Discovery of New Potent Positive Allosteric Modulators of Dopamine D2 Receptors: Insights into the Bioisosteric Replacement of Proline to 3-Furoic Acid in the Melanostatin Neuropeptide.

The control of Parkinson's disease (PD) is challenged by the motor and non-motor fluctuations as well as dyskinesias associated with levodopa long-term therapy. As such, pharmacological alternatives to reduce the reliance on this drug are needed. Melanostatin (MIF-1), a positive allosteric modulator (PAM) of D2 receptors (D2R), is being explored as a novel pharmacological approach focused on D2R potentiation. In this work, 3-furoic acid (3-Fu) was successfully employed as an l-proline (Pro) surrogate, affording two potent MIF-1 analogues, methyl 3-furoyl-l-leucylglycinate (4a) and 3-furoyl-l-leucylglycinamide (6a). In this series, the C-terminal carboxamide moiety was found crucial to enhancing the potency and toxicological profile, yet it is not considered a requisite for the PAM activity. Conformational analysis excludes 4a from adopting the claimed type II ß-turn. The discovery and validation of 6a as a lead compound open a new avenue for the development of a novel class of anti-Parkinson therapeutics targeting the D2R.

The Effect of PAOPA, a Novel Allosteric Modulator of Dopamine D2 Receptors, on Signaling Proteins Following Sub-Chronic Administration in Rats.

BACKGROUND: Allosteric modulators of G-protein coupled receptors regulate receptor activity by binding to sites other than the active site and have emerged as a new and highly desirable class of drugs. PAOPA (3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide), a peptidomimetic analog of L-prolyl-L-leucyl-glycinamide, is a potent dopamine D2 receptor allosteric modulator. PAOPA has shown therapeutic effects in pre-clinical models of schizophrenia and extrapyramidal dysfunction. OBJECTIVE: In this study, we sought to examine the biomolecular underpinnings of PAOPA's therapeutic outcomes in pre-clinical models of schizophrenia. METHODS: Following sub-chronic (daily for 7 days) administration of PAOPA, we assessed levels of dopamine D2 receptors, receptor kinases (GRK2 (G protein-coupled receptor kinase 2) and Arrestin- 3), and phosphorylated mitogen-activated protein kinase (MAPKs), namely, extracellular signal- regulated kinases (ERK1/2) in the hippocampus, medial pre-frontal cortex, nucleus accumbens, pre-frontal cortex, and dorsal striatum via protein quantification. RESULTS: Following 7 days of daily PAOPA treatment, we observed decreased GRK2 and increased dopamine D2 receptor expression in the dorsal striatum. These findings potentially underscore the therapeutic mechanism of action of PAOPA for the positive-like symptoms of schizophrenia in pre-clinical animal models. Additionally, we observed a decline in GRK2 in the hippocampus and an increase in phosphorylated ERK1 in the pre-frontal cortex, suggesting a role of PAOPA in treating cognitive and/or affective dysfunction in pre-clinical models. CONCLUSION: While further studies are required to elucidate the mechanism of action of PAOPA, this study discusses prior investigations and develops an early framework to describe the therapeutic mechanism of action of PAOPA.

Synthesis, Pharmacological, and Biological Evaluation of 2-Furoyl-Based MIF-1 Peptidomimetics and the Development of a General-Purpose Model for Allosteric Modulators (ALLOPTML).

This work describes the synthesis and pharmacological evaluation of 2-furoyl-based Melanostatin (MIF-1) peptidomimetics as dopamine D2 modulating agents. Eight novel peptidomimetics were tested for their ability to enhance the maximal effect of tritiated N-propylapomorphine ([3H]-NPA) at D2 receptors (D2R). In this series, 2-furoyl-l-leucylglycinamide (6a) produced a statistically significant increase in the maximal [3H]-NPA response at 10 pM (11 ± 1%), comparable to the effect of MIF-1 (18 ± 9%) at the same concentration. This result supports previous evidence that the replacement of proline residue by heteroaromatic scaffolds are tolerated at the allosteric binding site of MIF-1. Biological assays performed for peptidomimetic 6a using cortex neurons from 19-day-old Wistar-Kyoto rat embryos suggest that 6a displays no neurotoxicity up to 100 µM. Overall, the pharmacological and toxicological profile and the structural simplicity of 6a makes this peptidomimetic a potential lead compound for further development and optimization, paving the way for the development of novel modulating agents of D2R suitable for the treatment of CNS-related diseases. Additionally, the pharmacological and biological data herein reported, along with >20 000 outcomes of preclinical assays, was used to seek a general model to predict the allosteric modulatory potential of molecular candidates for a myriad of target receptors, organisms, cell lines, and biological activity parameters based on perturbation theory (PT) ideas and machine learning (ML) techniques, abbreviated as ALLOPTML. By doing so, ALLOPTML shows high specificity Sp = 89.2/89.4%, sensitivity Sn = 71.3/72.2%, and accuracy Ac = 86.1%/86.4% in training/validation series, respectively. To the best of our knowledge, ALLOPTML is the first general-purpose chemoinformatic tool using a PTML-based model for the multioutput and multicondition prediction of allosteric compounds, which is expected to save both time and resources during the early drug discovery of allosteric modulators.

Synthesis, Pharmacological, and Biological Evaluation of MIF-1 Picolinoyl Peptidomimetics as Positive Allosteric Modulators of D2R.

This work describes the synthesis and pharmacological evaluation of picolinoyl-based peptidomimetics of melanocyte stimulating hormone release inhibiting factor 1 (MIF-1) as dopamine modulating agents. Eight novel peptidomimetics were tested for their ability to enhance the maximal effect of tritiated N-propylapomorphine ([3H]-NPA) at dopamine D2 receptors (D2R). Methyl picolinoyl-l-valyl-l-alaninate (compound 6b) produced a statistically significant increase in the maximal [3H]-NPA response at 0.01 nM (11.9 ± 3.7%), which is close to the effect of MIF-1 in this assay at same concentration (18.3 ± 9.1%). Functional assays measuring cAMP mobilization in the presence of dopamine corroborate the activity of peptidomimetic 6b as a positive allosteric modulator (PAM) of D2R. In this assay, 6b produced a typical bell-shaped dose-response curve similar to that of the parent neuropeptide (18.3 ± 7.1% for 6b vs 15.4 ± 5.5% for MIF-1, both at 0.1 nM). Dose-response curves for dopamine in the presence of 6b show EC50 (0.33 ± 0.21 µM for 6b vs 0.17 ± 0.07 µM for MIF-1) and Emax (86.0 ± 5.4% for 6b vs 93.6 ± 4.4% for MIF-1) comparable to those of MIF-1, both at 0.01 nM. Furthermore, peptidomimetic 6b was tested for agonist activity at the human D2R and the results show that it displays no intrinsic agonism effect, endorsing its activity as a PAM of D2R. Cytotoxic and neurotoxic assays were performed for peptidomimetic 6b using HEK 293T cells and cortex neurons from 19 day old Wistar-Kyoto rat embryos, respectively, suggesting this analogue displays no toxicity effect in these assays up to 100 µM. Conformational energy minimization for 6b shows that this peptidomimetic cannot adopt the postulated type-II ß-turn bioactive conformation, endorsing the possibility of an extended bioactive conformation as claimed by other researchers as a second bioactive conformation of MIF-1. Overall, the pharmacological and toxicological profile of peptidomimetic 6b together with its favorable druglike properties and structural simplicity makes it a potential lead compound for further development and optimization.

NrCAM-regulating neural systems and addiction-related behaviors.

We have previously shown that a haplotype associated with decreased NrCAM expression in brain is protective against addiction vulnerability for polysubstance abuse in humans and that Nrcam knockout mice do not develop conditioned place preferences for morphine, cocaine or amphetamine. In order to gain insight into NrCAM involvement in addiction vulnerability, which may involve specific neural circuits underlying behavioral characteristics relevant to addiction, we evaluated several behavioral phenotypes in Nrcam knockout mice. Consistent with a potential general reduction in motivational function, Nrcam knockout mice demonstrated less curiosity for novel objects and for an unfamiliar conspecific, showed also less anxiety in the zero maze. Nrcam heterozygote knockout mice reduced alcohol preference and buried fewer marbles in home cage. These observations provide further support for a role of NrCAM in substance abuse including alcoholism vulnerability, possibly through its effects on behavioral traits that may affect addiction vulnerability, including novelty seeking, obsessive compulsion and responses to aversive or anxiety-provoking stimuli. Additionally, in order to prove glutamate homeostasis hypothesis of addiction, we analyzed glutamatergic molecules regulated by NRCAM expression. Glutaminase appears to be involved in NrCAM-related molecular pathway in two different tissues from human and mouse. An inhibitor of the enzyme, prolyl-leucyl-glycinamide, treatment produced, at least, some of the phenotypes of mice shown in alcohol preference and in anxiety-like behavior. Thus, NrCAM could affect addiction-related behaviors via at least partially modulation of some glutamatergic pathways and neural function in brain.

Synthesis of an MIF-1 analogue containing enantiopure (S)-α-trifluoromethyl-proline and biological evaluation on nociception.

The synthesis and the effect of a novel MIF-1 analogue on nociception during acute pain in rat model are reported. The synthesis of this enantiopure trifluoromethyl group containing tripeptide was performed through a peptide coupling reaction between the HCl. Leu-Gly-NH2 and the (S)-α-Tfm-proline. The analgesic effect of the CF3-(MIF-1) 2 has been evaluated in vivo on rat model by paw pressure (PP) and hot plate (HP) tests and compared to the native peptide MIF-1. Highest analgesic effect was observed with CF3-(MIF-1) 2 only in PP test. In order to study the mechanisms of nociception induced by the studied peptides, the involvement of the opioid and the nitric oxideergic systems was investigated. The results are in favor of a participation of both system since pretreatment, 20 min before injection of the CF3-(MIF-1) 2, with the non-competitive antagonist of opiate receptors naloxone, the nitric oxide synthase (NOS) inhibitor l-N(G)-nitroarginine ester (l-NAME) or the nitric oxide (NO) donor l-arginine (l-Arg) significantly decreased the pain perception in PP and HP tests.

Transformation of Pro-Leu-Gly-NH2 peptidomimetic positive allosteric modulators of the dopamine D2 receptor into negative modulators.

The synthesis of dimethyl derivatives of 5.6.5 spiro bicyclic lactam Pro-Leu-Gly-NH(2) peptidomimetics was carried out to test the hypothesis that by placing methyl groups on the ß-methylene carbon of the thiazolidine ring steric bulk would be introduced into the topological space that the ß-methylene carbon is believed to occupy in the negative allosteric modulators of the dopamine D(2) receptor. With such a modification, a positive allosteric modulator would be converted into a negative allosteric modulator. This hypothesis was shown to be correct as 3a and 4a where found to be negative allosteric modulators, whereas their unmethylated derivatives were positive allosteric modulators of the dopamine D(2) receptor.

Behavioral effects of neuropeptides in rodent models of depression and anxiety.

In recent years, studies have advocated neuropeptide systems as modulators for the behavioral states found in mood disorders such as depression and anxiety disorders. Neuropeptides have been tested in traditional animal models and screening procedures that have been validated by known antidepressants and anxiolytics. However, it has become clear that although these tests are very useful, neuropeptides have distinct behavioral effects and dose-dependent characteristics, and therefore, use of these tests with neuropeptides must be done with an understanding of their unique characteristics. This review will focus on the behavioral actions of neuropeptides and their synthetic analogs, particularly in studies utilizing various preclinical tests of depression and anxiety. Specifically, the following neuropeptide systems will be reviewed: corticotropin-releasing factor (CRF), urocortin (Ucn), teneurin C-terminal associated peptide (TCAP), neuropeptide Y (NPY), arginine vasopressin (AVP), oxytocin, the Tyr-MIF-1 family, cholecystokinin (CCK), galanin, and substance P. These neuropeptide systems each have a unique role in the regulation of stress-like behavior, and therefore provide intriguing therapeutic targets for mood disorder treatment.

Effect of Tyr-MIF-1 peptides on blood ACTH and corticosterone concentration induced by three experimental models of stress.

1. Studies, using a wide variety of stressors, have clearly indicated that the pattern of neuroendocrine response is dependent upon the stress stimulus applied. 2. The Tyr-MIF-1 family of peptides (Tyr-MIF-1s) includes MIF-1, Tyr-MIF-1, Tyr-W-MIF-1 and Tyr-K-MIF-1. These neuropeptides, neuromodulators are able to inhibit the expression of some forms of stress-induced analgesia. 3. The aim of this study was to compare changes in ACTH and corticosterone (CORT) concentration after various stressors (immobilization, cold and heat), as well as after injection of investigated Tyr-MIF-1s peptides. 4. According to our results, hypothalamic-pituitary-adrenal (HPA) system was activated by all the stressors applied. Heat and immobilization are stronger stressors, as the exposure of animals to a high ambient temperature and immobilization resulted in the highest rise of plasma ACTH and CORT concentration when compared with cold stress. Moreover, all the investigated peptides from Tyr-MIF-1 family, administered after application of stressors, inhibited the elevations in adrenocorticotropic hormone (ACTH) and corticosterone (CORT) plasma concentrations significantly. 5. In conclusion, the various stressors applied seem to induce a different response of the HPA system as judged by quantitative changes in ACTH and CORT release. We suggest that Tyr-MIF-1 peptides may possess anti-stressor effects, as they inhibited stress-induced rising in two hormones that were investigated.

Synthesis and evaluation of novel pyridine based PLG tripeptidomimetics.

Analogues of the pyridine based PLG (Pro-Leu-Gly-NH(2)) peptidomimetic were synthesized and evaluated as dopamine modulating agents. Modifications in the position corresponding to the leucine side chain in PLG afforded derivatives , and , substituted with H, Me and Bn instead of the isobutyl group, respectively. Changes in the proline residue produced derivative , substituted with a symmetrical piperidine ring instead of the pyrrolidine ring and , in which the pyrrolidine ring is connected to the pyridine ring via a hydroxymethyl group instead of a keto function. The peptidomimetics were tested for their ability to enhance the maximal effect of N-propylapomorphine (NPA) at dopamine D2 receptors in the functional cell-based R-SAT assay. Compounds , , and , produced a statistically significant increase in the maximal NPA response at 10 nM (117 +/- 6%, 118 +/- 6%, and 116 +/- 3%, respectively), which is similar to the effect of PLG in this assay, whereas was able to potentiate the response to a similar extent at 1 nM concentration (115 +/- 5%). All derivatives produced a bell-shaped dose-response curve and none of the compounds were active at the D2 receptor alone, which indicates that the mechanism behind the activity of both the pyridine based mimetics and PLG is the same. Interestingly, l-Pro-d-Leu-Gly-NH(2) was found to be more potent than PLG and produced a 119 +/- 1% increase in the NPA response at 1 nM.

From MIF-1 to endomorphin: the Tyr-MIF-1 family of peptides.

The Tyr-MIF-1 family of small peptides has served a prototypic role in the introduction of several novel concepts into the peptide field of research. MIF-1 (Pro-Leu-Gly-NH(2)) was the first hypothalamic peptide shown to act "up" on the brain, not just "down" on the pituitary. In several situations, including clinical depression, MIF-1 exhibits an inverted U-shaped dose-response relationship in which increasing doses can result in decreasing effects. This tripeptide also can antagonize opiate actions, and the first report of such activity also correctly predicted the discovery of other endogenous antiopiate peptides. The tetrapeptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH(2)) not only shows antiopiate activity, but also considerable selectivity for the mu-opiate binding site. Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH(2)) is an even more selective ligand for the mu receptor, leading to the discovery of two more Tyr-Pro tetrapeptides that have the highest specificity and affinity for this site. These are the endomorphins: endomorphin-1 is Tyr-Pro-Trp-Phe-NH(2) and endomorphin-2 is Tyr-Pro-Phe-Phe-NH(2). Tyr-MIF-1 proved, contrary to the then prevailing dogma, that peptides can be saturably transported across the blood-brain barrier by a quantifiable transport system. Unexpectedly, the Tyr-MIF-1 transporter is shared with Met-enkephalin. In the era in which it was doubtful whether a peripheral peptide could exert CNS effects, the Tyr-MIF-1 family of peptides also explicitly showed that they can exert more than one central action that persists longer than their half-lives in blood. These peptides clearly illustrate that the name of a peptide restricts neither its actions nor its conceptual implications.

MIF-1 and its peptidomimetic analogs attenuate haloperidol-induced vacuous chewing movements and modulate apomorphine-induced rotational behavior in 6-hydroxydopamine-lesioned rats.

Two melanocyte-stimulating hormone release inhibiting factor-1 (MIF-1) also known as L-prolyl-L-leucyl-glycinamide (PLG) peptidomimetic analogs, 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]-amino]-3-(butyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (A) and 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]amino]-3-(benzyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (B), were evaluated for their ability to modulate dopaminergic activity by measuring apomorphine-induced rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats, and haloperidol (HP)-induced vacuous chewing movements (VCMs) in rats; animal models of Parkinson's disease (PD) and human tardive dyskinesia (TD), respectively. In the 6-OHDA model, both analogs were found to potentiate the contralateral rotational behavior induced by apomorphine dose-dependently and with approximately the same potency. Furthermore, each analog was able to significantly attenuate HP-induced VCMs with almost equal efficacy. The potency and efficacy of these analogs were significantly greater than their parent compound, PLG. These results suggest that both analogs can modulate dopaminergic activity in vivo, likely by the same mechanisms recruited by PLG previously reported.

A Tyr-W-MIF-1 analog containing D-Pro2 discriminates among antinociception in mice mediated by different classes of mu-opioid receptors.

The antagonism by Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1) analog, of the antinociception induced by the mu-opioid receptor agonists Tyr-W-MIF-1, [D-Ala2,NMePhe4,Gly(ol)5]-enkephalin (DAMGO), Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2) was studied with the mouse tail-flick test. D-Pro2-Tyr-W-MIF-1 (0.5-3 nmol) given intracerebroventricularly (i.c.v.) had no effect on the thermal nociceptive threshold. High doses of D-Pro2-Tyr-W-MIF-1 (4-16 nmol) administered i.c.v. produced antinociception with a low intrinsic activity of about 30% of the maximal possible effect. D-Pro2-Tyr-W-MIF-1 (0.25-2 nmol) co-administered i.c.v. showed a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 or DAMGO without affecting endomorphin-2-induced antinociception. A 0.5 nmol dose of D-Pro2-Tyr-W-MIF-1 significantly attenuated Tyr-W-MIF-1-induced antinociception but not DAMGO- or endomorphin-1-induced antinociception. The highest dose (2 nmol) of D-Pro2-Tyr-W-MIF-1 almost completely attenuated Tyr-W-MIF-1-induced antinociception. However, that dose of D-Pro2-Tyr-W-MIF-1 significantly but not completely attenuated endomorphin-1 or DAMGO-induced antinociception, whereas the antinociception induced by endomorphin-2 was still not affected by D-Pro2-Tyr-W-MIF-1. Pretreatment i.c.v. with various doses of naloxonazine, a mu1-opioid receptor antagonist, attenuated the antinociception induced by Tyr-W-MIF-1, endomorphin-1, endomorphin-2, or DAMGO. Judging from the ID50 values for naloxonazine against the antinociception induced by the mu-opioid receptor agonists, the antinociceptive effect of Tyr-W-MIF-1 is extremely less sensitive to naloxonazine than that of endomorphin-1 or DAMGO. In contrast, endomorphin-2-induced antinociception is extremely sensitive to naloxonazine. The present results clearly suggest that D-Pro2-Tyr-W-MIF-1 is a selective antagonist for the mu2-opioid receptor in the mouse brain. D-Pro2-Tyr-W-MIF-1 may also discriminate between Tyr-W-MIF-1-induced antinociception and the antinociception induced by endomorphin-1 or DAMGO, which both show a preference for the mu2-opioid receptor in the brain.

Study on the antinociceptive action of Tyr-K-MIF-1, a peptide from the MIF family.

1. Tyr-K-MIF-1 is a melanocyte inhibiting factor (MIF) neuropeptide, isolated from the brain. Opposite to other MIFs (Tyr-MIF-1, Tyr-W-MIF-1), it has a very low affinity for opiate mu-receptors, but interacts with Tyr-MIF-1 specific binding sites. Tyr-MIF-1 and Tyr-W-MIF-1 evoke antinociception mainly by activating opioid receptors. We investigated the possible antinociceptive effect of Tyr-K-MIF-1 and the involvement of histaminergic system in its mechanism of action. 2. Tested on rats by paw-pressure test, Tyr-K-MIF-1 (0.5, 1 and 2 mg kg(-1)) was associated with short-lasting analgesia, which was abolished by naloxone (1 mg kg(-1)). 3. Injected intraperitoneally (i.p.) 15 min before Tyr-K-MIF-1, antagonists of H(1) (diphenhydramine, 100 mg kg(-1)) or H(2) (famotidine, 0.3 and 0.6 mg kg(-1)) histamine receptors diminished peptide antinociceptive effect. Simultaneous H(1)- and H(2) blockade, as well as pretreatment with 5 mg kg(-1) dimaprit (H(2) agonist) abolished Tyr-K-MIF-1-induced analgesia. Tyr-K-MIF-1-induced analgesia was also abolished by treatment with R-(alpha)-methylhistamine (10 mg kg(-1), i.p.), an H(3) histamine receptor agonist that acts to inhibit histamine release. 4. Our results together with data reported in the literature support the conclusion that activation of the histaminergic system is involved in the mechanism of Tyr-K-MIF-1-induced antinociception.

Involvement of spinal mu1-opioid receptors on the Tyr-d-Arg-Phe-sarcosine-induced antinociception.

The involvement of spinal mu-opioid receptor subtypes on the antinociception induced by i.t.-administered Tyr-D-Arg-Phe-sarcosine (TAPS), a N-terminal tetrapeptide analog of dermorphin, was determined in mice tail-flick test. Intrathecal administration of TAPS produced the marked inhibition of the tail-flick response in a dose-dependent manner. The antinociception induced by TAPS was completely eliminated by i.t.-co-administration of Tyr-D-Pro-Phe-Phe-NH2 (D-Pro2-endomorphin-2), the mu1-opioid receptor antagonist, whereas i.t. co-treatment with Tyr-D-Pro-Trp-Phe-NH2 (D-Pro2-endomorphin-1) or Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), the mu2-opioid receptor antagonists, did not affect the TAPS-induced antinociception. In contrast, the antinociception induced by i.t.-administered [D-Ala2,N-MePhe4,Gly-ol5]enkephalin was significantly attenuated by i.t.-co-administration of D-Pro2-endomorphin-1 or D-Pro2-Tyr-W-MIF-1, but not D-Pro2-endomorphin-2. These results suggest that TAPS may stimulate spinal mu1-opioid receptors to produce the antinociception.

Design and synthesis of photoaffinity-labeling ligands of the L-prolyl-L-leucylglycinamide binding site involved in the allosteric modulation of the dopamine receptor.

Pro-Leu-Gly-NH(2) (PLG), in addition to its endocrine effects, possesses the ability to modulate dopamine D(2) receptors within the central nervous system. However, the precise binding site of PLG is unknown. Potential photoaffinity-labeling ligands of the PLG binding site were designed as tools to be used in the identification of the macromolecule that possesses this binding site. Six different photoaffinity-labeling ligands were designed and synthesized on the basis of the gamma-lactam PLG peptidomimetic 1. The 4-azidobenzoyl and 4-azido-2-hydroxybenzoyl photoaffinity-labeling moieties were placed at opposite ends of PLG peptidomimetic 1 to generate a series of ligands that potentially could be used to map the PLG binding site. All of the compounds that were synthesized possessed activity comparable to or better than PLG in enhancing [(3)H]-N-propylnorapomorphine agonist binding to dopamine receptors. Photoaffinity ligands that were cross-linked to the receptor preparation produced a modulatory effect that was either comparable to or greater than the increase in agonist binding produced by the respective ligands that were not cross-linked to the dopamine receptor. The results indicate that these photoaffinity-labeling agents are binding at the same allosteric site as PLG and PLG peptidomimetic 1.

Modulation of agonist binding to human dopamine receptor subtypes by L-prolyl-L-leucyl-glycinamide and a peptidomimetic analog.

The present study was undertaken to investigate the role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) and its conformationally constrained analog 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) in modulating agonist binding to human dopamine (DA) receptor subtypes using human neuroblastoma SH-SY5Y cells stably transfected with respective cDNAs. Both PLG and PAOPA enhanced agonist [3H]N-propylnorapomorphine (NPA) and [3H]quinpirole binding in a dose-dependent manner to the DA D2L,D2S, and D4 receptors. However, agonist binding to the D1 and D3 receptors and antagonist binding to the D2L receptors by PLG were not significantly affected. Scatchard analysis of [3H]NPA binding to membranes in the presence of PLG revealed a significant increase in affinity of the agonist binding sites for the D2L, D2S, and D4 receptors. Analysis of agonist/antagonist competition curves revealed that PLG and PAOPA increased the population and affinity of the high-affinity form of the D2L receptor and attenuated guanosine 5'-(beta,gamma-imido)-triphosphate-induced inhibition of high-affinity agonist binding sites for the DA D2L receptor. Furthermore, direct NPA binding with D2L cell membranes pretreated with suramin, a compound that can uncouple receptor/G protein complexes, and incubated with and without DA showed that both PLG and PAOPA had only increased agonist binding in membranes pretreated with both suramin and DA, suggesting that PLG requires the D2L receptor/G protein complex to increase agonist binding. These results suggest that PLG possibly modulates DA D2S, D2L, and D4 receptors in an allosteric manner and that the coupling of D2 receptors to the G protein is essential for this modulation to occur.

A Tyr-W-MIF-1 analog containing D-Pro2 acts as a selective mu2-opioid receptor antagonist in the mouse.

The antagonistic properties of Tyr-d-Pro-Trp-Gly-NH(2) (d-Pro(2)-Tyr-W-MIF-1), a Tyr-Pro-Trp-Gly-NH(2)(Tyr-W-MIF-1) analog, on the antinociception induced by the mu-opioid receptor agonists Tyr-W-MIF-1, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), Tyr-Pro-Trp-Phe-NH(2) (endomorphin-1), and Tyr-Pro-Phe-Phe-NH(2) (endomorphin-2) were studied in the mouse paw-withdrawal test. d-Pro(2)-Tyr-W-MIF-1 injected intrathecally (i.t.) had no apparent effect on the thermal nociceptive threshold. d-Pro(2)-Tyr-W-MIF-1 (0.1-0.4 nmol) coadministered i.t. showed a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 without affecting endomorphin- or DAMGO-induced antinociception. However, higher doses of d-Pro(2)-Tyr-W-MIF-1 (0.8-1.2 nmol) significantly attenuated endomorphin-1- or DAMGO-induced antinociception, whereas the antinociception induced by endomorphin-2 was still not affected by d-Pro(2)-Tyr-W-MIF-1. Pretreatment i.t. with various doses of naloxonazine, a mu(1)-opioid receptor antagonist, attenuated the antinociception induced by Tyr-W-MIF-1, endomorphin-1, endomorphin-2, or DAMGO. Judging from the ID(50) values for naloxonazine against the antinociception induced by the mu-opioid receptor agonists, the antinociceptive effect of Tyr-W-MIF-1 is extremely less sensitive to naloxonazine than those of endomorphin-1 or DAMGO. In contrast, endomorphin-2-induced antinociception is extremely sensitive to naloxonazine. The present results clearly suggest that d-Pro(2)-Tyr-W-MIF-1 is the selective antagonist to be identified for the mu(2)-opioid receptor in the mouse spinal cord. d-Pro(2)-Tyr-W-MIF-1 may also discriminate between Tyr-W-MIF-1-induced antinociception and the antinociception induced by endomorphin-1 or DAMGO, all of which show a preference for the mu(2)-opioid receptor in the spinal cord.

Prolyl-leucyl-glycinamide shares some effects with oxytocin but decreases oxytocin levels.

Oxytocin treatment in rats induces long-lasting antistress and growth promoting effects. This study investigated whether prolyl-leucyl-glycinamide (PLG) (the c-terminal tripeptide of oxytocin) or tocinoic acid (the ring structure of oxytocin) could induce some of these effects in male rats. For this purpose, PLG (2 or 10 mg/kg, s.c.) or tocinoic acid (1 mg/kg, s.c.) was administered to rats once a day for 3 or 5 days. Blood pressure, heart rate, spontaneous motor activity, nociceptive thresholds, and the survival of ischaemic musculocutaneous flaps were measured. In addition, endogenous oxytocin levels and plasma levels of some hormones known to be influenced by oxytocin were determined. PLG (2 mg/kg, s.c., but not 10 mg/kg, s.c.) decreased diastolic blood pressure (p<0.05) and locomotor activity (p<0.05). PLG (10 mg/kg, s.c.) decreased gastrin (p<0.05) and endogenous oxytocin levels in plasma (p<0.01). Tocinoic acid decreased locomotor activity (p<0.05), but did not affect any of the other parameters measured. In conclusion, this study showed that both PLG and tocinoic acid decrease locomotor activity. In addition, PLG also induced some other effects similar to those induced by oxytocin treatment but when administered in high doses it decreased oxytocin levels.

Effects of Tyr-MIF's family of peptides on immobilization stress-induced antinociception in rats.

UNLABELLED: The Tyr-MIF-1 family of peptides includes MIF-1, Tyr-MIF-1, Tyr-W-MIF-1 and Tyr-K-MIF-1, which have been isolated from bovine hypothalamus and cortex of human brain. Data in the literature suggest that peptides of Tyr-MIF-1's family have opioid-like and anti-opioid actions. All these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides and able to inhibit the expression of some forms of stress-induced analgesia (SIA) in various species. AIM: To examined the effects of the Tyr-MIF-1's peptides on immobilization stress-induced antinociception. METHODS: Tyr-MIF-1's peptides were given to male Wistar rats intraperitoneally before or after 1 hour of restraint. The changes in the mechanical nociceptive threshold of the animals were measured by the Randall-Selitto paw pressure test. RESULTS: Immobilization of the rats increased the pain threshold at least 1 h. Tyr-MIF-1's peptides have contrasting effects on immobilization stress-induced antinociception in paw-pressure test in rats. When administered before immobilization procedure they potentiated the immobilization stress-induced antinociception, while if given after immobilization, they reduced it. Antinociceptive effects of Tyr-MIF-1, Tyr-W-MIF-1 and Tyr-K-MIF-1 were reduced in condition of stress. CONCLUSION: Tyr-MIF-1's peptides exerted antiopioide effects under condition of stress in paw-pressure test. These antiopioide effects were more pronounced when peptides were injected after stress exposure.