Opiate alkaloids and their synthetic derivatives are still widely used in pain management, drug a... more Opiate alkaloids and their synthetic derivatives are still widely used in pain management, drug addiction, and abuse. To avoid serious side effects, compounds with properly designed pharmacological profiles at the opioid receptor subtypes are long needed. Here a series of 17-N-substituted derivatives of normorphine and noroxymorphone analogues with five- and six-membered ring substituents have been synthesized for structure–activity study. Some compounds showed nanomolar affinity to MOR, DOR and KOR in in vitro competition binding experiments with selective agonists [3H]DAMGO, [3H]Ile5,6-deltorphin II and [3H]HS665, respectively. Pharmacological characterization of the compounds in G-protein signaling was determined by [35S]GTPγS binding assays. The normorphine analogues showed higher affinity to KOR compared to MOR and DOR, while most of the noroxymorphone derivatives did not bind to KOR. The presence of 14-OH substituent resulted in a shift in the pharmacological profiles in the a...
The use of α-amino-γ lactam of Freidinger (Agl) may serve as an impressive method to increase the... more The use of α-amino-γ lactam of Freidinger (Agl) may serve as an impressive method to increase the biological stability of peptides and an appropriate tool to elucidate their structure-activity relationships. The endomorphin-2 (EM-2) and [D-Ala2, des-Leu5] enkephalin amide (DAPEA) are two linear opioid tetrapeptides agonists of MOR and MOR/DOR respectively. Herein, we investigated the influence of the incorporation of (R/S)-Agl in position 2 and 3 on the biological profile of the aforementioned products in vitro and in vivo. Receptor radiolabeled displacement and functional assays were used to measure in vitro the binding affinity and receptors activation of the novel analogues. The mouse tail flick and formalin tests allowed to observe their antinociceptive effect in vivo. Data revealed that peptide A2D was able to selectively bind and activate MOR with a potent antinociceptive effect after intracerebroventricular (i.c.v.) administration, performing better than the parent compounds EM-2 and DAPEA. Molecular docking calculations helped us to understand the key role exerted by the Freidinger Agl moiety in A2D for the interaction with the MOR binding pocket.
Despite the large arsenal of analgesic medications, neuropathic pain (NP) management is not solve... more Despite the large arsenal of analgesic medications, neuropathic pain (NP) management is not solved yet. Angiotensin II receptor type 1 (AT1) has been identified as a potential target in NP therapy. Here, we investigate the antiallodynic effect of AT1 blockers telmisartan and losartan, and particularly their combination with morphine on rat mononeuropathic pain following acute or chronic oral administration. The impact of telmisartan on morphine analgesic tolerance was also assessed using the rat tail-flick assay. Morphine potency and efficacy in spinal cord samples of treated neuropathic animals were assessed by [35S]GTPγS-binding assay. Finally, the glutamate content of the cerebrospinal fluid (CSF) was measured by capillary electrophoresis. Oral telmisartan or losartan in higher doses showed an acute antiallodynic effect. In the chronic treatment study, the combination of subanalgesic doses of telmisartan and morphine ameliorated allodynia and resulted in a leftward shift in the d...
The low efficacy of antipsychotic drugs (e.g., clozapine) for negative symptoms and cognitive imp... more The low efficacy of antipsychotic drugs (e.g., clozapine) for negative symptoms and cognitive impairment has led to the introduction of adjuvant therapies. Because previous data suggest the procognitive potential of the antidiabetic drug metformin, this study aimed to assess the effects of chronic clozapine and metformin oral administration (alone and in combination) on locomotor and exploratory activities and cognitive function in a reward-based test in control and a schizophrenia-like animal model (Wisket rats). As impaired dopamine D1 receptor (D1R) function might play a role in the cognitive dysfunctions observed in patients with schizophrenia, the second goal of this study was to determine the brain-region-specific D1R-mediated signaling, ligand binding, and mRNA expression. None of the treatments affected the behavior of the control animals significantly; however, the combination treatment enhanced D1R binding and activation in the cerebral cortex. The Wisket rats exhibited im...
Despite the progress in early diagnostic and available treatments, pancreatic cancer remains one ... more Despite the progress in early diagnostic and available treatments, pancreatic cancer remains one of the deadliest cancers. Therefore, there is an urgent need for novel anticancer agents with a good safety profile, particularly in terms of possible side-effects. Recently dopaminergic receptors have been widely studied as they were proven to play an important role in cancer progression. Although various synthetic compounds are known for their interactions with the dopaminergic system, peptides have recently made a great comeback. This is because peptides are relatively safe, easy to correct in terms of the improvement of their physicochemical and biological properties, and easy to predict. This paper aims to evaluate the anticancer activity of a naturally existing peptide—ranatensin, toward three different pancreatic cancer cell lines. Additionally, since there is no sufficient information confirming the exact character of the interaction between ranatensin and dopaminergic receptors,...
Journal of enzyme inhibition and medicinal chemistry, 2017
Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavio... more Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH2. The bivalent compound (9) has been used for in vitro binding assays, for in vivo antinociception models and in vitro hypothalamic perfusion test, to evaluate the neurotransmitters release.
The aim of the study was to investigate the roles of α1-adrenoceptor subtypes in the last-day pre... more The aim of the study was to investigate the roles of α1-adrenoceptor subtypes in the last-day pregnant rat uterus in vitro by the administration of subtype-specific antagonists (the α1A-adrenoceptor antagonist WB 4101 and the α1D-adrenoceptor antagonist BMY 7378) after 17β-estradiol or progesterone pretreatment. In isolated organ bath studies, contractions were elicited with (-)-noradrenaline (10(-8)-10(-5)M) in the presence of propranolol (10(-5)M) and yohimbine (10(-6)M) in order to avoid β-, and α2-adrenergic action. The myometrial expressions of the α1-adrenoceptor subtypes were determined by means of the real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques. The activated G protein levels were investigated through radiolabelled GTP binding assays. Both 17β-estradiol and progesterone pretreatment changed the myometrial contracting effect of (-)-noradrenaline. In the presence of WB 4101, progesterone pretreatment decreased the (-)-nora...
The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) with the sequence of YGGFMRF is a potent endogen... more The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) with the sequence of YGGFMRF is a potent endogenous opioid located at the C-terminus of proenkephalin-A (PENK), the common polypeptide precursor of Met- and Leu-enkephalin. Our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different PENK prepropeptides among 56 vertebrate animals. Four orthologous heptapeptides with single or double amino acid replacements were identified among 15 animals, such as YGGFMGY (zebrafish), YGGFMRY (newt), YGGFMKF (hedgehog tenrek) and YGGFMRI (mudpuppy). Each novel heptapeptide, together with the mammalian consensus MERF and Met-enkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and G-protein activation assays in rat brain membranes. Equilibrium binding affinities changed from good to modest as measured by receptor type selective [3H]opioid radioligands. The relative affinities of the heptapeptides reveal slight mu-receptor (MOP) preference over the delta-receptors (DOP). [35S]GTPγS assay, which measures the agonist-mediated G-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory G-proteins. All peptides were effective in promoting the agonist induced internalization of the green fluorescence protein-tagged human mu-opioid receptor (hMOP-EGFP) stably expressed in HEK293 cells. Thus, the C-terminally processed PENK heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution.
Enkephalins are active in regulation of nociception in the body and are key in development of new... more Enkephalins are active in regulation of nociception in the body and are key in development of new synthetic peptide analogs that target centrally located opioid receptors. In this study, we investigated the in vivo blood-brain barrier (BBB) penetration behavior and antinociceptive activity of two cyclic enkephalin analogs with a thiourea (CycS) or a N-methyl-guanidine bridge (CycNMe), and their linear counterparts (LinS and LinNMe) in mice, as well as their in vitro metabolic stability. (125)I-LinS had the highest blood-brain clearance (K1=3.46μL/gmin), followed by (125)I-LinNMe, (125)I-CycNMe, and (125)I-CycS (K1=1.64, 0.31, and 0.11μL/gmin, respectively). Also, these peptides had a high metabolic stability (t1/2>1h) in mouse serum and brain homogenate, and half-inhibition constant (Ki) values in the nanomolar range with predominantly μ-opioid receptor selectivity. The positively charged NMe-enkephalins showed a higher antinociceptive activity (LinNMe: 298% and CycNMe: 205%), ex...
There is no information concerning signal transduction mechanisms downstream of the opioid/nocice... more There is no information concerning signal transduction mechanisms downstream of the opioid/nociceptin receptors in the human epileptic brain. The aim of this work was to evaluate the level of G-proteins activation mediated by DAMGO (a mu receptor selective peptide) and nociceptin, and the binding to mu and nociceptin (NOP) receptors and adenylyl cyclase (AC) in neocortex of patients with pharmacoresistant temporal lobe epilepsy. Patients with temporal lobe epilepsy associated with mesial sclerosis (MTLE) or secondary to tumor or vascular lesion showed enhanced [3H]DAMGO and [3H]forskolin binding, lower DAMGO-stimulated [35S]GTPgammaS binding and no significant changes in nociceptin-stimulated G-protein. [3H]Nociceptin binding was lower in patients with MTLE. Age of seizure onset correlated positively with [3H]DAMGO binding and DAMGO-stimulated [35S]GTPgammaS binding, whereas epilepsy duration correlated negatively with [3H]DAMGO and [3H]nociceptin binding, and positively with [3H]forskolin binding. In conclusion, our present data obtained from neocortex of epileptic patients provide strong evidence that a) temporal lobe epilepsy is associated with alterations in mu opioid and NOP receptor binding and signal transduction mechanisms downstream of these receptors, and b) clinical aspects may play an important role on these receptor changes.
Opiate alkaloids and their synthetic derivatives are still widely used in pain management, drug a... more Opiate alkaloids and their synthetic derivatives are still widely used in pain management, drug addiction, and abuse. To avoid serious side effects, compounds with properly designed pharmacological profiles at the opioid receptor subtypes are long needed. Here a series of 17-N-substituted derivatives of normorphine and noroxymorphone analogues with five- and six-membered ring substituents have been synthesized for structure–activity study. Some compounds showed nanomolar affinity to MOR, DOR and KOR in in vitro competition binding experiments with selective agonists [3H]DAMGO, [3H]Ile5,6-deltorphin II and [3H]HS665, respectively. Pharmacological characterization of the compounds in G-protein signaling was determined by [35S]GTPγS binding assays. The normorphine analogues showed higher affinity to KOR compared to MOR and DOR, while most of the noroxymorphone derivatives did not bind to KOR. The presence of 14-OH substituent resulted in a shift in the pharmacological profiles in the a...
The use of α-amino-γ lactam of Freidinger (Agl) may serve as an impressive method to increase the... more The use of α-amino-γ lactam of Freidinger (Agl) may serve as an impressive method to increase the biological stability of peptides and an appropriate tool to elucidate their structure-activity relationships. The endomorphin-2 (EM-2) and [D-Ala2, des-Leu5] enkephalin amide (DAPEA) are two linear opioid tetrapeptides agonists of MOR and MOR/DOR respectively. Herein, we investigated the influence of the incorporation of (R/S)-Agl in position 2 and 3 on the biological profile of the aforementioned products in vitro and in vivo. Receptor radiolabeled displacement and functional assays were used to measure in vitro the binding affinity and receptors activation of the novel analogues. The mouse tail flick and formalin tests allowed to observe their antinociceptive effect in vivo. Data revealed that peptide A2D was able to selectively bind and activate MOR with a potent antinociceptive effect after intracerebroventricular (i.c.v.) administration, performing better than the parent compounds EM-2 and DAPEA. Molecular docking calculations helped us to understand the key role exerted by the Freidinger Agl moiety in A2D for the interaction with the MOR binding pocket.
Despite the large arsenal of analgesic medications, neuropathic pain (NP) management is not solve... more Despite the large arsenal of analgesic medications, neuropathic pain (NP) management is not solved yet. Angiotensin II receptor type 1 (AT1) has been identified as a potential target in NP therapy. Here, we investigate the antiallodynic effect of AT1 blockers telmisartan and losartan, and particularly their combination with morphine on rat mononeuropathic pain following acute or chronic oral administration. The impact of telmisartan on morphine analgesic tolerance was also assessed using the rat tail-flick assay. Morphine potency and efficacy in spinal cord samples of treated neuropathic animals were assessed by [35S]GTPγS-binding assay. Finally, the glutamate content of the cerebrospinal fluid (CSF) was measured by capillary electrophoresis. Oral telmisartan or losartan in higher doses showed an acute antiallodynic effect. In the chronic treatment study, the combination of subanalgesic doses of telmisartan and morphine ameliorated allodynia and resulted in a leftward shift in the d...
The low efficacy of antipsychotic drugs (e.g., clozapine) for negative symptoms and cognitive imp... more The low efficacy of antipsychotic drugs (e.g., clozapine) for negative symptoms and cognitive impairment has led to the introduction of adjuvant therapies. Because previous data suggest the procognitive potential of the antidiabetic drug metformin, this study aimed to assess the effects of chronic clozapine and metformin oral administration (alone and in combination) on locomotor and exploratory activities and cognitive function in a reward-based test in control and a schizophrenia-like animal model (Wisket rats). As impaired dopamine D1 receptor (D1R) function might play a role in the cognitive dysfunctions observed in patients with schizophrenia, the second goal of this study was to determine the brain-region-specific D1R-mediated signaling, ligand binding, and mRNA expression. None of the treatments affected the behavior of the control animals significantly; however, the combination treatment enhanced D1R binding and activation in the cerebral cortex. The Wisket rats exhibited im...
Despite the progress in early diagnostic and available treatments, pancreatic cancer remains one ... more Despite the progress in early diagnostic and available treatments, pancreatic cancer remains one of the deadliest cancers. Therefore, there is an urgent need for novel anticancer agents with a good safety profile, particularly in terms of possible side-effects. Recently dopaminergic receptors have been widely studied as they were proven to play an important role in cancer progression. Although various synthetic compounds are known for their interactions with the dopaminergic system, peptides have recently made a great comeback. This is because peptides are relatively safe, easy to correct in terms of the improvement of their physicochemical and biological properties, and easy to predict. This paper aims to evaluate the anticancer activity of a naturally existing peptide—ranatensin, toward three different pancreatic cancer cell lines. Additionally, since there is no sufficient information confirming the exact character of the interaction between ranatensin and dopaminergic receptors,...
Journal of enzyme inhibition and medicinal chemistry, 2017
Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavio... more Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH2. The bivalent compound (9) has been used for in vitro binding assays, for in vivo antinociception models and in vitro hypothalamic perfusion test, to evaluate the neurotransmitters release.
The aim of the study was to investigate the roles of α1-adrenoceptor subtypes in the last-day pre... more The aim of the study was to investigate the roles of α1-adrenoceptor subtypes in the last-day pregnant rat uterus in vitro by the administration of subtype-specific antagonists (the α1A-adrenoceptor antagonist WB 4101 and the α1D-adrenoceptor antagonist BMY 7378) after 17β-estradiol or progesterone pretreatment. In isolated organ bath studies, contractions were elicited with (-)-noradrenaline (10(-8)-10(-5)M) in the presence of propranolol (10(-5)M) and yohimbine (10(-6)M) in order to avoid β-, and α2-adrenergic action. The myometrial expressions of the α1-adrenoceptor subtypes were determined by means of the real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques. The activated G protein levels were investigated through radiolabelled GTP binding assays. Both 17β-estradiol and progesterone pretreatment changed the myometrial contracting effect of (-)-noradrenaline. In the presence of WB 4101, progesterone pretreatment decreased the (-)-nora...
The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) with the sequence of YGGFMRF is a potent endogen... more The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) with the sequence of YGGFMRF is a potent endogenous opioid located at the C-terminus of proenkephalin-A (PENK), the common polypeptide precursor of Met- and Leu-enkephalin. Our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different PENK prepropeptides among 56 vertebrate animals. Four orthologous heptapeptides with single or double amino acid replacements were identified among 15 animals, such as YGGFMGY (zebrafish), YGGFMRY (newt), YGGFMKF (hedgehog tenrek) and YGGFMRI (mudpuppy). Each novel heptapeptide, together with the mammalian consensus MERF and Met-enkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and G-protein activation assays in rat brain membranes. Equilibrium binding affinities changed from good to modest as measured by receptor type selective [3H]opioid radioligands. The relative affinities of the heptapeptides reveal slight mu-receptor (MOP) preference over the delta-receptors (DOP). [35S]GTPγS assay, which measures the agonist-mediated G-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory G-proteins. All peptides were effective in promoting the agonist induced internalization of the green fluorescence protein-tagged human mu-opioid receptor (hMOP-EGFP) stably expressed in HEK293 cells. Thus, the C-terminally processed PENK heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution.
Enkephalins are active in regulation of nociception in the body and are key in development of new... more Enkephalins are active in regulation of nociception in the body and are key in development of new synthetic peptide analogs that target centrally located opioid receptors. In this study, we investigated the in vivo blood-brain barrier (BBB) penetration behavior and antinociceptive activity of two cyclic enkephalin analogs with a thiourea (CycS) or a N-methyl-guanidine bridge (CycNMe), and their linear counterparts (LinS and LinNMe) in mice, as well as their in vitro metabolic stability. (125)I-LinS had the highest blood-brain clearance (K1=3.46μL/gmin), followed by (125)I-LinNMe, (125)I-CycNMe, and (125)I-CycS (K1=1.64, 0.31, and 0.11μL/gmin, respectively). Also, these peptides had a high metabolic stability (t1/2>1h) in mouse serum and brain homogenate, and half-inhibition constant (Ki) values in the nanomolar range with predominantly μ-opioid receptor selectivity. The positively charged NMe-enkephalins showed a higher antinociceptive activity (LinNMe: 298% and CycNMe: 205%), ex...
There is no information concerning signal transduction mechanisms downstream of the opioid/nocice... more There is no information concerning signal transduction mechanisms downstream of the opioid/nociceptin receptors in the human epileptic brain. The aim of this work was to evaluate the level of G-proteins activation mediated by DAMGO (a mu receptor selective peptide) and nociceptin, and the binding to mu and nociceptin (NOP) receptors and adenylyl cyclase (AC) in neocortex of patients with pharmacoresistant temporal lobe epilepsy. Patients with temporal lobe epilepsy associated with mesial sclerosis (MTLE) or secondary to tumor or vascular lesion showed enhanced [3H]DAMGO and [3H]forskolin binding, lower DAMGO-stimulated [35S]GTPgammaS binding and no significant changes in nociceptin-stimulated G-protein. [3H]Nociceptin binding was lower in patients with MTLE. Age of seizure onset correlated positively with [3H]DAMGO binding and DAMGO-stimulated [35S]GTPgammaS binding, whereas epilepsy duration correlated negatively with [3H]DAMGO and [3H]nociceptin binding, and positively with [3H]forskolin binding. In conclusion, our present data obtained from neocortex of epileptic patients provide strong evidence that a) temporal lobe epilepsy is associated with alterations in mu opioid and NOP receptor binding and signal transduction mechanisms downstream of these receptors, and b) clinical aspects may play an important role on these receptor changes.
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