Development Of Potent Endomorphins Analogs Using Atypical Conformationally Constrained C-Terminus And Characterization Of Opioid Activities Of Endomorphins And Their Analogs Both In Vivo And In Vitro

The C-terminus of endomorphin-1 (Tyr¹-Pro²-Trp³-Phe⁴-NH₂, EM1) analogs [Xaa⁴-R]EM1 modified with substitution of non-aromatic residue for Phe⁴ and ended up with -NH-benzyl (X= Gly, Ala, p-Ala, Sar, D-Pro, D-Val or D-Ala, R=-NH-benzyl;2-8) were designed to generate a conformationally constrained peptide set. Radioligand binding assay indicated that several of the novel analogs exhibited μ opioid receptor affinity in the nanomolar range ([K_i (μ)= 2.13-4.9nM], which were similar to or more potent than the parent peptide. Interestingly, these EM1-derivatives, elimination of the C-terminal carboxamide group and address sequence (Phe⁴-NH₂) changed significantly, still behave high μ opioid receptor affinity and selectivity. Replacement -benzyl by -[S]-a-methyl-benzyl or -Phe-NH₂ (X=D-Ala, R=-[S]-a-methyl-benzyl or -Phe-NH₂;9-10) slightly decreased μ opioid receptor affinity than parent, however, a drastic loss of activity was found with the substitution of-NH-Ph, -Phg-NH₂ or -D-Phg-NH₂ for -NH-benzyl in analogs 11-13 (X=GIy, R=Ph;X=D-Val, R=-Phg-NH₂or-D-Phg-NH₂). It indicated that topographical constraint of the third aromatic ring properly was the key factor of EM1 interaction with μ-opioid receptor. Moreover, similar bioactivity changes were observed when all of above C-terminus modifications were applied to endomorphin2 (Tyr¹-Pro²-Phe³ -Phe⁴-NH₂, EM2) (16-27). The most promising derivatives-such as 7 ([D-V^4-NH-Bzl]EMl), 8 ([D-A⁴-NH-Bzl]EMl), 21 ([D-V-NH-Bzl] EM2)-displayed the following characteristics: (i) high μ-opioid receptor affinity [K_i (μ)= 2.13-4.97nM] and selectivity [K[ (δ) /K_i (μ)=200-1280]. (ii) potent functional μ-opioid receptor agonism (IC₅₀= 3.09-5.02 nM, 2-4 times more potent than parent ) in GPI assays (iii) intracerebroventricular administration of analogs 7, 21 produced dose-related antinociception, which was more potent than parent at the same dose. (iv).NMR spectroscopy investigations of these compounds suggested that the chiral of Xaa⁴ played important role of spatial disposition of the benzyl group and other crucial recognition groups correctly. Thus, these endomorphins analogs with atypical constrained C terminus provided model compounds with potent μ-opioid receptor agonism and provided evidences that proper spatial arrangement of the third aromaticpharmacophore was crucial for bioactivity.Endomorphinl-ol (Tyr-Pro-Trp-Phe-ol, EMl-ol) and endomorphin2-ol (Tyr-Pro-Phe-Phe-ol, EM2-ol), with C-terminal alcohol (-ol) containing, have been shown to exhibit higher affinity and 2-4 times lower intrinsic efficacy in vitro compared with endomorphins. In the present study, in order to investigate the alterations of systemic hemodynamic effects induced by C-terminal amide to alcohol conversion, responses to i.v or i.c.v injection of EMl-ol, EM2-ol and their parents were compared in the system arterial pressure (SAP) and heart rate (HR) of anesthetized rats. Both EMl-ol and EM2-ol induced dose-related decrease in SAP when injected in doses of 3-100nmol/kg i.v. In terms of relative vasodepressor activity, it's interesting to note that EM2-ol was more potent than endomorphin2 [DD25 (the dose of 25% decrease in SAP) =6.01±3.19 and 13.99±1.56 nmol/kg i.v., respectively] at a time when responses to EMl-ol were less potent than endomorphinl (DD25=29.04±2.75 and 6.14± 1.21 nmol/kg i.v., respectively). Moreover, decreases in SAP in response to EMl-ol and EM2-ol were reduced by naloxone (2mg/kg, i.v.), atropine sulfate (2mg/kg, i.v.), L-NAME (50mg/kg, i.v.) and bilateral vagotomy. It indicated that the vasodepressor responses were possibly mediated by a naloxone-sensitive, nitric oxide release, vagus-activated mechanism, which was similar to that of endomorphins. At the same time, the -ol derivatives-induced hypotension was associated with decrease in HR. It's noteworthy that i.c.v injections of -ol derivatives produced dose-related decreases in SAP and HR, which were significantly less potent than endomorphins and were attenuated by naloxone (2mg/kg, i.v.) and atropine sulfate (2mg/kg, i.v.). Furthermore, the duration of hypotension evoked by i.v. and i.c.v. injections of -ol derivatives was slightly shorter than that of endomorphins. In summary, the results of the present study indicated that the C-terminal amide to alcohol conversion produced different effects on the vasodepressor activity of endomorphinl and endomorphin2 and endowed EM2-ol distinctive hypotension characters in peripheral (i.v.) and central (i.c.v.) tissues. Moreover, these results provided indirect evidences that C-terminus of endomorphins was crucial for the bioactivity and amidated C-termius might play an important role inthe regulation of the cardiovascular system.EMI and EM2 are newly discovered endogenous MOR ligands. However, the functional role of these EMs in colonic mobility remains unknown. The present investigation was undertaken to evaluate the effects of the EMs on the colonic mobility in vitro and to determine the mechanisms responsible for the responses induced by EMs. EMs (KT'-IO^M) caused significantly contractions in longitudinal and circular muscle of isolated colon but not in the stomach and small intestine. It's noteworthy that the contractile actions of EMs varied slightly among different regions of colonic longitudinal muscle layers, whereas the contractile responses induced by EMs were significantly different among circular muscle layers of proximal colon, mid colon and distal colon. EMs-induced longitudinal or circular muscle contractions were not significantly affected by atropine, JV^-nitro-L-arginine methyl ester (z-NAME), indomethacin, phentolamine, propranolol and methysergide. Tetrodotoxin and naloxone completely abolished EMs-induced longitudinal or circular muscle contractions without affecting contractions in response to carbachol. Surprisingly, EMs (10"7M)-induced longitudinal muscle contractions were significantly attenuated by wor-binaltorphimine (nor-BNl) (3x10"^). By contrast, pretreatment with naltrindole (NTI) (10"6M) did not significantly affect EMs-induced longitudinal or circular muscle contractions. Interestingly, the circular muscle contractions in response to EM2 (10"7M) were not fully blocked by /?-funaltrexamine (/7-FNA) (6xlO'6M). Naloxonazine (lO^M) almost fully abolished the EMs-induced longitudinal or circular muscle contractions, and these effects could be only partially reversed by extensive washing. All the results indicated that the mechanisms and sites of actions of EMs were region specific. Furthermore, these finding showing that activation of multiple subtypes of opioid receptors, possibly /