Erkki Nissinen

Cardiac troponin C as a target protein for a novel calcium sensitizing drug, levosimendan

The role of cardiac troponin C (cTnC) as a target protein for the calcium sensitization by levosimendan, pimobendan, MCI-154 and EMD 53998 was evaluated using purified recombinant human cTnC. For determination of calcium- and magnesium-dependent binding of the compounds to cTnC a new type of cTnC-HPLAC column was used. Furthermore, dansylated cTnC was utilized to study the effect of the calcium sensitizing compounds on calcium-induced conformation of cTnC. Only levosimendan showed calcium-dependent and to a lesser extent magnesium-dependent retention in the cTnC column. The findings indicate that levosimendan binds both to the N-terminal and C-terminal domains of cTnC. In agreement with this, only levosimendan shifted the calcium-induced fluorescence curve of dansylated cTnC to the left. In the control experiments Ca50 and KCa2+ were calculated to be 2.73 microM and 4 x 10(5) M-1, respectively. Levosimendan at 3 microM decreased the value of Ca50 to 1.19 microM. In conclusion, it is suggested that the mechanism of calcium sensitizing effect of levosimendan, unlike that of the other calcium sensitizers, is based on calcium-dependent binding to the N-terminal domain of cTnC. This is proposed to amplify the trigger of contraction induced by cTnC in the cardiac muscle.

Troponin C-mediated calcium sensitization induced by levosimendan does not impair relaxation

Levosimendan is a novel positive inotropic drug targeted to increase contraction force of the heart through its calcium-dependent binding to troponin C (cTnC). We investigated the calcium-sensitizing effect of levosimendan on contractile proteins as well as its positive inotropic and lusitropic effects in paced guinea pig papillary muscle. We also studied the effect on energy consumption of myosin–actin crossbridges in a myosin ATPase assay. The calcium sensitization induced by levosimendan in fibers skinned with saponin was dependent on the perforation velocity of cell membranes. Levosimendan was almost ineffective in slowly perforated fibers, but was the most potent calcium sensitizer in fibers with rapidly perforated cells. The perforation-dependent calcium sensitization was probably due to changes in phosphorylation state of contractile proteins during the slow dissection of fibers. It is noteworthy that the calcium-sensitizing effect of levosimendan was not affected by acidic pH. Levosimendan at therapeutically relevant (0.3–10 μM) concentrations markedly increased calcium sensitivity both at pH 6.7 and 7.0, being more potent than EMD 53998, pimobendan, and MCI-154. The lack of effect of levosimendan on maximum tension supports the hypothesis that levosimendan increases calcium sensitivity through its action on cTnC. Unlike EMD 53998, levosimendan did not increase myosin ATPase activity, indicating that it did not increase the cycling rate of myosin–actin crossbridges. In paced papillary muscles, levosimendan induced positive inotropic effect without changing relaxation time. Thus, levosimendan was devoid of the main negative factors described for calcium sensitizers.

Biochemical and pharmacological properties of a peripherally acting catechol-O-methyltransferase inhibitor entacapone

SummaryEntacapone, OR-611, was found to be a potent peripherally acting inhibitor of catechol-O-methyl-transferase (COMT). IC50 values of 10 nmol/1 and 160 nmol/1 were obtained for rat duodenum and liver-soluble COMT, respectively. There were no effects on other catecholamine metabolizing enzymes. Entacapone showed reversible, tight-binding type of inhibition of soluble rat liver COMT with a K; value of 14 nmol/1 and it also caused 50% inhibition of rat duodenal, erythrocyte, liver and striatal COMT activity 1 h after oral dosing with 1.1, 5.4, 6.7 and 24.2 mg/kg, respectively. However, penetration of entacapone into the brain was poor, since the formation of homovanillic acid (HVA), the O-methyl metabolite of dopamine in the striatum, was not reduced, even after the highest dose of 30 mg/kg.In rat blood serum, the concentration of 3-0-methyldopa (3OMD), the O-methylated product of l-dopa, was reduced in a dose-dependent manner, and the concentration of l-dopa was increased after the administration of entacapone (3 - 30 mg/kg p. o.) together with l-dopa + carbidopa. These changes were reflected, in the striatum, by a significant rise in the dopamine concentration and a reduction in the 30MD concentration.Consequently, when entacapone was added to the treatment with l-dopa + carbidopa, the dose of l-dopa could be lowered from 50 mg/kg to 15 mg/kg in order to produce the same striatal dopamine concentrations as with 50 + 50 mg/kg of l-dopa + carbidopa alone.

Inhibition of catechol-O-methyltransferase activity by two novel disubstituted catechols in the rat

Catechol-O-methyltransferase (COMT) has an important role in the extraneuronal inactivation of catecholamine neurotransmitters and drugs with a catechol structure. Two novel COMT inhibitors, OR-462 and OR-486, were highly effective (IC50 = 18 and 12 nM, respectively) and selective in inhibiting COMT activity in vitro. Tyrosine hydroxylase was not inhibited until micromolar concentrations of these compounds were used: the IC50 values for OR-462 and OR-486 were 10 and 14 microM, respectively. The IC50 values for dopamine-beta-hydroxylase, dopa-decarboxylase and monoamine oxidase forms A and B were greater than 50 microM. In studies ex vivo oral OR-462 inhibited mainly the COMT activity in the duodenum while OR-486 inhibited COMT activity in the liver and red blood cells as well. Oral OR-462 did not penetrate into the brain in doses up to 30 mg/kg while the same dose of OR-486 had some effect on striatal COMT activity. When tested in combination with levodopa-carbidopa, orally administered OR-462 and OR-486 were more effective in reducing the formation of 3-O-methyldopa from levodopa than was the levodopa-carbidopa treatment alone. These results indicate that OR-462 and OR-486 are effective and long-lasting inhibitors of COMT activity.

Effects of entacapone and tolcapone on mitochondrial membrane potential

Catechol-O-methyl transferase (COMT) inhibitors, entacapone and tolcapone, are used as an adjunctive treatment to L-dopa in Parkinsons disease. Based on their catechol structure, both inhibitors are potential uncoupling agents, but only tolcapone shows this effect in vitro at clinically relevant concentrations. This study was designed to evaluate the direct uncoupling effects of the two COMT inhibitors in vitro and in vivo. In isolated rat liver mitochondria, entacapone had no effect on the membrane potential at therapeutical concentrations, but both tolcapone and the reference compound 2,4-dinitrophenol disrupted the potential at low microM concentrations. Since protein binding is speculated to decrease the uncoupling effects in vivo, the COMT inhibitory effect of entacapone and tolcapone as a surrogate for the overall activity of these inhibitors was evaluated in vitro with or without serum. The COMT inhibitory activity of entacapone was reduced to half, while tolcapone had only about 1/10 of its activity left in the presence of serum. Further, uncoupling is known to induce an increase in the body temperature in vivo, and these effects were evaluated in the rat by a possible hyperthermic response to the treatment with entacapone or tolcapone in combination with L-dopa (10 mg/kg) and carbidopa (20 mg/kg). This combination with entacapone (400 mg/kg) had no effect on the rectal body temperature. In contrast, tolcapone (50 mg/kg) caused an elevation in the body temperature together with L-dopa and carbidopa (P < 0.01). Both in vitro and in vivo results indicate that entacapone does not impair energy metabolism related to uncoupling of oxidative phosphorylation.

Properties of novel effective and highly selective inhibitors of catechol-O-methyltransferase

Novel bisubstituted catechols were found to be potent and highly selective COMT inhibitors in vitro. One of them, OR-462 (3-(3,4-dihydroxy-5-benzylidene)-2,4-pentanedione), was studied also in vivo. When administered to rats orally together with levodopa and carbidopa, OR-462 greatly improved the bioavailability of levodopa and effectively reduced the formation of 3OMD. The levels of levodopa and dopamine were increased also in the striatum, and the 3OMD levels were decreased. The metabolic profile of dopamine demonstrated that COMT inhibition occurred in the peripheral tissues but not in the striatum. OR-462 thus resembled the peripheral inhibitors of dopadecarboxylase. These potent, selective and orally active COMT inhibitors offer a new tool for interfering in the metabolism of various COMT substrates.

Entacapone, a novel catechol-O-methyltransferase inhibitor for Parkinson's disease, does not impair mitochondrial energy production

Entacapone, a novel mainly peripherally acting catechol-O-methyltransferase inhibitor used in the treatment of Parkinsons disease, was evaluated for its possible uncoupling activity in cell culture, in rat liver mitochondria, and in isolated guinea-pig heart. Entacapone did not stimulate respiration in the L1210 murine T cell lymphoma cell line at the concentrations studied (5-40 microM). Furthermore, entacapone neither increased mitochondrial respiration nor impaired cardiac function at pharmacologically relevant concentrations (< 10 microM). In fact, the threshold concentration for increased mitochondrial oxygen consumption was 20 microM and half-maximal stimulation of respiration was not detected until 58 microM. Surprisingly, tolcapone, another catechol-O-methyltransferase inhibitor, which acts both peripherally and centrally, stimulated respiration in L1210 cells at the lowest concentration studied (5 microM). In addition, 1 microM tolcapone increased mitochondrial respiration, indicating that it caused uncoupling at a much lower concentration than that of 2,4-dinitrophenol, a well-known uncoupler of oxidative phosphorylation. Tolcapone also impaired the mechanical function and oxygen consumption of the isolated guinea-pig heart at 1 microM. These results show that peripherally acting entacapone, unlike the brain-penetrating tolcapone, is a safe catechol-O-methyltransferase inhibitor for the treatment of Parkinsons disease, since it does not interfere with mitochondrial energy metabolism at pharmacologically effective concentrations.

Determination of catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection

A rapid and simple assay for catechol-O-methyltransferase (COMT) activity by high-performance liquid chromatography with electrochemical detection is described. The method is based on the measurement of 3-O- and 4-O-methylated products (3-methoxy-4-hydroxy- and 4-methoxy-3-hydroxybenzylamine, respectively) of the substrate 3,4-dihydroxybenzylamine. These compounds are determined from the incubation mixture after removal of protein injecting aliquot into the liquid chromatograph. The detection limit is 1 pmol of product. This method is very suitable for screening of COMT activity as well as for determining the meta/para product ratios.

Inhibition of rat liver and duodenum soluble catechol-O-methyltransferase by a tight-binding inhibitor OR-462

The inhibition kinetics of rat liver and duodenum soluble catechol-O-methyltransferase (COMT) with a disubstituted catechol OR-462 was studied. After preincubation of the enzyme and inhibitor in the presence of magnesium and S-adenyosylmethionine, an inhibition about thirty times greater than that without preincubation was observed. Reversible tight-binding inhibition was demonstrated with Ki values of 0.7 nM and 1.0 nM for liver and duodenum enzyme, respectively. Km values of 53.4 microM and 56.9 microM for substrate 3,4-dihydroxybenzoic acid and 23.0 microM and 17.5 microM for S-adenosylmethionine were calculated for liver and duodenum enzyme, respectively. A catalytic number of 24/min for liver soluble COMT was calculated.

PLS modelling of structure—activity relationships of catechol O-methyltransferase inhibitors

SummaryQuantitative structure-activity analysis was carried out for in vitro inhibition of rat brain soluble catechol O-methyltransferase by a series (N=99) of 1,5-substituted-3,4-dihydroxybenzenes using computational chemistry and multivariate PLS modelling of data sets. The molecular structural descriptors (N=19) associated with the electronics of the catecholic ring and sizes of substituents were derived theoretically. For the whole set of molecules two separate PLS models have to be used. A PLS model with two significant (crossvalidated) model dimensions describing 82.2% of the variance in inhibition activity data was capable of predicting all molecules except those having the largest R1 substituent or having a large R5 substituent compared to the NO2 group. The other PLS model with three significant (crossvalidated) model dimensions described 83.3% of the variance in inhibition activity data. This model could not handle compounds having a small R5 substituent, compared to the NO2 group, or the largest R1 substituent. The predictive capability of these PLS models was good. The models reveal that inhibition activity is nonlinearly related to the size of the R5 substituent. The analysis of the PLS models also shows that the binding affinity is greatly dependent on the electronic nature of both R1 and R5 substituents. The electron-withdrawing nature of the substituents enhances inhibition activity. In addition, the size of the R1 substituent and its lipophilicity are important in the binding of inhibitors. The size of the R1 substituent has an upper limit. On the other hand, ionized R1 substituents decrease inhibition activity.