Structure-activity relationship of rubiscolins as delta opioid peptides

To study the structure-activity relationship of rubiscolins (YPLDLF and YPLDL), delta opioid peptides derived from the spinach Rubisco, we substituted the amino acid residues and evaluated their activities by mouse vas deferens (MVD) and guinea pig ileum (GPI) assays as well as receptor affinity. Replacement of Leu(3) with Ile and Met in rubiscolin-6 potentiated the delta opioid activity by about four times in MVD assay. Asp(4) cannot be replaced by Ala, Glu or His. The original Leu(5) was optimal, while substitution of Phe(6) with Val potentiated its delta opioid activity by more than 10 times. The most potent derivative we obtained was YPMDLV, which was nearly 20 times more potent than rubiscolin-6 in MVD assay. The derivatives thus obtained showed higher delta receptor affinity and more potent antinociceptive activity than rubiscolins.     

Soymorphins, novel mu opioid peptides derived from soy beta-conglycinin beta-subunit, have anxiolytic activities

Based on the amino acid sequence YPFV found in the soy beta-conglycinin beta-subunit, which is common to an opioid peptide human beta-casomorphin-4, peptides YPFVV, YPFVVN, and YPFVVNA were synthesized according to their primary structure. On guinea pig ileum (GPI) assay, they showed opioid activity (IC50 = 6.0, 9.2 and 13 microM respectively) more potent than human beta-casomorphins, and were named soymorphins-5, -6, and -7, respectively. Their opioid activities on mouse vas deferens (MVD) assay were less potent than on GPI assay, suggesting that they are selective for the mu opioid receptor. Human beta-casomorphin-4 and soymorphin-5 were released from the soy 7S fraction (beta-conglycinin) by the action of gastrointestinal proteases. Soymorphins-5, -6, and -7 had anxiolytic activities after oral administration at doses of 10-30 mg/kg in the elevated plus-maze test in mice.     

Rubiscolin-6, a delta opioid peptide derived from spinach Rubisco, has anxiolytic effect via activating sigma1 and dopamine D1 receptors

Rubiscolin-6 (Tyr-Pro-Leu-Asp-Leu-Phe) is a delta opioid peptide derived from the large subunit of spinach d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We previously reported that rubiscolin-6 had an analgesic effect and stimulated memory consolidation. Here we show that intraperitoneally (i.p.) or orally administered rubiscolin-6 has an anxiolytic effect at a dose of 10 mg/kg or 100 mg/kg, respectively, in the elevated plus-maze test in mice. The anxiolytic effects of rubscolin-6 after i.p. (10 mg/kg) and oral (100 mg/kg) administration were blocked by a delta opioid receptor antagonist, naltrindole (1 mg/kg, s.c.), suggesting that the anxiolytic activity of rubiscolin-6 is mediated by delta opioid receptor. The anxiolytic effect of rubiscolin-6 (10 mg/kg, i.p.) was also blocked by a dopamine D(1) antagonist, SCH23390 (30 microg/kg, i.p.), but not by a dopamine D(2) antagonist, raclopride (15 microg/kg, i.p.). The anxiolytic effect of rubiscolin-6 (10 mg/kg, i.p.) was blocked by sigma(1) receptor antagonist, BMY14802 (0.5 mg/kg, i.p.) or BD1047 (10 mg/kg, i.p.). Taken together, the anxiolytic effect of rubiscolin-6 is mediated by sigma(1) and dopamine D(1) receptors downstream of delta opioid receptor.     

Effect of rubiscolin,a delta opioid peptide derived from Rubisco,on memory consolidation

Rubiscolin-6 (YPLDLF) is a delta selective opioid peptide isolated from the enzymatic digests of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves. In a step-through type passive avoidance test in ddY mice, rubiscolin-6 enhanced memory consolidation at doses of 3nmol/mouse after intracerebroventricular administration, and at 100mg/kg after oral administration. These doses are smaller than the optimal doses for an analgesic effect. The memory enhancing effect of rubiscolin-6 was blocked by pretreatment with the delta antagonist naltrindole, suggesting the involvement of the delta opioid receptor.     

Delta opioidmimetic antagonists: prototypes for designing a new generation of ultraselective opioid peptides.

BACKGROUND: Tyr-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and Tyr-Tic-Ala were the first peptides with delta opioid antagonist activity lacking Phe, considered essential for opioid activity based on the N-terminal tripeptide sequence (Tyr-D-Xaa-Phe) of amphibian skin opioids. Analogs were then designed to restrain the rotational flexibility of Tyr by the substitution of 2,6-dimethyl-L-tyrosine (Dmt). MATERIALS AND METHODS: Tyr and Dmt peptides were synthesized by solid phase and solution methods using Fmoc technology or condensing Boc-Dmt-OH or Boc-Tyr(But)-OH with H-L-Tic-OBut or H-D-Tic-OBut, respectively. Peptides were purified (> 99%) by HPLC and characteristics determined by 1H-NMR, FAB-MS, melting point, TLC, and amino acid analyses. RESULTS: H-Dmt-Tic-OH had high affinity (Ki delta = 0.022 nM) and extraordinary selectivity (Ki mu/Ki delta = 150,000); H-Dmt-Tic-Ala-OH had a Ki delta = 0.29 nM and delta selectivity = 20,000. Affinity and selectivity increased 8700- and 1000-fold relative to H-Tyr-Tic-OH, respectively. H-Dmt-Tic-OH and H-Dmt-Tic-NH2 fitted one-site receptor binding models (eta = 0.939-0.987), while H-Dmt-Tic-ol, H-Dmt-Tic-Ala-OH and H-Dmt-Tic-Ala-NH2 best fitted two-site models (eta = 0.708-0.801, F 18.9-26.0, p < 0.0001). Amidation increased mu affinity by 10- to 100-fold and acted synergistically with D-Tic2 to reverse selectivity (delta-->mu). Dmt-Tic di- and tripeptides exhibited delta antagonist bioactivity (Ke = 4-66 nM) with mouse vas deferens and lacked agonist mu activity (> 10 microM) in guinea-pig ileum preparations. Dmt-Tic analogs weakly interacted with kappa receptors in the 1 to > 20 microM range. CONCLUSIONS: Dmt-Tic opioidmimetic peptides represent a highly potent class of opioid peptide antagonists with greater potency than the nonopioid delta antagonist naltrindole and have potential application as clinical and therapeutic compounds.     

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.     

Modifications of the cyclic mu receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et): effects on opioid receptor binding and activation.

The previously described cyclic mu opioid receptor-selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et) (JOM-6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [35S]-GTPgammaS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr1 with conformationally restricted phenolic amino acids. In the [35S]-GTPgammaS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range 0.4-9nM. However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC50 value of less than 100nM. Lastly, we have identified a peptide, D-Hat-c[D-Cys-Phe-D-Pen]NH2 (Et), with high potency and > 1,000-fold functional selectivity for the mu over delta opioid receptor as measured by the [35S]-GTPgammaS assay.     

Opioid activities of beta-casomorphins

β-Casomorphin-7 (H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH) and its analogues: β-casomorphin-6, (-5) and (-4) (derived by sequential removal of respectively one, two or three amino acid residues from the C-terminus), were tested for their opioid activities in a variety of assay systems. Each of the four peptides displayed opioid activity in an opiate receptor binding assay, the isolated mouse vas deferens (MVD), the guinea-pig ileum longitudinal muscle myenteric plexus preparation (GPI) and produced naloxone-reversible analgesia after intracerebroventricular injection into rats. In contrast, none of the peptides displayed opioid activity in the isolated rat vas deferens preparation (RVD). β-Casomorphin-5 was the most potent compound in all the assays employed. Each β-casomorphin was more potent on the GPI than on the MVD. In view of the fact that the GPI, MVD and RVD are populated predominantly by μ-, δ- and ε-receptors, respectively, the β-casomorphins probably represent μ-type opiate receptor agonists.     

De novo identification of highly active fluorescent kappa opioid ligands from a rhodamine labeled tetrapeptide positional scanning library

Highly active fluorescent compounds having kappa opioid activity were identified following the screening in a kappa-specific radioligand binding assay of a positional scanning tetrapeptide combinatorial library in which every tetrapeptide was fluorescently labeled. Lissamine rhodamine B sulfonyl chloride was coupled to the N terminal of a mixture-based tetrapeptide positional scanning library made up of over 7.3 million tetrapeptides. Upon determination of the most active mixtures for each position of the library in the kappa binding assay, individual rhodamine labeled tetrapeptides were then synthesized and tested to determine their activities. Eight individual rhodamine labeled peptides were identified that were specific for the kappa opioid receptor, having binding affinities ranging from 5-20 nM. These peptides were poor inhibitors at the mu and delta receptors (K(i)>5,000 nM). Furthermore, neither rhodamine itself nor these same tetrapeptides lacking the N-terminal rhodamine had any significant activity at the kappa receptor, indicating that both the tetrapeptide sequence and the rhodamine moiety are required for receptor binding. This study has demonstrated that novel fluorescent compounds with intrinsic activity can be identified through the use of combinatorial chemistry.     

A novel opioid structure which accepts protonated as well as non-protonated nitrogen: a family of pure, delta receptor selective antagonists.

Conventional opioids including opioid peptides require an "opioid" nitrogen which exists in protonated state while interacting with the receptor. In the present paper we demonstrate that the Tyr-Pro-Gly-Phe-Leu-Thr hexapeptide sequence accepts N-terminal substituents such as N-t-Boc, N-phenylacetyl and N-diphenylacetyl where the N cannot become protonated, as well as "traditional" substitutions such as N,N-diallyl, where protonation is likely under physiological conditions. The opioid peptides bearing these substituents are pure antagonists of medium affinity (Ke values in the mouse vas deferens bioassay against [Met5]-enkephalin are in the 3 x 10(-7)-4 x 10(-6) M range) with a high delta receptor preference (50-350-fold delta over mu selectivity ratios).     

Evidence for coupling of the kappa opioid receptor to brain GTPase

In membranes from guinea pig cerebellum, a tissue which predominantly contains kappa opioid receptors, exogenous and endogenous kappa-selective opioid agonists stimulated low-km GTPase activity by 11-20% with concentrations for half-maximal stimulation of 3-23 microM. Opioid ligands of the mu and delta type had no effect on GTPase in these membranes. Similar stimulation of GTPase by kappa opiates was obtained in rat and monkey brain membranes pretreated with beta-funaltrexamine (beta-FNA) and cis-(+/-)-3-methylfentanyl isothiocyanate (superfit) to alkylate the mu and delta receptors, respectively. The stimulation of brain GTPase by kappa opiates in both types of membranes was inhibited by naloxone with IC50's of 0.35 microM and 0.40 microM. The results demonstrate the coupling of the kappa opioid receptor to high affinity GTPase, the Ni regulatory protein of the adenylate cyclase complex.     

Kinin and opioid receptors in the paratrigeminal nucleus modulate the somatosensory reflex to rat sciatic nerve stimulation

The influence of kinin and opioid receptor blockade in the paratrigeminal nucleus (Pa5) on the somatosensory reflex (SSR) to sciatic nerve stimulation (SNS) was assessed in anaesthetized-paralyzed rats. SNS (square 1 ms pulses at 0.6 mA and 20 Hz for 10s) increased mean arterial pressure from 87+/-3 to 106+/-3 mmHg. Pressor responses to SNS were reduced 40-60% by HOE-140 and LF 16-0687 (B2 receptor antagonists; 20 and 100 pmol respectively), CTOP or nor-binaltorphimine (mu and kappa opioid receptor antagonists, respectively; 1 microg) but potentiated by naltrindole (delta opioid receptor antagonist) receptor antagonist microinjections into the contralateral (but not ipsilateral) Pa5. The SSR to sciatic nerve stimulation was not changed by B1 kinin receptor or NK1, NK2 and NK3 tachykinin receptor antagonists administered to the Pa5. Capsaicin pretreatment (40 mg/kg/day, 3 days) abolished the effects of the opioid receptor antagonists, but did not change the effect of kinin B2 receptor blockade on the SSR. Thus, the activity of B2 and opioid receptor-operated mechanisms in the Pa5 contribute to the SSR in the rat, suggesting a role for these endogenous peptides in the cardiovascular responses to SNS.     

Soymorphins,Novel μ Opioid Peptides Derived from Soy β-Conglycinin β-Subunit,Have Anxiolytic Activities

Based on the amino acid sequence YPFV found in the soy β-conglycinin β-subunit, which is common to an opioid peptide human β-casomorphin-4, peptides YPFVV, YPFVVN, and YPFVVNA were synthesized according to their primary structure. On guinea pig ileum (GPI) assay, they showed opioid activity (IC₅₀ = 6.0, 9.2 and 13 μM respectively) more potent than human β-casomorphins, and were named soymorphins-5, -6, and -7, respectively. Their opioid activities on mouse vas deferens (MVD) assay were less potent than on GPI assay, suggesting that they are selective for the μ opioid receptor. Human β-casomorphin-4 and soymorphin-5 were released from the soy 7S fraction (β-conglycinin) by the action of gastrointestinal proteases. Soymorphins-5, -6, and -7 had anxiolytic activities after oral administration at doses of 10–30 mg/kg in the elevated plus-maze test in mice.     

Light-dependent changes in ribulose bisphosphate carboxylase activase activity in leaves

An assay for the activity of ribulose bisphosphate carboxylase (Rubisco) activase in crude leaf extracts was developed. The assay is based on a spectrophotometric assay of Rubisco, and activase activity (in nanomoles activated Rubisco per minute per milligram chlorophyll) was calculated from the rate of increase in Rubisco activity over time. Activase activity measurements were made using samples from spinach (Spinacia oleracea) leaves undergoing (a) steady-state photosynthesis at various photon flux density (PFD) values and (b) nonsteady-state photosynthesis following an increase from darkness to a high PFD. Analysis of these samples showed that steady-state Rubisco activase activity was relatively low in darkness, increased with PFD, and saturated below 300 micromoles per square meter per second. Rubisco activity (measured spectrophotometrically) was also found to be low in darkness and to increase with PFD, but it saturated at much higher PFD values (approximately 1000 micromoles per square meter per second) along with the rate of photosynthesis. Following an increase in PFD from darkness to 650 micromoles per square meter per second, activase activity increased more or less linearly over a period of 5 to 6 minutes, after which it was constant. Rubisco activity, however, increased more slowly. The light-dependence of Rubisco activase is consistent with previous gas-exchange data showing two interdependent processes in the activation of Rubisco following an increase in PFD.     

Met-Arg-Trp derived from Rubisco lowers blood pressure via prostaglandin D(2)-dependent vasorelaxation in spontaneously hypertensive rats

Met-Arg-Trp (MRW) has been isolated as an inhibitor for angiotensin I-converting enzyme (ACE) from a pepsin-pancreatin digest of spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco) (IC(50)=0.6 microM). It has been reported that hypotensive activity of ACE-inhibitory peptides derived from food proteins are weakened in spontaneously hypertensive rats older than 25 weeks (old SHR). However, MRW reduced blood pressure after oral administration at a dose of 5 mg/kg in old SHR as well as in younger SHR. MRW exhibited vasorelaxing activity above 1 microM in isolated mesenteric artery from adult and old SHR. The vasorelaxing activity of MRW was blocked by indomethacin and BW A868C, a cyclooxygenase inhibitor and an antagonist for DP(1) receptor, respectively. However, N(G)-nitro-L-arginine methyl ester, an inhibitor for nitric oxide synthase, had no effect on the relaxation. The hypotensive activity of MRW was also blocked by indomethacin and BW A868C, respectively, in adult and old SHR. Taken together, the vasorelaxing and hypotensive activities of MRW may be mediated by prostaglandin D(2) and the DP(1) receptor. These findings suggest that the hypotensive activity of MRW is mainly caused by vasorelaxation rather than by ACE-inhibition.     

Opioid receptor selectivity of peptide models of beta-endorphin

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic alpha-helical domain (residues 13-29) in beta-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for mu- and delta-opioid receptors than for kappa opioid receptors. Relative to beta-endorphin, the peptide models were 2-3 times more potent in the mu and kappa receptor binding assays, and about equipotent in the delta receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to beta-endorphin and the other was twice as potent. Like beta-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by mu receptors and not kappa receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of beta-endorphin.     

Effects of opioid peptides on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of opioid peptides on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. beta-Endorphin (0.3 - 30 nM), dynorphin (0.3 - 30 nM) and FK 33-824 (1 - 10 microM) suppressed basal I-CRF release in a dose-dependent fashion. At 2.2 nM concentrations of these peptides, mean percent inhibition was 56% for beta-endorphin; less than 5% for alpha-endorphin; 44% for dynorphin; 23% for leucine-enkephalin; 6% for methionine-enkephalin; less than 5% for FK 33-824; and less than 5% for D-ala2, D-leu5-enkephalin. The inhibitory effects of beta-endorphin and enkephalins were completely blocked by naloxone, but those of dynorphin were only partially blocked. These results suggest that opioid peptides act through opioid receptors and inhibit I-CRF release from the hypothalamus under our conditions. Therefore, endogenious opioid peptides may have a physiological role in the CRF-releasing mechanism of the hypothalamus.     

Arylacetamide kappa opioid receptor agonists with reduced cytochrome P450 2D6 inhibitory activity

Some kappa opioid receptor agonists of the arylacetamide class, for example, ICI 199441 (1), were found to strongly inhibit the activity of cytochrome P450 2D6 (CYP2D6) (1: CYP2D6 IC50=26 nM). Certain analogs bearing a substituted sulfonylamino group, for example, 13, were discovered to have significantly reduced CYP2D6 inhibitory activity (13: CYP2D6 IC50>10 microM) while displaying high affinity toward the cloned human kappa opioid receptor, good kappa/delta and kappa/mu selectivity, and potent in vitro and in vivo agonist activity.     

The Effects of Chilling in the Light on Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Tomato (Lycopersicon esculentum Mill.)

Photosynthesis rate, ribulsoe-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation state, and ribulose bisphosphate concentration were reduced after exposing tomato (Lycopersicon esculentum Mill.) plants to light at 4[deg]C for 6 h. Analysis of lysed and reconsituted chloroplasts showed that activity of the thylakoid membrane was inhibited and that Rubisco, Rubisco activase, and other soluble factors were not affected. Leaf photosynthesis rates and the ability of chilled thylakoid membranes to promote Rubisco activation recovered after 24 h at 25[deg]C. Thylakoid membranes from control tomato plants were as effective as spinach thylakoids in activating spinach Rubisco in the presence of spinach Rubisco activase. This observation is in sharp contrast to the poor ability of spinach Rubisco activase to activate tomato Rubisco (Z.-Y. Wang, G.W. Snyder, B.D. Esau, A.R. Portis, and W.L. Ogren [1992] Plant Physiol 100: 1858-1862). The ability of thylakoids from chilled tomato plants to activate Rubisco in the assay system was greatly inhibited compared to control plants. These experiments indicate that chilling tomato plants at 4[deg]C interferes with photosynthetic carbon metabolism at two sites, thioredoxin/ferredoxin reduction (G.F. Sassenrath, D.R. Ort, and A.R. Portis, Jr. [1990] Arch Biochem Biophys 282: 302-308), which limits bisphosphatase activity, and Rubisco activase, which reduces Rubisco activation state.     

Metabolism of opioid peptides by cerebral microvascular aminopeptidase M

Aminopeptidase M (EC 3.4.11.2), which can degrade low molecular weight opioid peptides, has been reported in both peripheral vasculature and in the CNS. Thus, we have studied the metabolism of opioid peptides by membrane-bound aminopeptidase M derived from cerebral microvessels of hog and rabbit. Both hog and rabbit microvessels were found to contain membrane-bound aminopeptidase M. At neutral pH, microvessels preferentially degraded low molecular weight opioid peptides by hydrolysis of the N-terminal Tyr1-Gly2 bond. Degradation was inhibited by amastatin (I50 = 0.2 microM) and bestatin (10 microM), but not by a number of other peptidase inhibitors including captopril and phosphoramidon. Rates of degradation were highest for the shorter peptides (Met5- and Leu5-enkephalin) whereas beta-endorphin was nearly completely resistant to N-terminal hydrolysis. Km values for the microvascular aminopeptidase also decreased significantly with increasing peptide length (Km = 91.3 +/- 4.9 and 28.9 +/- 3.5 microM for Met5-enkephalin and Met5-enkephalin-Arg6-Phe7, respectively). Peptides known to be present within or in close proximity to cerebral vessels (e.g., neurotensin and substance P) competitively inhibited enkephalin degradation (Ki = 20.4 +/- 2.5 and 7.9 +/- 1.6 microM, respectively). These data suggest that cerebral microvascular aminopeptidase M may play a role in vivo in modulating peptide-mediated local cerebral blood flow, and in preventing circulating enkephalins from crossing the blood-brain barrier.