Opioid peptides have the potential to be pharmaceutical agents for the treatment of pain because ... more Opioid peptides have the potential to be pharmaceutical agents for the treatment of pain because they modulate nociceptive pathways at supraspinal, spinal and peripheral levels. Unfortunately, peptides are generally hydrophilic compounds and therefore unable to cross the blood-brain barrier (BBB) by passive diffusion to reach the central nervous system (CNS) in an amount sufficient to activate appropriate receptors. Endomorphins (EMs) belong to the class of endogenous opioids eliciting the strongest analgesic effect, but only after direct administration to the CNS. Extensive research is in progress to better understand the relationships between EM structure and bioavailability. This article deals with the recent investigations that allow the design of stable and neuroactive EM analogs with enhanced brain passage and uptake.
A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-na... more A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-naphthyl)-alanine (1-Nal) or 3-(2-naphthyl)-alanine (2-Nal) in L- or D-configuration, was synthesized. The opioid activity profiles of these peptides were determined in the mu-opioid receptor representative binding assay and in the Guinea-Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot-plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the mu-opioid receptor was reduced compared with endomorphin-2. The two most potent analogs were [D-1-Nal(4)]- and [D-2-Nal4]endomorphin-2, with IC50 values 14 +/- 1.25 and 19 +/- 2.1 nM, respectively, compared with 1.9 +/- 0.21 nM for endomorphin-2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot-plate test. Antinociception induced by endomorphin-2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [D-1-Nal4]- and [D-2-Nal4]-endomorphin-2, indicating that these analogs were mu-opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 microg per animal naloxone almost completely inhibited antinociceptive action of endomorphin-2, while [D-1-Nal4]endomorphin-2 in about 46%.
In the present study, we reported on the synthesis of two new μ-opioid peptide analogs, [d-1-Nal3... more In the present study, we reported on the synthesis of two new μ-opioid peptide analogs, [d-1-Nal3]morphiceptin and [d-1-Nal4]-morphiceptin [1-Nal=3-(1-naphthyl)-alanine] which expressed receptor binding affinities at least at the level of the primary opioid ligands.The new analogs also labeled μ-opioid receptors on the cells of human breast cancer MCF-7 cell line with affinity much higher than that of endomorphins and morphiceptin,
In the present study we investigated and compared the in vivo analgesia of centrally administered... more In the present study we investigated and compared the in vivo analgesia of centrally administered endomorphin-2 and morphiceptin, and their analogs modified in position 3. Two series of analogs were synthesized by introducing unnatural aromatic amino acids in the d configuration: 3-(1-naphthyl)-d-alanine (d-1-Nal), 3-(2-naphthyl)-d-alanine (d-2-Nal), 3-(4-chlorophenyl)-d-alanine (d-ClPhe), 3-(3,4-dichlorophenyl)-d-alanine (d-Cl2Phe). Antinociceptive activity of endomorphin-2, morphiceptin, and their analogs was compared in
As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides... more As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides based on the structure of endomorphin-2 (EM-2), we report here the synthesis and biological activities of a new series of analogues of a general sequence Tyr/Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (where Dmt = 2',6'-dimethyltyrosine), incorporating fluorinated amino acids: 4-fluorophenylalanine (4-F-Phe), 2,4-difluorophenylalanine (2,4-F-Phe), or 4-trifluoromethylphenylalanine (4-CF3-Phe) instead of the Phe residue in position 3 or 4. Depending on the fluorinated amino acid residue and its position in the sequence, analogues were mixed, high affinity MOP/KOP receptor agonists, MOP/DOP/KOP agonists, or selective KOP agonists. The in vitro potencies and efficacies of all novel analogues were assessed in calcium mobilization assay. The most potent analogues, Dmt-c[d-Lys-Phe-4-F-Phe-Asp]NH2 and Dmt-c[d-Lys-Phe-2,4-F-Phe-Asp]NH2, were tested in vivo in the mouse hot-plate test. They produced strong antinociceptive effect not only after intracerebroventricular but also after intraperitoneal injection, indicating that they were able to cross the blood-brain barrier.
In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like act... more In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-β-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-β-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-β-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-β-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration...
The aim of the present study was to characterize the binding selectivity of the l-opioid receptor... more The aim of the present study was to characterize the binding selectivity of the l-opioid receptor ligands, endomorphin-1, endomorphin-2, and DAM-GO, in the in vitro functional assay, based on the changes in intracellular calcium levels. For the experiments Chinese hamster ovary cells, stably expressing human l-receptor, were used. The l-agonist-induced calcium responses were significantly inhibited by naloxone, an opioid antagonist with high preference for the l-opioid receptors. Naloxonazine, a l 1 -non-peptide antagonist, inhibited the effect of all tested l-agonists. However, there was no significant difference in the antagonist effect of naloxonazine on the calcium response induced by l 1 -(endomorphin-2) and l 2 -agonists (endomorphin-1, DAMGO). [D-Pro 2 ]endomorphin-1 and [D-Pro 2 ]endomorphin-2, putative peptide l 2 -and l 1 -antagonists, respectively, which had been shown in vivo to inhibit the antinociception induced by l-agonists, produced no inhibitory effect in our in vitro experiments. Our results demonstrated that there is only one population of the l-opioid receptors expressed in the Chinese hamster ovary cells. We suggest that the l-opioid receptors form a homogenous population in the in vitro systems. However, the existence of l-receptor subtypes in vivo is still pharmacologically possible.
Drosophila tachykinin receptor, a neurokinin receptor cloned from the fruit fly Drosophila melano... more Drosophila tachykinin receptor, a neurokinin receptor cloned from the fruit fly Drosophila melanogaster, is a G-protein-coupled receptor, which upon activation by a peptide agonist induces a transient increase in the concentration of intracellular calcium. The functional assay based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca 2+ levels was used to examine and compare the effect of tachykininrelated peptides from different species. Among the endogenous Drosophila peptides, Drm-TK I induced the strongest calcium response. The most potent tachykinin-related peptides from Leucophaea maderae, Locusta migratoria, and Calliphora vomitoria, were partial agonists at the Drosophila tachykinin receptor.
A series of luteinizing hormone-releasing hormone antagonists with new substitutions in position ... more A series of luteinizing hormone-releasing hormone antagonists with new substitutions in position 6 or positions 5 and 6 that included lysine acylated at the e-amino group with different heterocyclic carboxylic acids or amino-substituted heterocyclic carboxylic acids was Synthesized. These novel analogs were synthesized on a solid-phase support via the acylation of lysine residue in otherwise protected resin-bound peptides. All analogs were tested in the rat antiovulatory assay (AOA) and the best of them in in vitro histamine release assay. Introduction of lysine acylated with aminosubstituted heterocyclic carboxylic acids yielded several water-soluble antagonists with good therapeutic ratio (high AOA to low histamine releasing activity). The best antagonist in terms of activity, histamine release, and solubility was nictide: NAc~Nal-~cpa-~Pal-$er-PicLys-~(GANic)-Orn-Leu-ILys-Pro-~AlaNH2 (6ANic = 6-aminonicotinoyl).
Hot plate test Antinociception Solid phase peptide synthesis a b s t r a c t This study reports t... more Hot plate test Antinociception Solid phase peptide synthesis a b s t r a c t This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH 2 or Tyr-c(Xaa-Phe-D-Pro-Yaa)NH 2 , respectively, where Xaa and Yaa were L/D Asp or L/D Lys. Further modification of these analogs was achieved by introduction of 2 0 ,6 0 -dimethyl-L-tyrosine (Dmt) instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin by a microwave-assisted procedure.
Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generat... more Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generating analogs with improved bioactivities. Cyclization can be achieved through various bridging bonds between peptide ends or side-chains. In our earlier paper we have reported the synthesis and biological activity of a cyclic peptide, Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (1), which can be viewed as an analog of endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2). Cyclization was achieved through an amide bond between side-chains of D-Lys and Asp residues. Here, to increase rigidity of the cyclic structure, we replaced d-Lys with cis- or trans-4-aminocyclohexyl-D-alanine (D-ACAla). Two sets of analogs incorporating either Tyr or Dmt (2',6'-dimethyltyrosine) residues in position 1 were synthesized. In the binding studies the analog incorporating Dmt and trans-D-ACAla showed high affinity for both, μ- and δ-opioid receptors (MOR and DOR, respectively) and moderate affinity for the κ-opioid receptor (KOR), while analog with Dmt and cis-D-ACAla was exceptionally MOR-selective. Conformational analyses by NMR and molecular docking studies have been performed to investigate the molecular structural features responsible for the noteworthy MOR selectivity.
Synthesis and Cytotoxic Evaluation of β-Alkyl or β-Aryl-δ-methyl-α-methylene-δ-lactones. Comparis... more Synthesis and Cytotoxic Evaluation of β-Alkyl or β-Aryl-δ-methyl-α-methylene-δ-lactones. Comparison with the Corresponding γ-Lactones. -A simple and general strategy for the synthesis of title compounds (VII)/(VIII) is presented. The inseparable mixtures of δ-lactones are evaluated in vitro against mouse leukemia cell line L-1210 and the human leukemia cell lines HL-60 and NALM-6. The cytotoxicity of β-aryl-substituted derivatives is very high and comparable with that of previously tested β-aryl-γ-lactones. -(ALBRECHT, L.; WOJCIECHOWSKI, J.; ALBRECHT, A.; WOLF, W. M.; JANECKA, A.; STUDZIAN, K.; KRAJEWSKA, U.; ROZALSKI, M.; JANECKI, T.; KRAWCZYK*, H.; Eur. J. Med. Chem. 45 (2010) 2, 710-718; Inst. Org. Chem., Tech. Univ., PL-90-924 Lodz, Pol.; Eng.) -H. Haber 23-132
European journal of medicinal chemistry, Jan 14, 2015
Three series of new 4-methylidenepyrazolidin-3-ones with various substitution patterns were synth... more Three series of new 4-methylidenepyrazolidin-3-ones with various substitution patterns were synthesized and tested for the cytotoxic activity against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7 breast cancer cell line. Several obtained methylidenepyrazolidinones exhibited high cytotoxic activity with IC50 values below 10 μM, mainly against HL-60 leukemia cell line and two of them, 18d,e, displayed IC50 ≤ 5 μM, against all tested cell lines. Structure-activity relationship studies revealed that the presence of phenyl substituents on both ring nitrogen atoms and vinyl or phenyl substituents in position 5 are crucial for high activity. Selected methylidenepyrazolidinones were also tested on normal human umbilical vein endothelial cells (HUVEC) and pyrazolidinone 18a was found to be 5-fold more toxic against HL-60 than normal cells.
Somatostatin is a hypothalamic peptide hormone that inhibits the secretion of growth hormone, glu... more Somatostatin is a hypothalamic peptide hormone that inhibits the secretion of growth hormone, glucagon, insulin, gastrin and secretin, and also plays a role in neural transmission. Because of its wide range of possible clinical applications hundreds of somatostatin analogs have been synthesized and bioassayed to date. This review gives a historical perspective, summarizing approximately 30 years of research on somatostatin. The main focus is on the structure-activity relationships and conformational studies of the last generation of somatostatin agonists and their selectivity for five somatostatin receptor subtypes. Achievements in the synthesis of nonpeptide somatostatin analogs, as well as the first somatostatin antagonists, are also discussed. Finally, the use of a cyclic somatostatin scaffold to design ligands for other G-protein-coupled receptors, such as opioid and melanocortin receptors, is mentioned.
A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes... more A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca2+ levels, was used to examine relative potency and efficacy of the μ-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr–Pro–Phe–Phe–NH2) analogues containing d-3-(1-naphthyl)-alanine (d-1-Nal) or d-3-(2-naphthyl)-alanine (d-2-Nal), which were previously shown to reverse antinociception induced by endomorphin-2 in the in vivo hot-plate test in mice, was tested in the aequorin luminescence-based calcium assay to examine their μ-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the μ-opioid receptor together with a luminescent reporter protein, apoaequorin, was used in the study. The results obtained in this functional assay indicated that analogues with d-1-Nal or d-2-Nal substitutions in position 4 of endomorphin-2 are strong μ-opioid receptor antagonists, while those substituted in position 3 are partial agonist...
3-Aryl-2-diethoxyphosphoryl-4-nitrohexanoates 8, obtained by Michael addition of ethyl diethoxyph... more 3-Aryl-2-diethoxyphosphoryl-4-nitrohexanoates 8, obtained by Michael addition of ethyl diethoxyphosphorylacetate 6 to 1-aryl-2-nitro-1-butenes 7, were utilized as convenient common intermediates in the synthesis of β-aryl-γ-ethyl-α-methylidene-γ-lactones 17 and ...
The goal of this study was to determine whether opioid receptor antagonist naloxone abolishes the... more The goal of this study was to determine whether opioid receptor antagonist naloxone abolishes the influence of periaqueductal central gray (PAG) on nociceptive evoked tongue jerks (ETJ) -a trigemino-hypoglossal reflex induced by tooth pulp stimulation. In rats under chloralose anesthesia three series of experiments were performed. In the first two groups perfusions of lateral ventricles-cerebellomedullary cistern with McIlwain-Rodnight's solution and naloxone were carried out. In group 3 naloxone was infused through a catheter through the jugular vein. The amplitudes of tongue jerks induced by tooth pulp stimulation were recorded during subsequent 10 min perfusions. Mean amplitude of tongue movements induced by tooth pulp stimulation was regarded as the indicator of the magnitude of trigemino-hypoglossal reflex. We observed that perfusion of the cerebral ventricles with naloxone (100 nmol / ml) increased the trigemino-hypoglossal reflex up to 143%. The amplitude of ETJ was significantly reduced during PAG stimulation with a train of electrical impulses. After obtaining a significant -93% -inhibition of ETJ (7% of the control), naloxone (100 nmol / ml) was added to the perfusion fluid. This led to a significant increase of the reflex up to 68%. Infusion of naloxone through the jugular vein did not affect the reflex. The above results suggest that the inhibition of ETJ due to PAG stimulation is partially reversed by naloxone and mediated via interactions with endogenous opioid systems involved in modulation of nociception.
Opioid bowel dysfunction (OBD) summarizes common adverse side effects of opiate-based management ... more Opioid bowel dysfunction (OBD) summarizes common adverse side effects of opiate-based management of pain. A promising therapeutic approach to prevent OBD and other opioid-related disorders of the gastrointestinal (GI) tract is the co-administration of opiates with peripherally-restricted mu-opioid receptor (MOR)-selective antagonists. The aim of this study was to investigate the selectivity and efficacy of three novel peptide antagonists: antanal-1, antanal-2, and antanal-2A at MOR in the GI tract in vitro and in vivo. The effects of the antanals on GI motility were studied in vitro, using isolated preparations of mouse ileum and colon and in vivo, by measuring colonic propulsion in mice. Additionally, in vitro stability against enzymatic degradation and blood-brain barrier (BBB) permeability using the hot plate test in mice were examined. The antanals significantly reduced the inhibitory effect of the MOR agonists endomorphin-2, morphine, and loperamide on mouse ileum and colon contractions in vitro and blocked morphine-induced decrease of colonic bead expulsion in vivo. The hot plate test in mice showed that the antagonist activity of all antanals was restricted to the periphery. Antanal-1, antanal-2, and antanal-2A are promising MOR antagonists with limited BBB permeability, which may be developed into future therapeutics of opioid-related GI dysfunction.
Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are two endogenous op... more Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are two endogenous opioid peptides with high affinity and remarkable selectivity for the mu-opioid receptor. The neuroanatomical distribution of endomorphins reflects their potential endogenous role in many major physiological processes, which include perception of pain, responses related to stress, and complex functions such as reward, arousal, and vigilance, as well as autonomic, cognitive, neuroendocrine, and limbic homeostasis. In this review we discuss the biological effects of endomorphin-1 and endomorphin-2 in relation to their distribution in the central and peripheral nervous systems. We describe the relationship between these two mu-opioid receptor-selective peptides and endogenous neurohormones and neurotransmitters. We also evaluate the role of endomorphins from the physiological point of view and report selectively on the most important findings in their pharmacology.
Opioid peptides have the potential to be pharmaceutical agents for the treatment of pain because ... more Opioid peptides have the potential to be pharmaceutical agents for the treatment of pain because they modulate nociceptive pathways at supraspinal, spinal and peripheral levels. Unfortunately, peptides are generally hydrophilic compounds and therefore unable to cross the blood-brain barrier (BBB) by passive diffusion to reach the central nervous system (CNS) in an amount sufficient to activate appropriate receptors. Endomorphins (EMs) belong to the class of endogenous opioids eliciting the strongest analgesic effect, but only after direct administration to the CNS. Extensive research is in progress to better understand the relationships between EM structure and bioavailability. This article deals with the recent investigations that allow the design of stable and neuroactive EM analogs with enhanced brain passage and uptake.
A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-na... more A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-naphthyl)-alanine (1-Nal) or 3-(2-naphthyl)-alanine (2-Nal) in L- or D-configuration, was synthesized. The opioid activity profiles of these peptides were determined in the mu-opioid receptor representative binding assay and in the Guinea-Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot-plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the mu-opioid receptor was reduced compared with endomorphin-2. The two most potent analogs were [D-1-Nal(4)]- and [D-2-Nal4]endomorphin-2, with IC50 values 14 +/- 1.25 and 19 +/- 2.1 nM, respectively, compared with 1.9 +/- 0.21 nM for endomorphin-2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot-plate test. Antinociception induced by endomorphin-2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [D-1-Nal4]- and [D-2-Nal4]-endomorphin-2, indicating that these analogs were mu-opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 microg per animal naloxone almost completely inhibited antinociceptive action of endomorphin-2, while [D-1-Nal4]endomorphin-2 in about 46%.
In the present study, we reported on the synthesis of two new μ-opioid peptide analogs, [d-1-Nal3... more In the present study, we reported on the synthesis of two new μ-opioid peptide analogs, [d-1-Nal3]morphiceptin and [d-1-Nal4]-morphiceptin [1-Nal=3-(1-naphthyl)-alanine] which expressed receptor binding affinities at least at the level of the primary opioid ligands.The new analogs also labeled μ-opioid receptors on the cells of human breast cancer MCF-7 cell line with affinity much higher than that of endomorphins and morphiceptin,
In the present study we investigated and compared the in vivo analgesia of centrally administered... more In the present study we investigated and compared the in vivo analgesia of centrally administered endomorphin-2 and morphiceptin, and their analogs modified in position 3. Two series of analogs were synthesized by introducing unnatural aromatic amino acids in the d configuration: 3-(1-naphthyl)-d-alanine (d-1-Nal), 3-(2-naphthyl)-d-alanine (d-2-Nal), 3-(4-chlorophenyl)-d-alanine (d-ClPhe), 3-(3,4-dichlorophenyl)-d-alanine (d-Cl2Phe). Antinociceptive activity of endomorphin-2, morphiceptin, and their analogs was compared in
As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides... more As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides based on the structure of endomorphin-2 (EM-2), we report here the synthesis and biological activities of a new series of analogues of a general sequence Tyr/Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (where Dmt = 2',6'-dimethyltyrosine), incorporating fluorinated amino acids: 4-fluorophenylalanine (4-F-Phe), 2,4-difluorophenylalanine (2,4-F-Phe), or 4-trifluoromethylphenylalanine (4-CF3-Phe) instead of the Phe residue in position 3 or 4. Depending on the fluorinated amino acid residue and its position in the sequence, analogues were mixed, high affinity MOP/KOP receptor agonists, MOP/DOP/KOP agonists, or selective KOP agonists. The in vitro potencies and efficacies of all novel analogues were assessed in calcium mobilization assay. The most potent analogues, Dmt-c[d-Lys-Phe-4-F-Phe-Asp]NH2 and Dmt-c[d-Lys-Phe-2,4-F-Phe-Asp]NH2, were tested in vivo in the mouse hot-plate test. They produced strong antinociceptive effect not only after intracerebroventricular but also after intraperitoneal injection, indicating that they were able to cross the blood-brain barrier.
In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like act... more In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-β-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-β-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-β-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-β-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration...
The aim of the present study was to characterize the binding selectivity of the l-opioid receptor... more The aim of the present study was to characterize the binding selectivity of the l-opioid receptor ligands, endomorphin-1, endomorphin-2, and DAM-GO, in the in vitro functional assay, based on the changes in intracellular calcium levels. For the experiments Chinese hamster ovary cells, stably expressing human l-receptor, were used. The l-agonist-induced calcium responses were significantly inhibited by naloxone, an opioid antagonist with high preference for the l-opioid receptors. Naloxonazine, a l 1 -non-peptide antagonist, inhibited the effect of all tested l-agonists. However, there was no significant difference in the antagonist effect of naloxonazine on the calcium response induced by l 1 -(endomorphin-2) and l 2 -agonists (endomorphin-1, DAMGO). [D-Pro 2 ]endomorphin-1 and [D-Pro 2 ]endomorphin-2, putative peptide l 2 -and l 1 -antagonists, respectively, which had been shown in vivo to inhibit the antinociception induced by l-agonists, produced no inhibitory effect in our in vitro experiments. Our results demonstrated that there is only one population of the l-opioid receptors expressed in the Chinese hamster ovary cells. We suggest that the l-opioid receptors form a homogenous population in the in vitro systems. However, the existence of l-receptor subtypes in vivo is still pharmacologically possible.
Drosophila tachykinin receptor, a neurokinin receptor cloned from the fruit fly Drosophila melano... more Drosophila tachykinin receptor, a neurokinin receptor cloned from the fruit fly Drosophila melanogaster, is a G-protein-coupled receptor, which upon activation by a peptide agonist induces a transient increase in the concentration of intracellular calcium. The functional assay based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca 2+ levels was used to examine and compare the effect of tachykininrelated peptides from different species. Among the endogenous Drosophila peptides, Drm-TK I induced the strongest calcium response. The most potent tachykinin-related peptides from Leucophaea maderae, Locusta migratoria, and Calliphora vomitoria, were partial agonists at the Drosophila tachykinin receptor.
A series of luteinizing hormone-releasing hormone antagonists with new substitutions in position ... more A series of luteinizing hormone-releasing hormone antagonists with new substitutions in position 6 or positions 5 and 6 that included lysine acylated at the e-amino group with different heterocyclic carboxylic acids or amino-substituted heterocyclic carboxylic acids was Synthesized. These novel analogs were synthesized on a solid-phase support via the acylation of lysine residue in otherwise protected resin-bound peptides. All analogs were tested in the rat antiovulatory assay (AOA) and the best of them in in vitro histamine release assay. Introduction of lysine acylated with aminosubstituted heterocyclic carboxylic acids yielded several water-soluble antagonists with good therapeutic ratio (high AOA to low histamine releasing activity). The best antagonist in terms of activity, histamine release, and solubility was nictide: NAc~Nal-~cpa-~Pal-$er-PicLys-~(GANic)-Orn-Leu-ILys-Pro-~AlaNH2 (6ANic = 6-aminonicotinoyl).
Hot plate test Antinociception Solid phase peptide synthesis a b s t r a c t This study reports t... more Hot plate test Antinociception Solid phase peptide synthesis a b s t r a c t This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH 2 or Tyr-c(Xaa-Phe-D-Pro-Yaa)NH 2 , respectively, where Xaa and Yaa were L/D Asp or L/D Lys. Further modification of these analogs was achieved by introduction of 2 0 ,6 0 -dimethyl-L-tyrosine (Dmt) instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin by a microwave-assisted procedure.
Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generat... more Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generating analogs with improved bioactivities. Cyclization can be achieved through various bridging bonds between peptide ends or side-chains. In our earlier paper we have reported the synthesis and biological activity of a cyclic peptide, Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (1), which can be viewed as an analog of endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2). Cyclization was achieved through an amide bond between side-chains of D-Lys and Asp residues. Here, to increase rigidity of the cyclic structure, we replaced d-Lys with cis- or trans-4-aminocyclohexyl-D-alanine (D-ACAla). Two sets of analogs incorporating either Tyr or Dmt (2',6'-dimethyltyrosine) residues in position 1 were synthesized. In the binding studies the analog incorporating Dmt and trans-D-ACAla showed high affinity for both, μ- and δ-opioid receptors (MOR and DOR, respectively) and moderate affinity for the κ-opioid receptor (KOR), while analog with Dmt and cis-D-ACAla was exceptionally MOR-selective. Conformational analyses by NMR and molecular docking studies have been performed to investigate the molecular structural features responsible for the noteworthy MOR selectivity.
Synthesis and Cytotoxic Evaluation of β-Alkyl or β-Aryl-δ-methyl-α-methylene-δ-lactones. Comparis... more Synthesis and Cytotoxic Evaluation of β-Alkyl or β-Aryl-δ-methyl-α-methylene-δ-lactones. Comparison with the Corresponding γ-Lactones. -A simple and general strategy for the synthesis of title compounds (VII)/(VIII) is presented. The inseparable mixtures of δ-lactones are evaluated in vitro against mouse leukemia cell line L-1210 and the human leukemia cell lines HL-60 and NALM-6. The cytotoxicity of β-aryl-substituted derivatives is very high and comparable with that of previously tested β-aryl-γ-lactones. -(ALBRECHT, L.; WOJCIECHOWSKI, J.; ALBRECHT, A.; WOLF, W. M.; JANECKA, A.; STUDZIAN, K.; KRAJEWSKA, U.; ROZALSKI, M.; JANECKI, T.; KRAWCZYK*, H.; Eur. J. Med. Chem. 45 (2010) 2, 710-718; Inst. Org. Chem., Tech. Univ., PL-90-924 Lodz, Pol.; Eng.) -H. Haber 23-132
European journal of medicinal chemistry, Jan 14, 2015
Three series of new 4-methylidenepyrazolidin-3-ones with various substitution patterns were synth... more Three series of new 4-methylidenepyrazolidin-3-ones with various substitution patterns were synthesized and tested for the cytotoxic activity against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7 breast cancer cell line. Several obtained methylidenepyrazolidinones exhibited high cytotoxic activity with IC50 values below 10 μM, mainly against HL-60 leukemia cell line and two of them, 18d,e, displayed IC50 ≤ 5 μM, against all tested cell lines. Structure-activity relationship studies revealed that the presence of phenyl substituents on both ring nitrogen atoms and vinyl or phenyl substituents in position 5 are crucial for high activity. Selected methylidenepyrazolidinones were also tested on normal human umbilical vein endothelial cells (HUVEC) and pyrazolidinone 18a was found to be 5-fold more toxic against HL-60 than normal cells.
Somatostatin is a hypothalamic peptide hormone that inhibits the secretion of growth hormone, glu... more Somatostatin is a hypothalamic peptide hormone that inhibits the secretion of growth hormone, glucagon, insulin, gastrin and secretin, and also plays a role in neural transmission. Because of its wide range of possible clinical applications hundreds of somatostatin analogs have been synthesized and bioassayed to date. This review gives a historical perspective, summarizing approximately 30 years of research on somatostatin. The main focus is on the structure-activity relationships and conformational studies of the last generation of somatostatin agonists and their selectivity for five somatostatin receptor subtypes. Achievements in the synthesis of nonpeptide somatostatin analogs, as well as the first somatostatin antagonists, are also discussed. Finally, the use of a cyclic somatostatin scaffold to design ligands for other G-protein-coupled receptors, such as opioid and melanocortin receptors, is mentioned.
A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes... more A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca2+ levels, was used to examine relative potency and efficacy of the μ-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr–Pro–Phe–Phe–NH2) analogues containing d-3-(1-naphthyl)-alanine (d-1-Nal) or d-3-(2-naphthyl)-alanine (d-2-Nal), which were previously shown to reverse antinociception induced by endomorphin-2 in the in vivo hot-plate test in mice, was tested in the aequorin luminescence-based calcium assay to examine their μ-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the μ-opioid receptor together with a luminescent reporter protein, apoaequorin, was used in the study. The results obtained in this functional assay indicated that analogues with d-1-Nal or d-2-Nal substitutions in position 4 of endomorphin-2 are strong μ-opioid receptor antagonists, while those substituted in position 3 are partial agonist...
3-Aryl-2-diethoxyphosphoryl-4-nitrohexanoates 8, obtained by Michael addition of ethyl diethoxyph... more 3-Aryl-2-diethoxyphosphoryl-4-nitrohexanoates 8, obtained by Michael addition of ethyl diethoxyphosphorylacetate 6 to 1-aryl-2-nitro-1-butenes 7, were utilized as convenient common intermediates in the synthesis of β-aryl-γ-ethyl-α-methylidene-γ-lactones 17 and ...
The goal of this study was to determine whether opioid receptor antagonist naloxone abolishes the... more The goal of this study was to determine whether opioid receptor antagonist naloxone abolishes the influence of periaqueductal central gray (PAG) on nociceptive evoked tongue jerks (ETJ) -a trigemino-hypoglossal reflex induced by tooth pulp stimulation. In rats under chloralose anesthesia three series of experiments were performed. In the first two groups perfusions of lateral ventricles-cerebellomedullary cistern with McIlwain-Rodnight's solution and naloxone were carried out. In group 3 naloxone was infused through a catheter through the jugular vein. The amplitudes of tongue jerks induced by tooth pulp stimulation were recorded during subsequent 10 min perfusions. Mean amplitude of tongue movements induced by tooth pulp stimulation was regarded as the indicator of the magnitude of trigemino-hypoglossal reflex. We observed that perfusion of the cerebral ventricles with naloxone (100 nmol / ml) increased the trigemino-hypoglossal reflex up to 143%. The amplitude of ETJ was significantly reduced during PAG stimulation with a train of electrical impulses. After obtaining a significant -93% -inhibition of ETJ (7% of the control), naloxone (100 nmol / ml) was added to the perfusion fluid. This led to a significant increase of the reflex up to 68%. Infusion of naloxone through the jugular vein did not affect the reflex. The above results suggest that the inhibition of ETJ due to PAG stimulation is partially reversed by naloxone and mediated via interactions with endogenous opioid systems involved in modulation of nociception.
Opioid bowel dysfunction (OBD) summarizes common adverse side effects of opiate-based management ... more Opioid bowel dysfunction (OBD) summarizes common adverse side effects of opiate-based management of pain. A promising therapeutic approach to prevent OBD and other opioid-related disorders of the gastrointestinal (GI) tract is the co-administration of opiates with peripherally-restricted mu-opioid receptor (MOR)-selective antagonists. The aim of this study was to investigate the selectivity and efficacy of three novel peptide antagonists: antanal-1, antanal-2, and antanal-2A at MOR in the GI tract in vitro and in vivo. The effects of the antanals on GI motility were studied in vitro, using isolated preparations of mouse ileum and colon and in vivo, by measuring colonic propulsion in mice. Additionally, in vitro stability against enzymatic degradation and blood-brain barrier (BBB) permeability using the hot plate test in mice were examined. The antanals significantly reduced the inhibitory effect of the MOR agonists endomorphin-2, morphine, and loperamide on mouse ileum and colon contractions in vitro and blocked morphine-induced decrease of colonic bead expulsion in vivo. The hot plate test in mice showed that the antagonist activity of all antanals was restricted to the periphery. Antanal-1, antanal-2, and antanal-2A are promising MOR antagonists with limited BBB permeability, which may be developed into future therapeutics of opioid-related GI dysfunction.
Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are two endogenous op... more Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are two endogenous opioid peptides with high affinity and remarkable selectivity for the mu-opioid receptor. The neuroanatomical distribution of endomorphins reflects their potential endogenous role in many major physiological processes, which include perception of pain, responses related to stress, and complex functions such as reward, arousal, and vigilance, as well as autonomic, cognitive, neuroendocrine, and limbic homeostasis. In this review we discuss the biological effects of endomorphin-1 and endomorphin-2 in relation to their distribution in the central and peripheral nervous systems. We describe the relationship between these two mu-opioid receptor-selective peptides and endogenous neurohormones and neurotransmitters. We also evaluate the role of endomorphins from the physiological point of view and report selectively on the most important findings in their pharmacology.
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Papers by Anna Janecka