Discovery of Potent ALK Inhibitors Using Pharmacophore-Informatics Strategy.

Anaplastic lymphoma kinase is a tyrosine kinase receptor protein belonging to insulin receptor superfamily. Gene fusions in anaplastic lymphoma kinase are associated with non-small cell lung cancer development. Hence, they are of immense importance in targeted therapies. Thus, for the treatment of non-small cell lung cancer, effective anaplastic lymphoma kinase inhibitors are of great significance. Therefore, our objective is to find hit compounds that could have better inhibitory activity than the existing anaplastic lymphoma kinase inhibitors. Keeping this in mind, in the present study pharmacophore based virtual screening was performed to identify possible anaplastic lymphoma kinase inhibitors. Initially, a five-point common pharmacophore hypothesis was generated based on twelve anaplastic lymphoma kinase inhibitors using PHASE module of Schrödinger. Subsequently, common pharmacophore hypothesis-based screening was conducted against in-trials subset of ZINC database and a total of 1000 hits were identified. The molecules obtained were further screened by three stages of docking using GLIDE software. The docking results reveal that six hit molecules showed higher glide score in comparison with the reference molecules. Finally, pharmacokinetic properties of the hit molecules were also analysed using QikProp programme. The results indicate that molecules namely videx, dexecadotril, chloramphenicol, naficillin were found to have good pharmacokinetic properties and human oral absorption. Moreover, videx, naficillin and chloramphenicol were found to have significant inhibitory activity for mutant (F1174L) anaplastic lymphoma kinase. It was also found that videx exhibited crucial interactions with the Met1199 residue of the native and mutant anaplastic lymphoma kinase protein. Furthermore, PASS algorithm predicted anti-neoplastic activity for all the four molecules. Thus these hits are found to be promising leads for anaplastic lymphoma kinase inhibitors. We believe that this study will be useful for the discovery and designing of more potent anaplastic lymphoma kinase inhibitors in the near future.

Effects of Racecadotril on Weight Loss and Diarrhea Due to Human Rotavirus in Neonatal Gnotobiotic Pigs (Sus scrofa domesticus).

Diarrheal disease is the second leading cause of death in children younger than 5 y, and the most common cause of acute watery diarrhea in young children worldwide is rotaviral infection. Medicines to specifically reduce diarrhea would be a desirable adjunctive treatment to supportive fluid therapy to decrease the mortality rate of diarrheal diseases. In this study, we evaluated the efficacy of an antisecretory drug, racecadotril, in treating human rotavirus (HRV)-induced diarrhea in a neonatal gnotobiotic pig model. In total, 27 gnotobiotic pigs were randomly assigned (n = 9 per group) to receive either racecadotril, chlorpromazine (positive-control drug), or PBS (mock treatment) after inoculation with HRV. Pigs were weighed daily and rectal swabs were collected to determine fecal consistency scores and virus shedding. Rotaviral infection was confirmed by ELISA and cell culture immunofluorescence. Overall, the racecadotril-treated pigs had less severe illness than either the chlorpromazine- or mock-treated groups; this conclusion was supported by the lower fecal-consistency scores, shorter duration of diarrhea, and significant gain in body weight during the course of the study of the racecadotril-treated pigs. Through its influence on decreasing intestinal hypersecretion, racecadotril was better able to control the clinical signs of rotaviral infection in the gnotobiotic pigs. These results lend support for using racecadotril as a treatment for rotaviral diarrhea.

Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea.

Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil-treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy.

[des-Arg(1)]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom.

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

Colon in acute intestinal infection.

The colon is actively implicated in intestinal infections not only as a target of enteric pathogens and their products but also as a target organ for treatment. In the presence of diarrhea, both of osmotic and secretory nature, the colon reacts with homeostatic mechanisms to increase ion absorption. These mechanisms can be effectively exploited to decrease fluid discharge. A model of intestinal infections using rotavirus (RV) in colonic cells was set up and used to define a dual model of secretory and osmotic diarrhea in sequence. Using this model, antidiarrheal drugs were tested, namely zinc and the enkephalinase inhibitor racecadotril. Zinc was able to decrease the enterotoxic activity responsible for secretory diarrhea. It also inhibited the cytotoxic effect of RV. The mechanism of zinc was related at least in part to the activation of MAPK activity, but also a direct antiviral effect was observed. Racecadotril showed a potent and selective inhibition of active secretion, being particularly effective in the first phase of RV diarrhea. The use of drugs active at the colonic level, therefore, offers effective options to treat intestinal infections in childhood. In addition, the colon is the natural site of colonic microflora, a target of probiotic therapy, which is the first line of approach recommended by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition to treat infectious diarrhea.

Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding.

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.

An automated method for predicting the positions of hydrogen-bonding atoms in binding sites.

Hydrogen bonds are the most specific, and therefore predictable of the intermolecular interactions involved in ligand-protein binding. Given the structure of a molecule, it is possible to estimate the positions at which complementary hydrogen-bonding atoms could be found. Crystal-survey data are used in the design of a program, HBMAP, that generates a hydrogen-bond map for any given ligand, which contains all the feasible positions at which a complementary atom could be found. On superposition of ligands, the overlapping regions of their maps represent positions of receptor atoms to which each molecule can bind. The certainty of these positions is increased by the incorporation of a larger number and diversity of molecules. In this work, superposition is achieved using the program HBMATCH, which uses simulated annealing to generate the correspondence between points from the hydrogen-bonding maps of the two molecules. Equivalent matches are distinguished on the basis of their steric similarity. The strategy is tested on a number of ligands for which ligand-protein complexes have been solved crystallographically, which allows validation of the techniques. The receptor atom positions of thermolysin are successfully predicted when the correct superposition is obtained.

Peptidase inhibitor-induced antidiuresis mediated through angiotensin and opioid receptors in the rat hypothalamus.

We examined the effects on urine outflow rate after microinjections of thiorphan (a carboxypeptidase inhibitor) and bestatin (an aminopeptidase inhibitor) into the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei of anesthetized hydrated rats to determine the possible role of neuropeptides in the regulation of urine production. After individual microinjection of the peptidase inhibitors into the nuclei, only thiorphan at 100 nmol administered into the PVN significantly decreased the urine outflow rate. Two consecutive microinjections of the peptidase inhibitors at 100 nmol each into the nuclei induced potent antidiuresis. These effects after microinjections of the peptidase inhibitors into the PVN and SON were diminished by pretreatment with [Sar1,Ile8]angiotensin (ANG) II (an ANG II receptor antagonist) and naloxone (an opioid receptor antagonist) in the PVN and with [Sar1,Ile8]ANG II in the SON, respectively. A vasopressin (AVP) receptor antagonist, d(CH2)5-D-Tyr(Et)VAVP (i.v.), completely blocked the antidiuresis by microinjections of the peptidase inhibitors into both the nuclei. Urinary osmotic pressure was significantly increased by consecutive microinjections of the peptidase inhibitors into the PVN and SON. These results suggest that endogenously-released ANG II and opioid peptides in the PVN and ANG II in the SON regulate urine production mediated through increased AVP release.

Involvement of B2 receptors in the bradykinin-induced relaxation of guinea-pig isolated trachea.

1. The aim of this study was to determine the receptor type and involvement of arachidonic acid metabolites in bradykinin-induced relaxation of the guinea-pig isolated trachea. 2. In the resting tracheal preparation, bradykinin (0.1 nM-30 microM induced a concentration-related contractile response (pD2 = 8.8 +/- 0.3). The maximal tension (1056 +/- 321 mg) was observed at 0.3 microM bradykinin. In contrast, when tracheal preparations were pre-contracted with histamine (30 microM leading to a half-maximum response), a concentration-related relaxation was observed with bradykinin. At the highest concentration of bradykinin used (3 microM), a reversal of 63 +/- 13% of the contractile response to histamine was observed. Both effects of bradykinin were inhibited by the cyclo-oxygenase inhibitor, indomethacin (1 microM). In concentration-response curves, melittin (10 nM-1 microM), a direct activator of phospholipase A2, mimicked both effects of bradykinin. The highest concentration of melittin used (1 microM), induced a tension of 813 +/- 120 mg and led to the reversal of 41 +/- 8% of the contractile response to histamine. The contractile effect of melittin was inhibited in the presence of both indomethacin (1 microM) and AA861 (1 microM), a 5-lipoxygenase inhibitor. 3. [Des Arg9]-bradykinin (1 nM-3 microM), a B1-receptor agonist, was unable to relax precontracted guinea-pig tracheal preparations. The relaxation induced by bradykinin was antagonized by the B2 receptor antagonists, Hoe 140 (D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin) and NPC 17761 (D-Arg0[Hyp3,D-HypE(trans-thiophenyl)7,Oic8]bradykinin ). Hoe 140 (0.1 microM to 0.6 microM) behaved as a non-competitive antagonist with an apparent pA2 = 7.2 +/- 0.4, whereas NPC 17761 (0.3 to 1 microM) competitively antagonized bradykinin-induced relaxation with a pKB = 7.3 +/- 0.2. The Schild regression slope did not differ from unity, 0.96 +/- 0.20, P<0.05.4. These data demonstrate that bradykinin-induced relaxation of guinea-pig trachea occurs via the activation of bradykinin B2-receptors. The stimulation of B2-bradykinin receptors induces the activation of the cyclo-oxygenase pathway, leading either to contraction or relaxation depending on the tone of the trachea.

Effects of thiorphan, bestatin and a novel metallopeptidase inhibitor JMV 390-1 on the recovery of neurotensin and neuromedin N released from mouse hypothalamus.

The effects of the endopeptidase 24.11 ('enkephalinase') inhibitor thiorphan, the aminopeptidase inhibitor bestatin and a novel metallopeptidase inhibitor JMV 390-1 on the K(+)-evoked release of immunoreactive neurotensin and neuromedin N (iNT and iNN) from mouse hypothalamic slices were examined. (JMV 390-1 inhibits several metallopeptidases including endopeptidases 24.11, 24.15 and 24.16, and aminopeptidase N equipotently with Ki values around 50 nM.) Thiorphan increased the recovery of released iNT nearly 2-fold and had no effect on iNN. Bestatin produced a 4-fold increase in iNN recovery and was inactive on iNT. Finally, iNT and iNN recoveries were increased up to 4- and 5-fold, respectively, by JMV 390-1. These results show that in the mouse hypothalamus endopeptidase 24.11 participates with other metalloendopeptidases to the degradation of endogenously released NT while endogenously released NN is principally degraded by aminopeptidase(s).

Effects of acetorphan, an enkephalinase inhibitor, on experimental and acute diarrhoea.

Acetorphan is an orally active inhibitor of enkephalinase (EC 3.4.24.11) with antidiarrhoeal activity in rodents apparently through protection of endogenous enkephalins and a purely antisecretory mechanism. Its antidiarrhoeal activity in man was assessed in an experimental model of cathartic induced secretory diarrhoea as well as in acute diarrhoea of presumed infectious origin. In six healthy volunteers receiving castor oil and pretreated with acetorphan or placebo in a crossover controlled trial, the drug significantly decreased the number and weight of stools passed during 24 hours. About 200 outpatients with severe acute diarrhoea (more than five stools per day) were included in a randomised double blind study of acetorphan against placebo. The significant antidiarrhoeal activity of acetorphan was established using a variety of criteria: (i) the duration of both diarrhoea and treatment were diminished; (ii) no acetorphan treated patient withdrew from the study whereas five dropped out because of worsening in the placebo group; (iii) the frequency of symptoms associated with diarrhoea--for example, abdominal pain or distension, nausea and anorexia--remaining after two weeks was nearly halved; (iv) using visual analogue scales acetorphan treatment was found more effective than placebo by both investigators and patients. There was statistically no significant difference between acetorphan and placebo in respect of side effects, particularly constipation, which often accompanies the antidiarrhoeal activity of mu opioid receptor agonists this difference is attributable to the lack of antipropulsive activity of acetorphan in man. The efficacy and tolerance of acetorphan suggest that enkephalinase inhibition may represent a novel therapeutic approach for the symptomatic management of acute secretory diarrhoea without impairing intestinal transit.

[Enkephelinase inhibitors acetorphan and SCH34826 potentiate the analgesic effect of low frequency electroacupuncture in rats].

Influence of two novel enkephelinase inhibitors, Acetorphan and SCH34826, on the effect of electroacupuncture analgesia (EAA) has been observed in rats. Administered Acetorphan (2.0-4.0 mg/kg, i.p.) or SCH34826 (30-100 mg/kg, p.o.) potentiated and prolonged the effect of 2 Hz EAA significantly in the nonresponder in which an evident analgesic effect can not be induced by 2 Hz electroacupuncture. This means that che non-responder of 2 Hz EAA can be converted into the responder with these two drugs. The non-responder of 100 Hz EAA, however, can not be changed into the responder with Acetorphan. These results again support our previous reports that enkephelin plays an important role in low frequency EAA. Meanwhile, this suggests us a probable route for potentiating acupuncture analgesia in clinical practice.

Augmented analgesic effects of enkephalinase inhibitors combined with transcranial electrostimulation.

The analgesic effects of very low current transcranial electrostimulation are naloxone-reversible and thus presumably mediated by endogenous opioid activity. The present experiments indicate that blocking enkephalinase activity by i.c.v. thiorphan or i.p. acetorphan results in an increased analgesic effect of electrostimulation as measured by the 50 degrees C wet tail flick test. In the case of each drug, rats receiving both drug and electrostimulation displayed significantly more analgesia than rats receiving electrostimulation and injection vehicle alone, rats receiving drug and sham stimulation or rats receiving vehicle and sham stimulation.

Naloxone-reversible antidiarrheal effects of enkephalinase inhibitors.

Thiorphan and acetorphan, two potent inhibitors of enkephalinase (EC 3.4.24.11 membrane-metalloendopeptidase) significantly reduced the castor oil-induced diarrhea in rats when administered intravenously (or orally, for acetorphan) but not when administered intracerebroventricularly. These effects were more marked during the 90 min period following the castor oil challenge but were still significant up to 4-8 h after the latter. Acetorphan was about 6 times more potent than thiorphan. The antidiarrheal activity of both compounds was completely prevented in rats receiving naloxone subcutaneously but not intracerebroventricularly (in the case of thiorphan). In contrast to loperamide, a peripherally acting opiate receptor agonist, the enkephalinase inhibitors did not significantly reduce gastrointestinal transit as measured in the charcoal meal test. The antidiarrheal activity of enkephalinase inhibitors therefore seems attributable to protection of endogenous opioids, presumably outside the brain, and to reduction of intestinal secretion rather than transit.

Electroacupuncture and morphine analgesia potentiated by bestatin and thiorphan administered to the nucleus accumbens of the rabbit.

The endogenous opioid peptide enkephalin (EK) is known to be degraded mainly by two enzymes, the dipeptidyl carboxypeptidase 'enkephalinase' and aminopeptidase. Microinjection of the enkephalinase inhibitor thiorphan or the aminopeptidase inhibitor bestatin into the nucleus accumbens of the rabbit produced a dose-dependent analgesic effect. This analgesic effect was totally reversed by the narcotic antagonist naloxone or by antibodies against [Met5]enkephalin (MEK) administered to the same site. Antibodies against [Leu5]enkephalin were not effective. Moreover, microinjection of thiorphan or bestatin into the nucleus accumbens resulted in a marked potentiation of the aftereffect of electroacupuncture (EA) produced analgesia, as well as the analgesia induced by a small dose of morphine. It is concluded that the analgesic effect elicited by EA and morphine is mediated, at least in part, by MEK-like immunoreactive substance(s) in the nucleus accumbens.

Effects of ES52, an enkephalinase inhibitor, on responses of ventrobasal thalamic neurons in rat.

The effects of ES52, a highly potent derivative of Thiorphan, an inhibitor of enkephalinase, at doses of 5 and 10 mg/kg IV were studied on the responses to cutaneous stimuli of 18 "nociceptive" (N), 10 "convergent" (NNn) and 4 "non-nociceptive" (Nn) neurons recorded in the ventrobasal (VB) complex of the rat. The responses of neurons exclusively driven by noxious mechanical and thermal stimuli (N neurons) were depressed by 56% by ES52 15 min after the injection of 5 or 10 mg/kg IV. This depressive effect was reversed by naloxone for half the neurons. For the ten neurons driven by both noxious and non-noxious stimuli (convergent NNn neurons), the responses to noxious heat were decreased by 42% at 15 min. By contrast, there was a marked enlargement of their receptive fields to light tactile stimuli, which was not naloxone-reversible. The receptive fields of neurons exclusively driven by non-noxious stimuli (Nn neurons) were also greatly expanded by ES52. These results show that ES52 can depress the responses of VB thalamic neurons to noxious stimuli; the effects on receptive field size underlines the complexity of the endogenous opiate systems.

Action of peptidase inhibitors on methionine5-enkephalin-arginine6-phenylalanine7 (YGGFMRF) and methionine5-enkephalin (YGGFM) metabolism and on electroacupuncture antinociception.

In the spinal cord Met5-enkephalin-Arg6-Phe7 (YGGFMRF) is located in small interneurons of the dorsal and ventral horns. From these storage sites, YGGFMRF can be released by perfusing the subarachnoidal spaces of the spinal cord with artificial spinal fluid containing substance P. In vitro YGGFMRF can be hydrolyzed readily by a dipeptidyl carboxypeptidase. In order to ascertain whether this reaction is physiologically relevant, we measured the content of YGGFMRF and Met5-enkephalin (YGGFM) in subarachnoidal space perfusate in presence and in absence of captopril, bestatin and thiorphan using substance P to activate the release of opioid peptides. Without peptidase inhibitors, the efflux of YGGFMRF and YGGFM was hardly detectable. The addition of captopril to the perfusion medium increased the substance P (10(-7) M)-induced release of YGGFMRF markedly but it increased the efflux of YGGFM to a much smaller extent. When captopril and bestatin were added together the amount of YGGFMRF present in the perfusate was further increased slightly. In contrast, the YGGFM content in the same perfusate was increased greatly by bestatin and only slightly by thiorphan. To characterize the pharmacological profile of these peptidase inhibitors, we compared electroacupuncture antinociception with and without intrathecal injections of captopril and bestatin. This antinociception, as measured by tail-flick latency, was potentiated by the intrathecal injection of captopril and bestatin. These results taken together suggest that YGGFMRF released in the perfusate of the arachnoidal space by substance P is metabolized by both dipeptidyl carboxypeptidase and aminopeptidase.

Depressive effects of morphine and of an enkephalinase inhibitor on responses of ventro-basal thalamic neurones to noxious stimuli.

It has been shown that the responses of VB thalamic neurones to noxious mechanical and thermal stimuli are strongly depressed by low doses of Morphine (0,03; 0,1; 1 mg/kg i.v.) and to a lesser extent by ES 52 (a highly potent Thiorphan derivative) injected at higher doses (5; 10 mg/kg i.v). This last effect was less easily reversed by Naloxone than was the depressive effect of Morphine. Moreover, ES 52 can facilitate activity of some thalamic neurones induced by non-noxious mechanical stimuli.