Dmt1, d-1-Nal3]morphiceptin, a novel opioid peptide analog with high analgesic activity

The morphiceptin-derived peptide [Dmt1, d-1-Nal3]morphiceptin, labeled mu-opioid receptor (MOP) with very high affinity and selectivity in the receptor binding assays. In the mouse hot plate test, [Dmt1, d-1-Nal3]morphiceptin given intracerebroventricularly (i.c.v.) produced profound supraspinal analgesia, being approximately 100-fold more potent than the endogenous MOP receptor ligand, endomorphin-2. The antinociceptive effect of this new analog lasted up to 120min. Thus, [Dmt1, d-1-Nal3]morphiceptin is an interesting and extraordinarily potent analgesic, raising the possibility of novel approaches in the design of clinically useful drugs for pain treatment.     

(35)S]GTPgammaS binding stimulated by endomorphin-2 and morphiceptin analogs

The ability of several mu-selective opioid peptides to activate G-proteins was measured in rat thalamus membrane preparations. The mu-selective ligands used in this study were three structurally related peptides, endomorphin-1, endomorphin-2 and morphiceptin, and their analogs modified in position 3 or 4 by introducing 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal). The results obtained for these peptides in [(35)S]GTPgammaS binding assay were compared with those obtained for a standard mu-opioid agonist DAMGO. [d-1-Nal(3)]Morphiceptin was more potent in G-protein activation (EC(50) value of 82.5+/-4.5 nM) than DAMGO (EC(50)=105+/-9 nM). [d-2-Nal(3)]Morphiceptin, as well as endomorphin-2 analogs substituted in position 4 by either d-1-Nal or d-2-Nal failed to stimulate [(35)S]GTPgammaS binding and were shown to be potent antagonists against DAMGO. It seems that the topographical location of the aromatic ring of position 3 and 4 amino acid residues can result in a completely different mode of action, producing either agonists or antagonists.     

Transformation of mu-opioid receptor agonists into biologically potent mu-opioid receptor antagonists

N-Allylation (-CH(2)-CHCH(2)) of [Dmt(1)]endomorphins yielded the following: (i) [N-allyl-Dmt(1)]endomorphin-2 (Dmt=2',6'-dimethyl-l-tyrosine) (12) and [N-allyl-Dmt(1)]endomorphin-1 (15) (K(i)mu=0.45 and 0.26nM, respectively) became mu-antagonists (pA(2)=8.59 and 8.18, respectively) with weak delta-antagonism (pA(2)=6.32 and 7.32, respectively); (ii) intracerebroventricularly administered 12 inhibited morphine-induced CNS-mediated antinociception in mice [AD(50) (0.148ng/mouse) was 16-fold more potent than naloxone], but not spinal antinociception, and (iii) 15 reversed the alcohol-elevated frequency in spontaneous inhibitory post-synaptic currents (IPSC) in hippocampal CA1 pyramidal cells in rat brain slices (P=0.0055). Similarly, N-allylation of the potent mu-opioidmimetic agonists, 1,6-bis-[H-Dmt-NH]-hexane and 3,6-bis-[Dmt-NH-propyl]-2(1H)-pyrazinone, converted them into mu-antagonists (pA(2)=7.23 and 7.17 for the N-allyl-derivatives 17 and 19, respectively), and exhibited weak delta-antagonism. Thus, N-allylation of Dmt containing opioid peptides or opioidmimetics continues to provide a facile means to convert selective mu-opioid agonists into potent mu-opioid antagonists.     

D-1-Nal4]endomorphin-2 is a potent micro-opioid receptor antagonist in the aequorin luminescence-based calcium assay

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca(2+) levels, was used to examine relative potency and efficacy of the micro-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) 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 micro-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the micro-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 micro-opioid receptor antagonists, while those substituted in position 3 are partial agonists. Exceptional antagonist potency in the calcium assay was observed for [D-1-Nal(4)]endomorphin-2. The pA(2) value for this analogue was 7.95, compared to the value of 8.68 obtained for the universal, non-selective opioid antagonist of the alkaloid structure, naloxone. The obtained results were compared with the data from the hot-plate test in mice. In that in vivo assay [D-1-Nal(4)]endomorphin-2 was also the most potent analogue of the series.     

Novel endomorphin-2 analogs with mu-opioid receptor antagonist activity.

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%.     

Structural studies of [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues: enhanced activity and cis orientation of the Dmt-Pro amide bond

Analogues of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) (1) were designed to examine the importance of each residue on mu-opioid receptor interaction. Replacement of Tyr(1) by 2',6'-dimethyl-L-tyrosine (Dmt) (9-12) exerted profound effects: [Dmt(1)]EM-2 (9) elevated mu-opioid affinity 4.6-fold (K(i mu=0.15 nM) yet selectivity fell 330-fold as delta-affinity rose (K(i)delta=28.2 nM). This simultaneous increased mu- and delta-receptor bioactivities resulted in dual agonism (IC(50)=0.07 and 1.87 nM, respectively). While substitution of Phe(4) by a phenethyl group (4) decreased mu affinity (K(i)mu=13.3 nM), the same derivative containing Dmt (12) was comparable to EM-2 but also acquired weak delta antagonism (pA(2)=7.05). 1H NMR spectroscopy revealed a trans configuration (1:2 to 1:3, cis/trans) in the Tyr-Pro amide bond, but a cis configuration (5:3 to 13:7, cis/trans) with Dmt-Pro analogues.     

Binding of endomorphin-2 to mu-opioid receptors in experimental mouse mammary adenocarcinoma.

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) binds with high affinity and selectivity to the mu-opioid receptor. In the present study, [125I]endomorphin-2 has been used to characterize mu-opioid-binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [125I]endomorphin-2 (1 nM) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K(d) = 18.79 +/- 1.13 nM, B(max) = 635 +/- 24 fmol/mg protein) and the other shows low affinity and higher capacity (K(d) = 7.67 +/- 0.81 microM, B(max) = 157 +/- 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [125I]endomorphin-2 binding site was [d-1-Nal3]morphiceptin > endomorphin-2 > [d-Phe3]morphiceptin > morphiceptin > [d-1-Nal3]endomorphin-2, indicating binding of these peptides to mu-opioid receptors. The uptake of 131I-labeled peptides administered intraperitoneally to tumor-bearing mice was also investigated. The highest accumulation in the tumor was observed for [d-1-Nal3)morphiceptin, which reached the value of 8.19 +/- 1.14% dose/g tissue.     

Synthesis and in vitro evaluation of a library of modified endomorphin 1 peptides

Endomorphin 1 (Endo-1=Tyr-Pro-Trp-Phe-NH(2)), an endogenous opioid with high affinity and selectivity for mu-opioid receptors, mediates acute and neuropathic pain in rodents. To overcome metabolic instability and poor membrane permeability, the N- and C-termini of Endo-1 were modified by lipoamino acids (Laa) and/or sugars, and 2',6'-dimethyltyrosine (Dmt) replacement of Tyr. Analogues were assessed for mu-opioid receptor affinity, inhibition of cAMP accumulation, enzymatic stability, and permeability across Caco-2 cell monolayers. C-terminus modification decreased receptor affinity, while N-terminus C8-Laa improved stability and permeability with slight change in receptor affinity. Dmt provided a promising lead compound: [C8Laa-Dmt[1]]-Endo-1 is nine times more stable (t(1/2)=43.5min), >8-fold more permeable in Caco-2 cell monolayers, and exhibits 140-fold greater mu-opioid receptor affinity (K(imu)=0.08nM).     

Type and location of fluorescent probes incorporated into the potent mu-opioid peptide [Dmt]DALDA affect potency, receptor selectivity and intrinsic efficacy.

The dermorphin-derived tetrapeptide H-Dmt-d-Arg-Phe-Lys-NH(2) (Dmt = 2',6'-dimethyltyrosine) ([Dmt(1)]DALDA) is a highly potent and selective mu-opioid agonist capable of crossing the blood-brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt(1)]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6-dimethylamino-2'-naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea-pig ileum and mouse vas deferens assays, and in mu-, delta- and kappa-opioid receptor-binding assays. The analogues showed various degrees of mu receptor-binding selectivity, but all of them were less mu-selective than the [Dmt(1)]DALDA parent peptide. Most analogues retained potent, full mu-agonist activity, except for one with fluorescein attached at the C-terminus (3a) (partial mu-agonist) and one containing beta-(6'-dimethylamino-2'-naphthoyl)alanine (aladan) in place of Phe(3) (4) (mu- and kappa-antagonist). The obtained data indicate that the receptor-binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence-labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group (2a) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.     

Activation of mu-opioid receptors inhibits lordosis behavior in estrogen and progesterone-primed female rats

The present study investigated the effect of highly selective mu-opioid receptor (OR) agonists on lordosis behavior in ovariectomized rats treated with 3 microg of estradiol benzoate followed 48 h later by 200 microg of progesterone. Ventricular infusion of the endogenous mu-OR agonists endomorphin-1 and -2 suppressed receptive behavior in a time- and dose-dependent fashion. At 6 microg, both endomorphin-1 and -2 inhibited lordosis behavior within 30 min. However, while the effect of endomorphin-1 lasted 60 min, endomorphin-2 inhibition lasted up to 120 min after infusion. Pretreatment with naloxone (5 mg/kg sc) was able to block both endomorphin-1 and endomorphin-2 effects on lordosis. Site-specific infusions of endomorphin-1 or endomorphin-2 into the medial preoptic area (mPOA), the ventromedial nucleus of the hypothalamus (VMH), or into the mesencephalic central gray did not affect receptivity. In contrast, infusion of 1 mug of either compound into the medial septum/horizontal diagonal band of Broca inhibited lordosis in a pattern very similar to that seen after intraventricular infusions. Infusion of the potent synthetic mu-OR agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (0.08 microg) into the VMH and mPOA inhibited lordosis behavior for at least 60 min after infusion. The nonspecific opioid receptor antagonist naloxone was able to facilitate lordosis in partially receptive female rats when infused into the mPOA but not when infused into the VMH. The behavioral effects of the agonists and antagonist used in this study suggest that the endogenous mu-opioid system modulates estrogen and progesterone-induced lordosis behavior.     

Structure-activity relationship of the novel bivalent and C-terminal modified analogues of endomorphin-2

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) is a putative endogenous mu-opioid receptor ligand. To develop potent analgesics with less side effects related to it, we used the methods of dimerization and C-terminal modification. Through dimerization we got the 'balanced agonists' with potent analgesic activity and we have developed the structure-activity relationship between the selectivity and the distance of the two tyrosine pharmacophores. Modification at the C-terminal increased the selectivity of endomorphin-2 to mu-opioid receptor with binding affinity conserved.     

Opioid receptor binding and in vivo antinociceptive activity of position 3-substituted morphiceptin analogs

Analogs of morphiceptin (Tyr-Pro-Phe-Pro-NH2), a mu-selective opioid receptor ligand, with position 3-modifications, including altered size, lipophilicity, and electronic character, while maintaining aromaticity were synthesized. The activity of the new analogs in in vitro assays and in in vivo hot-plate test of analgesia was compared and the results were consistent. [D-1-Nal3]Morphiceptin was the most potent analog of this series with a 26-fold increase in mu-opioid receptor affinity, a 15-fold potency increase in the GPI assay, and a significant potency increase in the hot-plate analgesic test, as compared with morphiceptin. [d-Qal3]Morphiceptin was found to be a weak antagonist in the GPI assay.     

Quantitative analysis of [Dmt(1)]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry

The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA; [Dmt= 2',6'-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt(1)]DALDA in ovine plasma, using deuterated [Dmt(1)]DALDA as the internal standard. The standard MS/MS spectra of d(0)- and d(5)-[Dmt(1)]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [ M + 2H-NH(3)](2+) (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt(1)]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.     

Dansylated analogues of the opioid peptide [Dmt1]DALDA: in vitro activity profiles and fluorescence parameters

Dansylated analogues of the potent and selective micro opioid peptide agonist [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) were prepared either by substitution of N(beta)-dansyl-alpha,beta-diaminopropionic acid or N(epsilon)-dansyllysine for Lys(4), or by attachment of a dansyl group to the C-terminal carboxamide function via a linker. All three analogues displayed high micro agonist potency in vitro and the C-terminally dansylated one retained significant micro receptor selectivity. The three analogues showed interesting differences in their fluorescence emission maxima and quantum yields, indicating that the dansyl group in two of them was engaged in intramolecular hydrophobic interactions. These dansylated [Dmt(1)]DALDA analogues represent valuable tools for binding studies, cellular uptake and intracellular distribution studies, and tissue distribution studies.     

Design and synthesis of opioidmimetics containing 2',6'-dimethyl-L-tyrosine and a pyrazinone-ring platform

Twelve 2',6'-dimethyl-L-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-NH(CH(2))(n)],3- or 6-R-2(1H)-pyrazinone (n=1-4). 3-[H-Dmt-NH-(CH(2))(4)]-6-beta-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to mu-opioid receptors with high affinity (K(i)mu=0.13 nM; K(i)delta/K(i)mu=447) with mu-agonism (GPI IC(50)=15.9 nM) and weak delta-antagonism (MVD pA(2)=6.35). Key factors affecting opioid affinity and functional bioactivity are the length of the aminoalkyl chain linked to Dmt and the nature of the R residue. These data present a simplified method for the formation of pyrazinone opioidmimetics and new lead compounds.     

Differential antagonism of endomorphin-1 and endomorphin-2 supraspinal antinociception by naloxonazine and 3-methylnaltrexone

To determine if different subtypes of mu-opioid receptors were involved in antinociception induced by endomorphin-1 and endomorphin-2, the effect of pretreatment with various mu-opioid receptor antagonists beta-funaltrexamine, naloxonazine and 3-methylnaltrexone on the inhibition of the paw-withdrawal induced by endomorphin-1 and endomorphin-2 given intracerebroventricularly (i.c.v.) were studied in ddY male mice. The inhibition of the paw-withdrawal induced by i.c.v. administration of endomorphin-1, endomorphin-2 or DAMGO was completely blocked by the pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine (40 mg/kg), indicating that the antinociception induced by all these peptides are mediated by the stimulation of mu-opioid receptors. However, naloxonazine, a mu1-opioid receptor antagonist pretreated s.c. for 24h was more effective in blocking the antinociception induced by endomorphin-2, than by endomorphin-1 or DAMGO given i.c.v. Pretreatment with a selective morphine-6 beta-glucuronide blocker 3-methylnaltrexone 0.25mg/kg given s.c. for 25 min or co-administration of 3-methylnaltrexone 2.5 ng given i.c.v. effectively attenuated the antinociception induced by endomorphin-2 given i.c.v. and co-administration of 3-methylnaltrexone shifted the dose-response curves for endomorphin-2 induced antinociception to the right by 4-fold. The administration of 3-methylnaltrexone did not affect the antinociception induced by endomorphin-1 or DAMGO given i.c.v. Our results indicate that the antinociception induced by endomorphin-2 is mediated by the stimulation of subtypes of mu-opioid receptor, which is different from that of mu-opioid receptor subtype stimulation by endomorphin-1 and DAMGO.     

G protein activation by endomorphins in the mouse periaqueductal gray matter

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.     

Biological activity of endomorphin and [Dmt1]endomorphin analogs with six-membered proline surrogates in position 2

Endogenous μ-opioid receptor (MOR) selective peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), unlike so called ‘typical opioids’, are characterized by the presence of Pro² residue, which is a spacer connecting aromatic pharmacophoric residues. In order to investigate structural requirements for position 2, we synthesized endomorphin analogs incorporating, instead of Pro, unnatural amino acids with six-membered heterocyclic rings, such as piperidine 2-, 3- or 4-carboxylic acids (Pip, Nip and Inp, respectively). (R)-Nip residue turned out to be favourable for improving MOR affinity. Introduction of 2′,6′-dimethyltyrosine (Dmt) instead of Tyr¹ led to obtaining [Dmt¹, (R)-Nip²]EM-2 which showed exceptional MOR affinity and high stability against enzymatic degradation in rat brain homogenate. In in vivo hot-plate test in mice, this analog given intracerebroventicularly (i.c.v.), produced profound supraspinal analgesia, being much more potent than EM-2. The antinociceptive effect of this analog lasted about 170 min and was almost completely reversed by β-funaltrexamine (β-FNA), a selective MOR antagonist.     

Structure-activity relationships of dynorphin analogs substituted in positions 2 and 3

Following up on the observation that the dynorphin analog [Pro(3)]Dyn A(1-11)-NH(2) 2 possesses high affinity and selectivity for the kappa opioid receptor, a number of related peptides were prepared and characterized by radioligand binding and [(35)S]GTPgammaS assays. While incorporation of 2-azetidine carboxylic acid in position 3 led to the equally potent analog 3, the corresponding analog containing piperidine-2-carboxylic acid showed a nearly 90-fold reduction in kappa affinity. Differential preferred bond angles phi in the three building blocks might account for these observations. Compounds 2 and 3 were kappa antagonists with IC(50) values of 380 and 350 nM, respectively. The Sar(3) analog 7 and the Sar(2) analog 8 were kappa agonists, with greater selectivity than Dyn A(1-11)-NH(2) 1. In view of their high kappa affinities (8: K(i) = 1.5 nM; 2: K(i) = 2.4 nM), the new analogs were surprisingly weak kappa agonists or antagonists, e.g., the EC(50) value for the agonist 8 was 280 nM. Different kappa receptor subtypes in binding vs functional assays can not account for these results, since both assays were performed using the same membrane preparation.     

Search of the human proteome for endomorphin-1 and endomorphin-2 precursor proteins

Based on the promising opioid pharmacological profile of the peptide, Tyr-Pro-Trp-Gly-NH(2) (Tyr-W-MIF), Zadina et al. [Zadina, J.E., Hackler, L., Ge, L.-J., Kastin, A.J., 1997. A potent and selective endogenous agonist for the mu-opiate receptor. Nature 386, 499-5502] synthesized and screened other Gly(4)-substituted peptides, culminating in the synthesis of Tyr-Pro-Trp-Phe-NH(2) (endomorphin-1), which displayed high affinity and selectivity for the mu-opioid receptor. The amidated peptide was then isolated from bovine brain frontal cortex, as was a related peptide, Tyr-Pro-Phe-Phe-NH(2) (endomorphin-2), that displayed similar high affinity and selectivity for the mu-opioid receptor. The biosynthesis of the endomorphins in the brain remains obscure, since the putative precursor proteins for the peptides have not been identified. With the completion of the human genome sequencing project, we hypothesized that we should uncover the biological precursors of the peptides using a bioinformatic approach to search the current human proteome for proteins that contained the endomorphin peptide sequences followed by Gly-Lys/Arg, the consensus sequence for peptide alpha-amidation and precursor cleavage. Twelve proteins were identified that contained the endomorphin-1 Tyr-Pro-Trp-Phe sequence, however none contained the Tyr-Pro-Trp-Phe-Gly sequence necessary for alpha-amidation. Twenty-two distinct proteins contained the endomorphin-2 tetrapeptide sequence, and two of those contained the sequence, Tyr-Pro-Phe-Phe-Gly, however, none contained the requisite peptide-Gly-Lys/Arg sequence. Western blot analysis using an endomorphin-2 antibody detected 4 prominent proteins in mouse brain, necessitating reinterpretation of previous immunocytolocalization studies in the brain. Screening of the current human proteome yielded no evidence for endomorphin precursor proteins based on accepted biochemical criteria.