A computer-aided drug design approach to discover tumour suppressor p53 protein activators for colorectal cancer therapy.

Colorectal cancer (CRC) is the third most detected cancer and the second foremost cause of cancer deaths in the world. Intervention targeting p53 provides potential therapeutic strategies, but thus far no p53-based therapy has been successfully translated into clinical cancer treatment. Here we developed a Quantitative Structure-Activity Relationships (QSAR) classification models using empirical molecular descriptors and fingerprints to predict the activity against the p53 protein, using the potency value with the active or inactive label, were developed. These models were built using in total 10,505 molecules that were extracted from the ChEMBL, ZINC and Reaxys® databases, and recent literature. Three machine learning (ML) techniques e.g., Random Forest, Support Vector Machine, Convolutional Neural Network were explored to build models for p53 inhibitor prediction. The performances of the models were successfully evaluated by internal and external validation. Moreover, based on the best in silico p53 model, a virtual screening campaign was carried out using 1443 FDA-approved drugs that were extracted from the ZINC database. A list of virtual screening hits was assented on base of some limits established in this approach, such as: (1) probability of being active against p53; (2) applicability domain; (3) prediction of the affinity between the p53, and ligands, through molecular docking. The most promising according to the limits established above was dihydroergocristine. This compound revealed cytotoxic activity against a p53-expressing CRC cell line with an IC50 of 56.8 µM. This study demonstrated that the computer-aided drug design approach can be used to identify previously unknown molecules for targeting p53 protein with anti-cancer activity and thus pave the way for the study of a therapeutic solution for CRC.

Mechanism for the inhibition of contractile activity of the gastric antrum and pylorus in rabbits during psychogenic stress.

Psychogenic stress in rabbits (fixation to a frame) was accompanied by the inhibition of contractile activity of the gastric antrum and pylorus. These changes persisted during blockade of muscarinic receptors, nicotinic receptors, alpha(2)-adrenoceptors, and beta(1)/beta(2) adrenoceptors. A stress-induced decrease in gastric motor activity was mediated by the nonadrenergic noncholinergic mechanism. It resulted from the influence of a hormonal stress factor on the stomach, which was probably realized through nonadrenergic inhibitory neurons of the enteric nervous system.

Expression of gamma-aminobutyric acid receptor (subtype A) in prostate cancer.

BACKGROUND: In prostate cancer, gamma-aminobutyric acid (GABA) has been previously reported to increase cellular proliferation via the ionotropic GABAa receptor (GABAar) and to promote cellular invasiveness via the metabotropic GABAb receptor. METHODS: In this study, we have investigated, by immunohistochemistry, GABAar levels in 12 normal human prostate, 13 benign prostatic hyperplasia (BPH) and 148 human prostate cancer specimens. We have also examined the effect of several GABA agonists and antagonists on the in vitro proliferation of four human prostate cancer cell lines: LNCaP, MDA-PCA-2b, DU145 and PC3. RESULTS: GABAar immunoreactivity was present in the stroma of ~75% of the normal and BPH specimens, and in 95% of the prostate cancer specimens. Also, low to moderate GABAar staining was observed in the acinar epithelium of 50 (33%) prostate cancer specimens. No correlation was observed between GABAar staining and patient age, Gleason Sum or TNM stage. A GABAa agonist isoguvacine, at doses between 5-50 microg/ml (31-310 microM), stimulated the proliferation of all four human prostate cancer cell lines, tested. Baclofen, a GABAb agonist (up to 50 microg/ml, 234 microM) had no effect on growth. Also, at concentrations up to 100 microg/ml, GABA antagonists, bicuculline (223 microM), picrotoxin (166 microM) and saclofen (400 microM), did not have significant growth-inhibitory effects. However, dihydroergotoxine, which binds the GABAar chloride ion-channel, inhibited cellular proliferation (IC(50) 18-38 microM). CONCLUSIONS: These data indicate frequent expression of GABAar in prostate cancer and support a role for GABAar in the proliferation of prostate cancer cells.

Identification of alpha-adrenergic receptors in human platelets by [3H]dihydroergocryptine binding.

Binding of [(3)H]dihydroergocryptine to platelet lysates appears to have all the characteristics of binding to alpha-adrenergic receptors. At 25 degrees C binding reaches equilibrium within 20 min and is reversible upon addition of excess phentolamine. Binding is saturable with 183+/-22 fmol of [(3)H]dihydroergocryptine bound per mg of protein at saturation, corresponding to 220+/-26 sites per platelet. Kinetic and equilibrium studies indicate the dissociation constant of [(3)H]dihydroergocryptine for the receptors is 1-3 nM. The specificity of the binding sites is typical of an alpha-adrenergic receptor. Catecholamine agonists compete for occupancy of the [(3)H]dihydroergocryptine binding sites with an order of potency (-)epinephrine> (-)norepinephrine>> (-)isoproterenol. Stereospecificity was demonstrated inasmuch as the (+)isomers of epinephrine and norepinephrine were 10-20-fold less potent than the (-)isomers. The potent alpha-adrenergic antagonists phentolamine, phenoxybenzamine, and yohimbine competed potently for the sites, whereas beta-antagonists such as propranolol and dichlorisoproterenol were quite weak. Dopamine and serotonin competed only at high concentrations (0.1 mM). The [(3)H]dihydroergocryptine binding sites could also be demonstrated in intact platelets where they displayed comparable specificity, stereospecificity, and saturability. Saturation binding studies with the intact platelets indicated 220+/-45 receptors per platelet, in good agreement with the value derived from studies with platelet lysates. Ability of alpha-adrenergic agonists to inhibit adenylate cyclase and of alpha-adrenergic antagonists to antagonize this inhibitory effect directly paralleled ability to interact with the [(3)H]dihydroergocryptine binding sites. These data demonstrate the feasibility of directly studying alpha-adrenergic receptor binding sites in human platelets with [(3)H]dihydroergocryptine.

The role of phosphorylation in the alpha-adrenergic-mediated inhibition of rat hepatic pyruvate kinase.

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40-50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2-3-fold increase in the phosphate content of the enzyme. These changes were blocked by the alpha-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the alpha-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by alpha-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.

Regulation of adrenergic stimulation of hepatic adenylate cyclase by divalent cations.

Liver plasma membrane adenylate cyclase was stimulated paradoxically by an alpha 2-adrenergic mechanism under conditions of low metal ion and low GTP concentrations. In untreated membranes, epinephrine stimulation was GTP-dependent and was mediated by beta-adrenergic receptors since it was completely blocked by propranolol, but unaffected by dihydroergocryptine. Pre-treatment of membranes to remove or reduce divalent cations and guanine nucleotides changed epinephrine stimulation to a form that was mediated by alpha 2-receptors since it was completely blocked by dihydroergocryptine, phenoxybenzamine and yohimbine, but not by propranolol or prazosin. The pre-treatment did not alter enzyme activation by isoproterenol or glucagon, alpha 2-Adrenergic stimulation of adenylate cyclase in depleted membranes required the presence in the assay of 1-2 mM Mg2+ and small amounts of exogenous GTP (less than or equal to 50 nM). Increasing the Mg2+ or GTP concentration in the assay produced a progressive reversal of epinephrine-stimulated activity from an alpha 2-adrenergic form to a predominantly beta-adrenergic form. Readdition of Ca2+ or Mg2+, but not Mn2+, into depleted membranes by incubation in the presence of metal reestablished the pattern of enzyme sensitivity to epinephrine to that seen with untreated membranes i.e., it changed from alpha 2- to beta-receptor mediation. Alterations in membrane and assay content of metal ions and GTP did not result in the activation of the enzyme by vasopressin or angiotensin II. These findings demonstrate the ability of Ca2+, Mg2+ and GTP to control the coupling of beta- and alpha 2-adrenergic receptors with liver adenylate cyclase. It is hypothesized that the cations act by regulating the interaction of the receptors with adrenergic agonists and/or the guanine nucleotide binding protein(s) which is postulated to be involved in control of the enzyme.

Characterization of dopamine receptors associated with aldosterone secretion in rat adrenal glomerulosa.

Dopamine (DA) may participate in the control of aldosterone secretion. We report that two different receptors for DA are present in rat adrenal glomerulosa: D-1, associated with stimulation of adenylate cyclase, and D-2, whose action inhibits adenylate cyclase. The adenylate cyclase system was stimulated by DA (EC50, 7.2 microM) and different DA agonists. When the D-1 receptor blocker SCH 23390 was added to the incubation medium, DA elicited a dose-dependent inhibition of adenylate cyclase (IC50, 10 microM); (-)sulpiride specifically blocked this effect. Furthermore, DA blocked angiotensin II-induced aldosterone release from glomerulosa slices in vitro. This effect was prevented by (-)sulpiride, but not by SCH 23390. The results suggest that the D-2 receptor acts to inhibit the cAMP-generating system and may be physiologically involved in the regulation of aldosterone secretion.

D-2 dopamine receptor activation reduces free [3H]arachidonate release induced by hypophysiotropic peptides in anterior pituitary cells.

Dopamine reduces the stimulation of intracellular [3H]arachidonate release produced by the two PRL-stimulating peptides angiotensin-II and TRH. This effect is concentration dependent and is mediated by stimulation of D-2 dopamine receptors. D-2 receptor agonists (bromocriptine, dihydroergocryptine, and dihydroergocristine) inhibit the release of fatty acid induced by angiotensin-II with a potency that parallels their ability to inhibit PRL release in vitro. Conversely, the selective D-2 receptor antagonist L-sulpiride completely prevents dopamine's effect, whereas SCH 23390 (a D-1 receptor antagonist) is ineffective. The inhibitory action of dopamine does not seem to be consequent to an action on the adenylate cyclase-cAMP system, as 8-bromo-cAMP (1 mM) does not affect either basal or dopamine-inhibited [3H]arachidonate release. However, a 24-h pertussis toxin pretreatment significantly reduces the action of dopamine on fatty acid release. Collectively, these results suggest that D-2 dopamine receptor-mediated inhibition of intracellular [3H]arachidonate release requires the action of a GTP-binding protein, but is not a consequence of an inhibitory action on cAMP levels.

Effect of dihydroergocristine on energy metabolism studied in the isolated perfused rat brain affected by ischemia and in neuroblastoma cells deprived of oxygen and glucose.

The effect of dihydroergocristine on energy metabolism was studied in the isolated perfused rat brain affected by ischemia and in cultivated C-1300 neuroblastoma cells deprived of oxygen and glucose. Creatine phosphate, ATP, ADP, AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, pyruvate, and lactate were measured enzymatically. After a perfusion period of 30 min, the cortex of the isolated perfused rat brain exhibited an energy state not different from that in vivo. Dihydroergocristine added to the perfusion medium (5 mumol/L) did not influence these substrate levels under normal perfusion conditions. However, this drug was able to retard the breakdown of high-energy phosphates during ischemia and to accelerate the restoration of the energy state during the postischemic reperfusion period. The perfusion rate was not changed by the drug, and therefore it was assumed that dihydroergocristine could act directly on cell metabolism. This view was supported by the results obtained from experiments using cultivated N-2a neuroblastoma cells. These cells were incubated in a buffered salt solution deprived of glucose and oxygen for 15 min. Under these conditions, dihydroergocristine (2 mumol/L) added to the incubation medium caused changes in the concentrations or the high-energy phosphates similar to those in the isolated brain preparation: It increased the ATP concentration and decreased the ADP concentration significantly.

Dihydroergocryptine improves behavioral deficits of aged male rats.

Dihydroergocryptine (DHECP), an ergot alkaloid with potent dopaminergic activity, was injected acutely or subchronically to aged (24 months old) male rats of the F344 strain, at the dose of 0.05 or 0.1 mg/kg. The immobility in the "despair" test (constrained swim) was longer in 24-month-old rats than in younger animals. This behavioral change was reduced in rats treated subchronically with DHECP, but no significant change was found in animals with acute treatment. Aged rats showed an increased haloperidol-induced catalepsy and a reduced motor performance and coordination as compared to animals of 2 and 10 months of age. Acute treatment with DHECP was followed by an attenuation of haloperidol-induced catalepsy and an improvement of motor performance and coordination of aged rats. Similar results were obtained after subchronic treatment with DHECP, but the effect on the rotorod behavior was more potent after subchronic treatment than after acute injection of the drug. It is possible that the improvement of behavioral deficits of aged rats after the treatment with DHECP depends on the facilitating effect of this drug on central dopamine transmission that may be impaired in these animals.

Influence of aging on the acute depletion of reduced glutathione induced by electrophilic agents.

A severe age-dependent depletion of reduced glutathione (GSH) occurs in rat forebrain at 1-3 h from intraperitoneal injection of the electrophilic agents cyclohexene-1-one and cycloheptene-1-one. Chronic pretreatment with central dopamine agonists (i.e., ergot alkaloids; particularly, dihydroergocriptine) partially counteracts the GSH depletion induced in 15-month-old forebrains by the prooxidants tested. In contrast, chronic pretreatment with a vasodilator agent (i.e., papaverine) magnifies the GSH depletion.

Comparison between radioreceptor assay and radioimmunoassay for the determination of dihydroergotoxine in rabbit plasma samples.

Two analytical methods, radioreceptor assay and radioimmunoassay, for the determination of dihydroergotoxine have been developed. Antiserum, providing sufficient sensitivity for the radioimmunoassay, was produced by immunizing rabbits with D-lysergic acid coupled to bovine serum albumin. Radioreceptor assay utilizing dopamine receptor was carried out to determine dihydroergotoxine and its pharmacologically active metabolites in rabbit plasma, and the result was compared with that obtained by radioimmunoassay. The values obtained in both assays were almost identical; it was, therefore, assumed that the plasma concentrations of dihydroergotoxine determined by the present radioimmunoassay reflects the amount of unchanged drug and its active metabolites.

Dihydroergocristine-induced stimulation of the 5-HT autoreceptor in the hypothalamus of the rat.

In slices of the hypothalamus of the rat, prelabelled with [3H]5-hydroxytryptamine ([3H]5-HT), dihydroergocristine (DHEC) decreased in a concentration-dependent manner (0.01-1 microM) the release of [3H]transmitter elicited by stimulation. In the presence of phentolamine (1 microM), sulpiride (1 microM), atropine (1 microM) or methiothepin (0.1 microM), the effect of DHEC remained unchanged. However, methiothepin at 1 microM and citalopram at 1 microM antagonized the inhibition induced by DHEC. Methiothepin at 0.1 microM has been shown to be sufficient to shift to the right the concentration-effect curve of D-lysergic acid diethylamide (LSD) on the stimulation-evoked release of [3H]5-HT by a factor of 10. However, in the present experiments, 1 microM methiothepin was required to antagonize the effect of DHEC. Thus, the alpha adrenergic, dopaminergic and cholinergic activities of DHEC do not seem to be responsible for its effect on the release of 5-HT. The lower potency of methiothepin suggests, however, that stimulation of the 5-HT autoreceptor by DHEC may not fully explain the results.

Immunoreactive endogenous ouabain in primary aldosteronism and essential hypertension: relationship with plasma renin, aldosterone and blood pressure levels.

OBJECTIVE: To investigate the role of ouabain in human hypertension and to establish whether immunoreactive endogenous ouabain is secreted by the adrenal gland under the influence of dopaminergic regulation. METHODS: We measured plasma levels of endogenous ouabain by immunoassay, together with other variables, including plasma renin activity and aldosterone levels, in 91 clinically selected hypertensives and 19 healthy volunteers. We also measured endogenous ouabain in adrenal venous blood and the effect of DA2 dopaminergic receptor blockade and stimulation. After a thorough clinical evaluation, 64 patients were diagnosed with essential hypertension and 24 with primary aldosteronism. RESULTS: Plasma levels of endogenous ouabain were higher in essential hypertensives than in controls. Multiple regression analysis showed a significant relationship of mean blood pressure with plasma endogenous ouabain, age and body mass index, but not with other measured parameters. The plasma levels of endogenous ouabain were more than two standard deviations above the mean value for normotensives in 45% of patients with essential hypertension in whom plasma renin activity was normal. Higher plasma levels of endogenous ouabain were found in patients with aldosterone excess, specifically affecting 56% of 17 patients with surgically confirmed adrenal cortical adenoma and one (14%) of seven patients with idiopathic causes. Removal of adenomas lowered blood pressure in half of the patients in whom plasma levels of endogenous ouabain normalized after surgery. Plasma endogenous ouabain levels were similar in venous blood from the adrenal and inferior vena cava, and plasma levels were not influenced by DA2 dopaminergic blockade and stimulation. CONCLUSION: Approximately half of Caucasian patients with essential hypertension and with hyperaldosteronism exhibit elevated circulating levels of endogenous ouabain. The latter do not appear to be secondary to hypertension, are unrelated to plasma renin activity, and may not involve adrenal type-2 dopaminergic receptors.

Potentiating effect of adrenaline on adenosine diphosphate-induced reduction in rabbit circulating platelet count: inhibition by dihydroergotoxine.

The number of circulating platelets was monitored in anaesthetized rabbits by a continuous flow technique, using a Technicon Autocounter. Transient reductions in circulating platelet count induced by a submaximal dose of adenosine diphosphate (ADP) were potentiated by concomitant infusion of adrenaline at doses (1-25 microgram/kg) i.v.) which did not influence platelet count when infused alone. The adrenaline effect was dose-dependent. Repeated infusions of adrenaline at 15 min intervals resulted in reproducible reductions in circulating platelet count during an observation period of at least 105 min. Dihydroergotoxine (DHET), administered either i.v. (2.5-10 microgram/kg) or intraduodenally (i.d.; 25-100 microgram/kg), inhibited adrenaline-induced potentiation dose-dependently; ADP-induced effects were not influenced. Duration of action was relatively long, and significant inhibitory activity was still apparent 50 (i.v.) and 115 (i.d.) min after drug administration. DHET doses inhibiting adrenaline-induced potentiation of platelet aggregation in the rabbit are similar to doses used in the treatment of impaired human cerebral function. It is conceivable that DHET could prevent activation of human platelets by catecholamines released into the blood stream in clinical stress situations.

Naloxone reverses the antihypertensive effect of clonidine.

In unanesthetized, spontaneously hypertensive rats the decrease in blood pressure and heart rate produced by intravenous clonidine, 5 to 20 micrograms/kg, was inhibited or reversed by nalozone, 0.2 to 2 mg/kg. The hypotensive effect of 100 mg/kg alpha-methyldopa was also partially reversed by naloxone. Naloxone alone did not affect either blood pressure or heart rate. In brain membranes from spontaneously hypertensive rats clonidine, 10(-8) to 10(-5) M, did not influence stereoselective binding of [3H]-naloxone (8 nM), and naloxone, 10(-8) to 10(-4) M, did not influence clonidine-suppressible binding of [3H]-dihydroergocryptine (1 nM). These findings indicate that in spontaneously hypertensive rats the effects of central alpha-adrenoceptor stimulation involve activation of opiate receptors. As naloxone and clonidine do not appear to interact with the same receptor site, the observed functional antagonism suggests the release of an endogenous opiate by clonidine or alpha-methyldopa and the possible role of the opiate in the central control of sympathetic tone.

'Cerebroactive' drugs. Clinical pharmacology and therapeutic role in cerebrovascular disorders.

While their importance in the market-place is steadily increasing in developed (mainly continental Europe) and even in developing countries, compounds included in the broad category of 'cerebroactive' drugs hardly rate a mention in reference pharmacology and therapeutics textbooks. It is an undeniable fact, however, that the principal users or targets of this drug class, mainly elderly people, represent an increasingly worrying problem, with their often puzzling cohort of ill-definable and even less predictable neurological and mental symptoms. The combination of the above factors cannot but produce a rather confused situation, in which the pressure to treat and the adherence to scientifically rigorous assessment are likely to prevail alternately, on a purely casual basis. This review aims to provide sound methodological guidelines for assessment of 'cerebroactive' drugs in a not always easily accessible literature. It covers firstly the general problems of stroke, dementia and 'common symptoms' of the elderly, and then looks in detail at those compounds which have to date attracted most attention (ergot derivatives, cinnarizine, flunarizine, vincamine, eburnamonine, naftidrofuryl, oxpentifylline, piracetam and citicoline), as well as those which are currently considered investigational (choline and lecithin). The pharmacology and available clinical studies of each drug are examined. No therapeutic indication can be derived from the available evidence, as the few positive results do not go beyond random improvement of symptoms. More fundamentally, the lines of research which need to be pursued most intensively relate to better preliminary definition of diagnostic and prognostic criteria and, with the establishment of adequate testing tools for the assessment of behaviour and neuropsychological performance, those basal conditions which are modified 'naturally' or by drugs.

Hypoxia-induced sleep disturbance in rats.

The effects of varying degrees of hypoxia on sleep-wake organization were studied in rats prepared for chronic electrophysiological recording. The influence of Piracetam (75, 50, and 500 mg/kg, i.p.) and Hydergine (0.5, 1, and 3 mg/kg, i.p.) on sleep-wake organization in 10.5% oxygen was also investigated. The sleep-wake organization of rats under the effect of 15.5% oxygen content was unchanged, compared to that of normoxic control. More extreme hypoxia (12.6 and 10.5% oxygen) produced dramatic changes in sleep organization without influencing gross behavior. Paradoxical sleep (PS) stages became less frequent and shortened and were totally absent in 10.5% oxygen. Frequent wakings caused disturbed and superficial sleep. Central biochemical mechanisms, peripheral chemoreceptor reflex pathways and, as a consequence, decrease of duration of deep sleep periods, may contribute to the development of hypoxic sleep disturbances. Piracetam alleviated and Hydergine moderately reversed the hypoxic sleep disturbance.

Influence of age upon the cerebral metabolic changes induced by acute hypoxia on the synaptosomes from dog brain.

The synaptosomal fraction obtained from the motor area of the cerebral cortex of normocapnic, normoxic or hypoxic "young adult," "mature" and "senescent" beagle dogs is incubated and analyzed for : ATP, ADP, AMP, creatine phosphate, pyruvate and lactate. The data are compared with those obtained from the whole controlateral cortical motor area, by the surface freezing technique. After hypoxic hypoxia /15 min; PaO2 = 17-19 mm Hg), the metabolite contents and ratios are differently affected by ageing when the evaluations are performed in the incubated synaptosomal preparation or in the controlateral whole cerebral tissue. In fact, ageing does not affect so much the cerebral changes that occur in the overall energetic state during the hypoxic assault in vivo, but rather those that the synaptosomes remember the tend to reverse during the subsequent incubation in vitro. The protective action of several drugs on the synaptosomal phosphorylation state is tested. Phenobarbital shows a quite broad, age-independent spectrum of action. (-)Eburnamonine and dihydroergocristine exhibits a more limited, age-dependent effectiveness, but are devoid of anesthetic action. Papaverine proves unable to affect the tested biochemical parameters.

Changes in motor activity with age and the effects of pharmacologic treatment.

In a previous study the decline in the motor performance of old rats was determined to be differential. In this study, whether, and to what extent, this decline can be pharmacologically influenced was tested. Therefore, 27 month old rats were orally treated with several nootropics and d-amphetamine for six weeks. Food and water intake were determined. The rats were tested on spontaneous activity and on the tilting plane, climbing, and rotarod tests. The results showed that the nootropics only effected pronounced improvements on complex motor tasks such as on the rotarod test. In contrast, amphetamine treatment caused rather negative effects. This could be observed in the motor performance as well as in the food and water intake. Young rats did not have this sort of reaction to amphetamine. The nootropics were all well tolerated. Additionally, it was obvious that the untreated rats also showed a slight improvement in motor performance due to repeated practice.