In Silico Design of Novel Sirtuin 1 Enzyme Activators for the Treatment of Age-related Diseases and Life Span.

AIM: The aim of the study was to develop new SIRT1 activator compounds, for this aim, we used virtual screening and molecular dynamics methods, which have been important tools for new hit compound searches. BACKGROUND: Recently, with the progress of computing technology, it has been possible to obtain higher efficiency and lower costs for drug discovery. With in silico research and drug design, there is a reduction in time-consuming and expensive experimental work. An NAD+ dependent histone deacetylase enzyme, Sirtuin 1 (SIRT1), is involved in a variety of human disorders such as type II diabetes, cancer, obesity, and aging. Activation of SIRT1 could be useful for longevity and treating metabolic disorders. OBJECTIVE: We used computational methods to develop new SIRT1 activator compounds. METHODS: Firstly, virtual screening studies on the human SIRT1 enzyme were carried out. We used approximately 150.000 commercially available compounds from the Zinc database, which include FDA-approved drugs. According to virtual screening results, we selected seven potent activators. Then we compared these hit compounds with known activators by using docking methods. One of these hit compounds, acebutolol, is an FDA-approved drug, and was selected for additional studies using molecular dynamics simulations. RESULTS: Seven hit compounds were identified with database screening. Each showed strong interactions with SIRT1, and acebutolol formed H-bonds with the important active site residues, Asn226 and/or Glu230 during the dynamics simulation. CONCLUSION: Based on our in silico studies, the seven most promising compounds, especially acebutolol, showed promising SIRT1 activator potency. The results may be used to design new selective and more potent SIRT1 activator drugs.

Stereoselective cell uptake of adrenergic agonists and antagonists by organic cation transporters.

Stereoselectivity is well described for receptor binding and enzyme catalysis, but so far has only been scarcely investigated in carrier-mediated membrane transport. We thus studied transport kinetics of racemic (anti)adrenergic drugs by the organic cation transporters OCT1 (wild-type and allelic variants), OCT2, OCT3, MATE1, and MATE2-K with a focus on stereospecificity. OCT1 showed stereoselective uptake with up to 2-fold higher vmax over their corresponding counterpart enantiomers for (R,R)-fenoterol, (R,R)-formoterol, (S)-salbutamol, (S)-acebutolol, and (S)-atenolol. Orciprenaline and etilefrine were also transported stereoselectively. The Km was 2.1-fold and 1.5-fold lower for the (S,S)-enantiomers of fenoterol and formoterol, while no significant difference in Km was seen for the other aforementioned drugs. Common OCT1 variants showed similar enantiopreference to wild-type OCT1, with a few notable exceptions (e.g. a switch in enantiospecificity for fenoterol in OCT1*2 compared to the wild-type). Other cation transporters showed strong differences to OCT1 in stereoselectivity and transport activity: The closely related OCT2 displayed a 20-fold higher vmax for (S,S)-fenoterol compared to (R,R)-fenoterol and OCT2 and OCT3 showed 3.5-fold and 4.6-fold higher vmax for the pharmacologically active (R)-salbutamol over (S)-salbutamol. MATE1 and MATE2-K generally mediated transport with a higher capacity but lower affinity compared to OCT1, with moderate stereoselectivity. Our kinetic studies showed that significant stereoselectivity exists in solute carrier-mediated membrane transport of racemic beta-adrenergic drugs with surprising, and in some instances even opposing, preferences between closely related organic cation transporters. This may be relevant for drug therapy, given the strong involvement of these transporters in hepatic and renal drug elimination.

Synthesis and In Vitro Pharmacological Evaluation of 5-(Alkoxymethyl)-2-(3-alkylamino-2-hydroxypropoxy)phenylethanones Related to Acebutolol and Celiprolol.

The structure-activity relationships of 13 analogs of aryloxyaminopropanol type derived from 2-hydroxyphenylethanone as potential ß-blockers are described. The synthesized compounds possess an isopropyl or a tert-butyl group in the hydrophilic part of the molecule and an alkoxymethyl substitution in the lipophilic moiety. The target compounds were prepared by an established four-step method and their structures were confirmed by interpretation of their UV, IR, (1) H NMR and (13) C NMR spectra, and by elemental analysis. The ß-adrenolytic efficacy of the prepared racemic compounds was determined on isolated guinea pig atria (ß1 ) and trachea (ß2 ) and expressed as pA2 values against isoprenaline tachycardia. The assumed cardioselectivity was expressed as ß1 /ß2 ratio and the values of compounds with an alkoxy group (CH3 O, iC3 H5 O, C5 H11 O, CH2 CHCH2 O, CH3 OCH2 CH2 O) in the lipophilic part and with tert-butyl in the hydrophilic part of the molecule were found to be comparable or higher than those of the standards acebutolol and celiprolol. All evaluated substances at a concentration of 10(-7) mol/dm(3) showed also negative chronotropic effects.

Upregulation of P-glycoprotein expression by ophthalmic drugs in different corneal in-vitro models.

OBJECTIVES: The purpose of this study was to analyse P-glycoprotein (P-gp) expression in different human in-vitro cornea models (HCE-T epithelial model and Hemicornea construct) after stimulation with P-gp substrates (rhodamine 123, levofloxacin and acebutolol). METHODS: The influence of P-gp substrates on mRNA expression was analysed using reverse transcriptase polymerase chain reaction (PCR) and real-time PCR. The effect of stimulation on the transporter functionality was estimated with a digoxin efflux assay. The Caco-2 cell line was used as positive control. KEY FINDINGS: The reverse transcriptase PCR results showed an increase in band intensity compared with the control medium for all substrates. The real-time PCR for the Caco-2 and HCE-T epithelial model yielded a similar outcome, in which all tested substrates upregulated P-gp. In contrast, the Hemicornea construct showed no significant increase in the mRNA expression after stimulation. Both in-vitro models possessed similar drug transport profiles after stimulation. A significantly increased efflux of digoxin was measured after 24 and 72 h of stimulation with levofloxacin and acebutolol. CONCLUSIONS: The expression and functionality of the P-gp in corneal tissue can be influenced through time exposure with specific substrates. However, the exact mechanism still requires further elucidation.

Active components of frequently used ß-blockers from the aspect of computational study.

The aim of this study is to investigate the active components of representative drugs for blood pressure regulation by applying quantum mechanical computer codes and comparison of the same for the sake of obtaining knowledge about the properties associated with the electronic structure of given molecules. The study included three well-known, but not theoretically investigated enough, active components of ß-blockers: acebutolol, metoprolol and atenolol. The results are in agreement with the experimental data and were used for initial assumptions concerning the degradation of these compounds.

Infantile haemangioma and ß-blockers in otolaryngology.

Infantile haemangioma (IH) is the most common tumour during early childhood. Although these benign lesions resolve spontaneously, up until recently laryngotracheal sites of IH required invasive management. The dramatic efficacy of ß-blockers on IH has radically changed the prognosis. Surgery is now no longer indicated as first-line therapy, but should only be performed for difficult, refractory cases, or in the presence of absolute contraindications to ß-blockers. Long-term steroid therapy is also no longer indicated. Propranolol can be used as first-line, single-agent therapy.

The effect of selective beta1-blockade on EMG signal characteristics during progressive endurance exercise.

This study analysed the effect of selective beta(1)-blockade on neuromuscular recruitment characteristics during progressive endurance exercise. Ten healthy subjects ingested a selective beta(1)-blocker, acebutolol (200 mg b.d.), for 7 days (for one of two cycling trials), with a 10-day wash-out period between trials. On the last day of acebutolol ingestion subjects performed three successive 15-min rides at 30%, 50% and 70% of their peak power output and then cycled at increasing (15 W min(-1)) work rates to exhaustion. Force output, heart rate, submaximal VO(2), rate of perceived exertion (RPE), electromyographic (EMG) data and blood lactate were captured during the cycling activity. Peak work rate [270 (111) W vs 197 (75) W, CON vs BETA, P <0.01], time to exhaustion [49.7 (23.2) min vs 40.3 (23.7) min, CON vs BETA, P <0.05] and heart rate [mean, for the full ride 135.5 (38.3) beats min(-1) vs 111.5 (30.0) beats min(-1) CON vs BETA, P <0.05] were significantly lower for the group who ingested beta(1)-blockade (BETA) compared to the control group (CON). Although not significant, submaximal VO(2)was reduced in BETA during the ride, while RPE was significantly higher during the ride for BETA (P <0.01). Mean integrated electromyography was higher in the BETA group although these differences were not significant. Mean power frequency values of the BETA group showed a significant (P <0.05) shift to the upper end of the spectrum in comparison to the control group. Lactate values [11.7 (3.5) mmol x l(-1) vs 7.1 (4.1) mmol x l(-1)CON vs BETA] were significantly lower (P <0.05) at exhaustion in BETA. Significant reductions in cycling performance were found when subjects ingested beta(1)-blockers. This study has shown significant shifts to the upper end of the EMG frequency spectrum after beta(1)-blocker ingestion, which could be caused by a change in neuromuscular recruitment strategy to compensate for the impaired submaximal exercise performance.

beta(1)-adrenergic antagonists improve sleep and behavioural disturbances in a circadian disorder, Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a clinically recognisable contiguous gene syndrome ascribed to interstitial deletions of chromosome 17p11.2. Patients have a phase shift of their circadian rhythm of melatonin with a paradoxical diurnal secretion of the hormone. Serum melatonin levels and day-night behaviour were studied in nine SMS children (aged 4 to 17 years) given acebutolol, a selective beta(1)-adrenergic antagonist (10 mg/kg early in the morning). Cardiac examination, serum melatonin, motor activity recordings, and sleep diaries were monitored before and after drug administration. The present study shows that a single morning dose of acebutolol suppressed the inappropriate secretion of melatonin in SMS. A significant improvement of inappropriate behaviour with increased concentration, delayed sleep onset, increased hours of sleep, and delayed waking were also noted. These results suggest that beta(1)-adrenergic antagonists help to manage hyperactivity, enhance cognitive performance, and reduce sleep disorders in SMS.

Effect of an alpha-blocker (Nicergoline) and of a beta-blocker (Acebutolol) on the in vitro biosynthesis of vascular extracellular matrix.

The effect of an alpha-blocking agent and of a beta-blocking agent on the biosynthesis of extracellular matrix macromolecules of the arterial wall was investigated. Rabbit aorta explants were cultured up to 48 hours with radioactive proline, lysine or glucosamine. In presence of these drugs, at concentration shown to be effective for the inhibition of platelet-endothelial cell interactions (10(-7) M), the incorporation of 14C proline in total macromolecular proline was higher than in macromolecular hydroxyproline suggesting a relatively higher rate of biosynthesis of non-collagenous proteins as compared to collagens. The alpha-blocking increased the incorporation of 14C proline in collagenous and non-collagenous proteins after 18 hours of incubation. beta-blocking also increased the incorporation of proline in macromolecular proline and hydroxyproline as compared to control cultures. Both increased the incorporation of 3H glucosamine in newly synthesised glycosaminoglycans. beta-blocking increased mainly the neosynthesis of heparan sulphate, alpha-blocking that of hyaluronan. The incorporation of 14C-lysine in crosslinked, insoluble elastin was not modified. These experiments confirm that alpha and beta-blocking agents can influence not only the tonus of aortic smooth muscle cells but also the relative rates of biosynthesis of extracellular matrix macromolecules. This effect should be taken in consideration for the evaluation of the long range effect of alpha and beta-blocking drugs on the vascular wall.

Influence of acebutolol and metoprolol on cardiac output and regional blood flow in rats.

Beta-adrenoceptor blocking drugs are widely used as effective antihypertensive and antianginal agents. We have determined the effect of beta-blockade in the rat to ascertain whether there are differences between metoprolol (MET) and acebutolol (AC) with respect to regional blood flow (RBF). Both AC and MET were administered as a single or multiple intravenous (iv) doses in Sprague-Dawley rats. Microspheres labelled with (85)Sr and (141)Ce were used to measure cardiac output (CO) and RBF before and after drug administration. CO and RBF were measured 1 and 10 min after the i.v. administration of AC (30 mg/kg) and MET (10 mg/kg). After acute administration of MET, CO decreased by 65% and 31% after 1 and 10 min measurements, respectively. These values were 54% and 28% for AC as compared with baseline values. After chronic administration of either AC or MET, however, there were no significant reductions in CO as compared with saline. Both MET and AC significantly reduced RBF in most organs either after 1 or 10 min measurements when compared with the baseline values. It is concluded that both AC and MET reduced CO and RBF after acute administration. The CO and RBF however, returned to normal after chronic administration.

Adrenoceptors modulate depressor responses of endothelin-1 into the superior colliculus of rats.

Endothelin-1 (ET-1) (10 pmol) microinjected into the superficial layer of superior colliculus induces decreases in blood pressure (control, 108 +/- 5 mmHg, n=6; ET-1, 71 +/- 4 mmHg, n=5). The effects on blood pressure induced by endothelin-1 were significantly (p<0.05) reduced by pre-administration into the superior colliculus of the alpha1-adrenoceptor agonist phenylephrine (1 nmol) (46 +/- 5%, n=5), beta1-adrenoceptor antagonist acebutolol (5 nmol) (51 +/- 6%, n=5) or beta1/beta2-adrenoceptor antagonist propranolol (3.4 nmol) (51 +/- 11%, n=5). In contrast, endothelin-1-induced effects were increased (p<0.05) by microinjections into the superior colliculus of prazosin (2.4 nmol) (49 +/- 7%, n=5), an alpha1-adrenoceptor antagonist; dobutamine (4 nmol) (51 +/- 9%, n=5), a beta1-adrenoceptor agonist or isoprenaline (1 nmol) (49 +/- 6%, n=5), a beta1/beta2-adrenoceptor agonist. No involvement of alpha2- or beta2-adrenoceptors has been detected. Therefore, ET-1 induces decreases in blood pressure with selective involvement of alpha1- and beta1-adrenoceptors.

Beta-blockers inhibit the modification of low-density lipoproteins by sodium hypochlorite in vitro.

The effect of beta-blockers (alprenolol, oxprenolol, atenolol, acebutolol) and the non-steroidal anti-inflammatory drug, diclofenac, on modification of low-density lipoproteins (LDL) by sodium hypochlorite (NaOCl) was investigated in vitro. Beta-blockers and diclofenac inhibit the formation of thiobarbituric acid reactive substances in LDL modified by NaOCl. Beta-blockers, but not diclofenac, inhibit the hypochlorite-induced aggregation of LDL which was determined by photon correlation spectroscopy. The intracellular accumulation of cholesterol esters in J774 macrophages is inhibited by addition of beta-blockers, but not diclofenac, to LDL prior to the addition of NaOCl. The modification inhibiting effect of beta-blockers is inversely correlated to the binding capabilities of these substances to LDL which were assessed by laser electrophoresis. Inhibition of LDL modification in vivo by beta-blockers may reduce the risk of atherosclerosis and, therefore, compensate for the cholesterol-raising effect of these drugs in human plasma.

Prolonged treatment with beta-adrenoceptor antagonists counteracts the aggression deficit induced by chronic stress.

Chronic stress-induced behavioral disturbances have been used as experimental models of depression. One of them is the deficit of fighting behavior induced by 16-day application of various unpredictable stressors. In the present study we investigated the effect of beta-adrenoceptor antagonists (propranolol, pindolol, nadolol and acebutolol) on electric footshock-induced fighting behavior in chronically stressed (14 various stressors over 16 days) male Wistar rats. It was found that the number of fighting attacks was reduced by about 50-80% in the rats submitted to chronic stress. Prolonged, 14-day, but not acute, treatment with propranolol, pindolol or nadolol (but not acebutolol) counteracted the deficit of aggression induced by chronic stress. It is suggested that beta-adrenoceptor antagonists which penetrate the blood-brain barrier may prevent the behavioral changes induced by chronic stress.

Relationship between beta adrenoceptor occupancy and receptor down-regulation induced by beta antagonists with intrinsic sympathomimetic activity.

The relationship between the Ki of the B2 adrenoceptor and EC50 values characterizing receptor down-regulation induced by isoproterenol and six beta antagonists classified as having weak to strong intrinsic sympathomimetic activity (ISA) was determined using L6 myoblasts. It was hypothesized that if receptor loss induced by beta antagonists with ISA was mediated through cAMP, EC50 = Ki. EC50/Ki ratios for (-)isoproterenol, (-) and (+) celiprolol were 0.006, 0.01 and 0.08, respectively (p < 0.05); ratios for (-)pindolol and dilevalol were 19 and 9.5, respectively (p < 0.05). EC50/Ki ratios for acebutalol and (-)alprenolol were not significantly different from 1.0. Isoproterenol and dilevalol maximally down-regulated receptor density 89 and 83%, respectively, followed by (+)celiprolol, 54%; (-)celiprolol, 53%; acebutalol, 41%; (-)pindolol, 36% and (-)alprenolol, 31%. Receptor loss was blocked in each case by ICI118,551 or sotalol. A sensitive radioimmunoassay failed to detect increased cAMP accumulation following pretreatment with concentrations of acebutalol, (-)alprenolol, celiprolol and (-)pindolol 100 times their respective Ki values. Isoproterenol and dilevalol stimulated cAMP accumulation 100- and 2-fold over basal, respectively. We conclude that receptor loss induced by beta antagonists with ISA is mediated through the beta 2 adrenoceptor and in at least some cases is cAMP-independent.

Beta-adrenoceptors regulate myoelectric activity in the small intestine of rats: stimulation by beta 2 and inhibition by beta 3 subtypes.

Using beta-adrenergic agonists and antagonists this study investigated the importance of three different adrenoceptor subtypes for the regulation of migrating myoelectric complexes (MMCs) of the upper small intestine in conscious, naive rats. After a control period of 60 min with four activity fronts, agonists were given as an intravenous infusion for another 60 min. The non-selective beta-adrenoceptor agonist isoprenaline (1 microgram kg-1 min-1) inhibited MMCs and induced irregular spiking during the infusion period. This effect was blocked by intravenous administration of a bolus dose of either the non-selective beta-adrenoceptor antagonist propranolol (1 mg kg-1), or the beta 2-antagonist ICI 118 551 (1 mg kg-1), both given prior to isoprenaline. However, acebutolol (1 mg kg-1), a selective beta 1-antagonist, failed to antagonize the effect of isoprenaline. Furthermore, prenalterol, a selective beta 1-agonist (12.5-800.0 micrograms kg-1 min-1), had no effect on the MMC pattern, whereas the beta 2-selective agonist ritodrine (25-100 micrograms kg-1 min-1) induced a myoelectric pattern similar to one induced by isoprenaline. The partial beta 3-adrenoceptor agonist D7114 (50-100 micrograms kg-1 min-1), disrupted the MMCs and induced quiescence. Neither of the antagonists, i.e. propranolol (1 mg kg-1), acebutolol (1 mg kg-1) nor ICI 118 551 (1 mg kg-1), given alone induced changes in the MMC pattern. In conclusion, beta 2-adrenoceptors in particular but also beta 3-adrenoceptors seem to be of importance in the regulation of small intestinal motility by disrupting the regular MMC pattern in rats.

Long-term effects on plasma lipids of diet and drugs to treat hypertension. Treatment of Mild Hypertension Study (TOMHS) Research Group.

OBJECTIVE: - To compare long-term plasma lipid changes among 6 antihypertensive treatment interventions for stage I (mild) hypertension. DESIGN: - Multicenter, randomized, double-blind, parallel-group clinical trial. SETTING: - Four academic clinical research units in the United States. PARTICIPANTS: - A total of 902 men and women, aged 45 to 69 years, with stage I diastolic hypertension (diastolic blood pressure <100 mm Hg), recruited from 11914 persons screened in their communities. INTERVENTIONS: - Participants were randomized to 1 of 6 treatment groups: (1) placebo, (2) beta-blocker (acebutolol), (3) calcium antagonist (amlodipine), (4) diuretic (chlorthalidone), (5) alpha1-antagonist (doxazosin), and (6) angiotensin-converting enzyme inhibitor (enalapril). All groups received intensive lifestyle counseling to achieve weight loss, dietary sodium and alcohol reduction, and increased physical activity. MAIN OUTCOME MEASURES: - Changes in plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides from baseline to annual visits through 4 years. RESULTS: - Mean changes in all plasma lipids were favorable in all groups. The degree of weight loss with fat-modified diet and exercise was significantly related to favorable lipid changes. Significant differences (P<.01) among groups for average changes during follow-up in each lipid were observed. Decreases in plasma total cholesterol and LDL cholesterol were greater with doxazosin and acebutolol (for plasma total cholesterol, 0.36 and 0.30 mmol/L [13.8 and 11.7 mg/dL], respectively), less with chlorthalidone and placebo (0.12 and 0.13 mmol/L [4.5 and 5.1 mg/dL], respectively). Decreases in triglycerides were greater with doxazosin and enalapril, least with acebutolol. Increases in HDL cholesterol were greater with enalapril and doxazosin, least with acebutolol. Significant relative increases in plasma total cholesterol with chlorthalidone compared with placebo at 12 months were no longer present at 24 months and beyond, when mean plasma total cholesterol for the chlorthalidone group fell below baseline. Analyses of participants continuing to receive chlorthalidone throughout the 4 years of follow-up indicated this was not due solely to an increasing percentage of participants changing or discontinuing use of medication during follow-up. CONCLUSIONS: - Weight loss with a fat-modified diet plus increased exercise produces favorable long-term effects on blood pressure and all plasma lipid fractions of adults with stage I hypertension; blood pressure reduction is enhanced to a similar degree by addition of a drug from any one of 5 classes of antihypertensive medication. These drugs differ quantitatively in influencing the degree of long-term favorable effects on blood lipids obtained with nutritional-hygienic treatment.

Arrhythmogenic effect of beta-adrenoceptor-blocking drugs in Purkinje fibres of guinea-pig hearts.

The association between torsade de pointes and experimentally induced early afterdepolarizations in isolated fibres is well documented. The effect of eight beta-adrenoceptor-blocking drugs (sotalol, nifenalol, acebutolol, dichloroisoproterenol, propranolol, oxprenolol, pindolol, atenolol), and of amiodarone, was studied in isolated spontaneously beating guinea-pig Purkinje fibres by the intracellular microelectrode technique. Phase 3 early afterdepolarizations were initiated by nifenalol hydrochloride (n = 18; 10 mumol/l: 0/18, 40 mumol/l: 3/18, 80 mumol/l: 8/18, 160 mumol/l: 11/18), rac.-(+/-)-sotalol hydrochloride (n = 28; 20 mumol/l: 0/28, 40 mumol/l: 9/28, 80 mumol/l: 20/28), (R)(+)-sotalol hydrochloride (n = 12; 40 mumol/l: 1/12, 80 mumol/l: 4/12), and (S)(-)-sotalol hydrochloride (n = 10, 40 mumol/l: 1/10, 80 mumol/l: 4/10). The arrhythmogenic effect was reversible after a washout period of one hour and early after-depolarizations could be terminated by tetrodotoxin (0.4-1.6 mumol/l, n = 6). Amiodarone only induced early afterdepolarizations at a low extracellular potassium concentration of [K+]o = 1.35 mmol/l (n = 5; 150 mumol/l: 0/5, 300 mumol/l: 1/5). The initiation of early after-depolarizations by sotalol and nifenalol might be induced by an imbalance of sodium inward current and potassium outward currents, and early afterdepolarizations are blocked by tetrodotoxin.

Influence of hypotension on cochlear blood flow in polycythemic condition.

We examined the influence of hypotension by infusion of acebutolol hydrochloride (AH), a cardioselective beta-receptor antagonist, on cochlear blood flow in guinea pigs with various hematocrit values. AH infusion lowered the mean blood pressure to almost the same degree in all animals, regardless of the hematocrit level. The degree of the concomitant decrease of CBF varied with the hematocrit, being greater in animals with a higher hematocrit. In those with the highest hematocrit CBF did not return to the initial level. From these values we calculated the O2 transport capacity after AH infusion and found it to be lower than in animals without AH infusion. The difference was greater at higher hematocrits. These findings suggest that the microcirculation of the inner ear is responsive to transient decreases of perfusion pressure at high hematocrits.