the cytotoxic effects of doxorubicin (DOX) in DOX-resistant (P388/DOX) P388 mouse leukemia cells by augmenting cellular accumulation and retention of drug. In P388/DOX cells which are cross-resistant to vinblastine (VLB) and vincristine (VCR), cell kill following treatment with VLB and VCR alone was evident only after 12 hr of treatment. Additionally, the 2- to 10-fold increase in cytotoxicity of the vinca alkaloids in the presence of 2 and 4 microM trifluoperazine (TFP) was observed only in P388/DOX cells treated for 12 hr, but not for 3 or 6 hr. However, in DOX-sensitive (P388/S) P388 mouse leukemia cells, cytotoxic effects of VCR but not VLB were apparent after treatment for 3 hr, and cell kill with VLB and VCR was enhanced 2- to 20-fold in the presence of 2 and 4 microM TFP following treatment for 12 hr. Cellular accumulation of [3H]VLB in P388/DOX cells was 12-fold lower than in similarly treated P388/S cells and, in the presence of 2 and 4 microM TFP, cellular VLB levels were enhanced 1.3- to 2.0-fold in P388/S cells and 2- to 8-fold in P388/DOX cells. The effect of TFP in increasing cellular retention of [3H]VLB was more apparent with P388/DOX cells, and retention of [3H]VLB in the presence of 4 microM TFP was enhanced less than 1.5-fold and greater than 4-fold in P388/S and P388/DOX cells respectively. Results from this study and our earlier observations with DOX and TFP in P388/DOX cells demonstrate that: (1) TFP potentiates the cytotoxicity of VLB and VCR in P388/S and P388/DOX cells by augmenting drug accumulation and retention; (2) enhanced cell kill in the presence of TFP with P388/DOX cells is apparent at 1 hr for DOX vs 12 hr for VLB and VCR; and (3) in P388/S cells, TFP has a more striking effect on the cellular accumulation, retention and cytotoxicity of VLB and VCR rather than DOX.     

Inhibition of mechanotransducer currents in crayfish sensory neuron by CGS 9343B, a calmodulin antagonist

The effects of CGS 9343B (zaldaride maleate), a calmodulin antagonist, on mechanosensitive channels were examined in crayfish slowly adapting sensory neurons using the two-electrode voltage clamp technique. In addition to its inhibition of voltage-gated Na(+) and K(+) currents, CGS 9343B (<30 microM) blocked reversibly the receptor current in a dose-dependent and voltage-dependent manner with a dissociation constant (K(d)) of 26.8 microM. The time course of the block was 265 s. Within the extension range of 3-30%, the reduction in receptor current was stimulus-independent and the gating mechanisms were not affected. Extracellular Ca(2+) was not necessary for its blocking effects. No changes in passive muscle tension were observed in the presence of 20 microM CGS 9343B. These results suggest that CGS 9343B, as a calmodulin antagonist, can also block mechanosensitive channels, possibly by being incorporated into the lipid membrane and/or interacting with the channel protein.     

Improvement of ischemia-reperfusion-induced myocardial dysfunction by modulating calcium-overload using a novel, specific calmodulin antagonist, CGS 9343B

The present paper explores the mechanism of calcium-overloaded cardiac cell exocytosis during reperfusion of ischemic myocardium. A novel specific inhibitor of calmodulin, CGS 9343B, was used to pretreat an ischemic heart in an effort to enhance myocardial preservation. The experimental model employed an isolated in situ pig heart subjected to 120 min of ischemic insult by reversibly occluding the left anterior descending coronary artery, the last 60 min being superimposed with global hypothermic cardioplegic arrest. This ischemic episode was followed by 60 min of revascularization. CGS 9343B enhanced post-ischemic myocardial recovery, as judged by improved regional as well as global myocardial functions, better preservation of high-energy phosphate compounds, and reduced release of creatine kinase. Since this compound blocks calmodulin without inhibiting protein kinase C, the results of this study suggest that calmodulin-dependent kinase, rather than protein kinase C, is primarily involved in expressing calcium-overloaded cell exocytosis, and a specific calmodulin antagonist such as CGS 9343B can be used to salvage an ischemic heart from reperfusion injury.     

Inhibition by adenosine A(2A) receptors of NMDA but not AMPA currents in rat neostriatal neurons

Whole-cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A(2A) receptors in modulating N-methyl-D-aspartate (NMDA)-induced currents in rat striatal brain slices. The A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current in a subset of striatal neurons. Lucifer yellow-filled pipettes in combination with immunostaining of A(2A) receptors were used to identify CGS 21680-sensitive cells as typical medium spiny striatal neurons. Dibutyryl cyclic AMP and the protein kinase A activator Sp-cyclic AMPs, but not the protein kinase A inhibitors Rp-cyclic AMPS or PKI(14 - 24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U-73122, but not the inactive structural analogue U-73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12-myristate 13-acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1, 4,5-trisphosphate (InsP(3)) and a more efficient buffering of intracellular Ca(2+) by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680-induced inhibition. The calmodulin antagonist W-7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281 - 309) and KN-93 but not the inactive structural analogue KN-92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680. It is suggested that the transduction mechanism of A(2A) receptors to inhibit NMDA receptor channels is the phospholipase C/InsP(3)/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.     

Quinoline antifolate thymidylate synthase inhibitors: variation of the C2- and C4-substituents.

Modifications to the bicyclic ring system of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (1, CB3717) have led to the synthesis of a series of quinoline antifolates bearing a variety of substituents at the C2 and C4 positions. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate with a disubstituted 6-(bromomethyl)quinoline followed by deprotection using mild alkali. The compounds were tested as inhibitors of partially purified L1210 TS. As a measure of cytotoxicity, the compounds were tested for their inhibition of the growth of L1210 cells in culture. Good enzyme inhibition and cytotoxicity were found for compounds containing chloro, amino, or methyl substituents at the C2 position with chloro or bromo substituents at C4. The effect on enzyme inhibition of varying the N10 substituent of 2h was similar to that observed in the quinazolinone-containing antifolates, indicating that the quinoline compounds may be interacting with the enzyme in a similar way to the quinazolinones. Also, the introduction of a 2'-fluoro substituent into the benzoyl ring of several of the quinoline antifolates led to an increase in both TS inhibition and the inhibition of L1210 cell growth. These data demonstrate that the N3-H of the pyrimidine ring of the quinazolinone antifolates is not required for binding to TS if appropriate substituents are placed at the C2 and C4 positions of the bicyclic ring system.     

Metofenazate as a more selective calmodulin inhibitor than trifluoperazine

The interaction of several phenothiazines, benzodiazepines, butyrophenones, polycyclic neuroleptics and tricyclic antidepressants with calmodulin and troponin C was investigated using the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide. In the presence of Ca2+, trifluoperazine (2-trifluoromethyl-10-[3-(1-methylpiperazinyl-4)propyl]-phenothiaz ine dihydrochloride, TFP), which is commonly used as a selective calmodulin inhibitor, half maximally increased the fluorescence of the complex formed of the fluorescent dye with calmodulin at a concentration of 4 mumol/l, and with troponin C at 24 mumol/l. TFP completely inhibited the calmodulin dependent stimulation of cyclic nucleotide phosphodiesterase with a Ki of 4 mumol/l and decreased the maximum Ca2+ dependent troponin C mediated activation of actomyosin ATPase by 35% at a concentration of 100 mumol/l. Metofenazate (3,4,5-trimethoxybenzoate-2-chlor-10-(3-[(beta-oxyethyl) piperazinyl-4]-propyl)phenothiazine diethanesulfonate, methophenazine, MP) produced half maximal fluorescence enhancement of the calmodulin dye complex at a concentration of 6 mumol/l and did not influence the fluorescence of the troponin C dye complex at concentrations of up to 1000 mumol/l. MP also completely inhibited the calmodulin dependent stimulation of phosphodiesterase with a Ki of 7 mumol/l but it had not effect on maximum Ca2+ stimulation of actomyosin ATPase. MP increased the Ca2+ sensitivity of skinned cardiac muscle with an about 10fold lower potency than TFP. In view of these results, we propose MP as a useful tool for distinction between processes mediated by either calmodulin or troponin C.     

三氟拉嗪对大鼠脑突触体内Ca2＋水平和Ca2＋，Mg2＋─ATP酶活性的作用

用Ｑｕｉｎ２法测得大鼠脑突触体内静息游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）为８５±１３μｍｏｌ·ｇ－１ｐｒｏｔｅｉｎ．三氟拉嗪（ＴＦＰ）１，５和１０μｍｏｌ·Ｌ－１对静息突触体［Ｃａ２＋］ｉ无明显影响，但能以剂量依赖方式增高６５ｍｍｏｌ·Ｌ－１ＫＣｌ所致突触体［Ｃａ２＋］ｉ升高，从１９２±５８μｍｏｌ·ｇ－１ｐｒｏｔｅｉｎ分别达到２３３±６３，４３１±９９和６６１±１７３μｍｏｌ·ｇ－１ｐｒｏｔｅｉｎ．ＴＦＰ５，１０和５０μｍｏｌ·Ｌ－１分别使突触体Ｃａ２＋，Ｍｇ２＋－ＡＴＰ酶活性降低３１％，４１％和４５％；使Ｍｇ２＋－ＡＴＰ酶活性降低３０％，３６％和３９％，提示ＴＦＰ可能是通过抑制钙调素，进而抑制Ｃａ２＋，Ｍｇ２＋－ＡＴＰ酶活性，使突触体［Ｃａ２＋］ｉ升高，促进神经末梢释放递质     

Quinazoline antifolate thymidylate synthase inhibitors: alkyl, substituted alkyl, and aryl substituents in the C2 position

Modification of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop-2- ynylamino]benzoyl]-L-glutamic acid (1a) has led to the synthesis of quinazoline antifolates bearing alkyl, substituted alkyl, and aryl substituents at C2. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(alkylamino)benzoyl]-L-glutamate with a C2-substituted 6-(bromo-methyl)-3,4-dihydro-4-oxoquinazoline followed by deprotection using mild alkali. Good enzyme inhibition and cytotoxicity were found with compounds containing small nonpolar groups in the C2 position with the 2-desamino-2-methyl analogue 3a being the most potent. Larger C2 substituents were tolerated by the enzyme, but cytotoxicity was reduced. Highly potent series were followed up by the synthesis of a number of analogues in which the N10 substituent was varied. In this manner a number of interesting TS inhibitors have been prepared. Although none of these was more potent than 1a against the isolated enzyme, over half of the compounds prepared were more potent as cytotoxic agents against L1210 cells in culture. The potential of such compounds as useful antitumor agents was further enhanced by the finding that the improved aqueous solubilities of compounds such as 3a over 1a were reflected in vivo in that 3a was at least 5 times less toxic to mice than 1a.     

Role of Ca2+/calmodulin signaling pathway on morphological development of Candida albicans

A human fungal pathogen, Candida albicans, varies from the yeast form to the hyphal form due to various external signals. This morphogenetic transformation has been implicated in the development of pathogenicity. In this report, we show that calmodulin inhibitors (TFP and W-7) and an adenylatecyclase inhibitor (MDL-12-330A) suppressed the hyphae formation of C. albicans. Furthermore, the expression of hyphae-specific mRNAs located downstream from the RAS1-cAMP pathway was inhibited by these inhibitors. Suppression of hyphae formation by TFP or W-7 was not inhibited by the addition of cAMP, and these inhibitors did not affect the amount of cAMP in C. albicans. These results suggest that the Ca2+/calmodulin pathway contributes to hyphae formation and is related to the RAS1-cAMP pathway.     

Inhibition of voltage-gated Ca2+ channels and insulin secretion in HIT cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62: comparison with antagonists of calmodulin and L-type Ca2+ channels.

To probe for the involvement of Ca2+/calmodulin-dependent protein kinase II in the regulation of insulin secretion, the effects of a specific inhibitor of this enzyme, KN-62, on secretagogue-stimulated insulin secretion, cytosolic Ca2+ concentration ([Ca2+]i) rise, membrane depolarization, and nutrient metabolism were examined in HIT-T15 cells. KN-62 dose-dependently inhibited insulin secretion induced by a nutrient mixture (10 mM glucose, 5 mM leucine, and 5 mM glutamine) alone or combined with either the Ca(2+)-mobilizing receptor agonist bombesin or the cAMP-raising agent forskolin in intact cells. KN-62 did not affect Ca(2+)- or GTP analogue-induced insulin secretion from permeabilized cells, indicating an action at a step before exocytosis. The stimulating effects of nutrients on insulin secretion, [Ca2+]i, and membrane depolarization were potentiated by bombesin. Similarly, bombesin promoted a larger depolarization and [Ca2+]i rise in the presence of nutrients. This was associated with enhanced Ca2+ mobilization and the appearance of sustained [Ca2+]i elevation. The bombesin-induced membrane depolarization, like the nutrient effect, was inhibited by diazoxide, suggesting that this is due to closure of ATP-sensitive K+ channels. Bombesin elicited Ca2+ influx by both membrane potential-sensitive and -insensitive conductance pathways. KN-62 did not affect Ca2+ mobilization and only partially reduced Ca2+ entry during the sustained [Ca2+]i rise in bombesin-stimulated cells. When added before or during the stimulation, KN-62 dose-dependently inhibited nutrient- and KCl-stimulated [Ca2+]i elevation and Mn2+ influx (reflecting Ca2+ entry). The calmodulin antagonist CGS 9343B and the L-type Ca2+ channel blocker SR-7037 mimicked the inhibitory effect of KN-62 on stimulated insulin secretion and [Ca2+]i elevation. Membrane depolarization and nutrient metabolism (reduction of a tetrazolium derivative), however, were not altered by KN-62 treatment, indicating that the early coupling events from nutrient metabolism to closure of ATP-sensitive K+ channels remain operative. These results suggest that KN-62 and the calmodulin antagonist CGS 9343B inhibit Ca2+ influx by means of direct interaction with L-type Ca2+ channels, which, in turn, causes inhibition of stimulated insulin secretion. Thus, it appears that Ca2+/calmodulin-dependent protein kinase II is not involved in the regulation of insulin secretion.     

Sesamin ameliorates doxorubicin-induced cardiotoxicity: Involvement of Sirt1 and Mn-SOD pathway

Oxidative stress caused by doxorubicin (DOX) is believed to be a major underlying molecular mechanism of DOX-induced cardiotoxicity. Sesamin (Ses), an active component extracted from sesame seeds, exhibits antioxidative and anti-inflammatory effects. In the present study, possible protective mechanisms of Ses on DOX-induced cardiotoxicity were investigated in rats and cultured H9C2 cells. We demonstrated that Ses exhibits a significant protective effect on cardiac tissue in animal and cell models of DOX-induced cardiac injury. Moreover, Ses can ameliorate DOX-induced oxidative stress and mitochondrial damage. Further studies suggested that Ses is able to up-regulate the protein expression of Mn-SOD in normal rats and to restore the decreased expression of Mn-SOD in DOX-induced cardiac injury rats. Exposure to Ses or DOX alone slightly increased the protein expression of Sirt1; however, a more remarkable increase in Sirt1 protein level was detected in the Ses + DOX group. Treatment with a pan-sirtuin inhibitor (nicotinamide) or a Sirt1-specific inhibitor (EX-527) partially antagonised the effect of Ses on DOX-induced mitochondrial damage and completely abolished the effect of Ses on Mn-SOD expression. These findings indicate that the protective mechanisms of Ses on DOX-induced cardiotoxicity are involved in the alleviation of oxidative stress injury and Mn-SOD dysfunction, partially via the activation of Sirt1.