Comparison of [Dmt1]DALDA and DAMGO in binding and G protein activation at mu, delta, and kappa opioid receptors

• Published in: The Journal of pharmacology and experimental therapeutics 2003-12, Vol.307 (3), p.947-954
• Main Authors: Zhao, Guo-Min, Qian, Xuanxuan, Schiller, Peter W, Szeto, Hazel H
• Format: Article
• Language: English
• Subjects: Analgesics, Opioid - metabolism; Animals; Binding, Competitive - drug effects; Biotransformation - physiology; Brain - metabolism; Cell Line; Cell Membrane - metabolism; Cloning, Molecular; Enkephalin, Ala-MePhe-Gly- - metabolism; Enkephalin, Ala-MePhe-Gly- - pharmacology; GTP-Binding Proteins - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Guanylyl Imidodiphosphate - pharmacology; Humans; Kinetics; Male; Mice; Oligopeptides - pharmacology; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta - metabolism; Receptors, Opioid, kappa - metabolism; Receptors, Opioid, mu - metabolism; Sodium - pharmacology; Spinal Cord - metabolism
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.103.054775

[Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine) binds with high affinity and selectivity to the mu opioid receptor and is a surprisingly potent and long-acting analgesic, especially after intrathecal administration. In an attempt to better understand the unique pharmacological profile of [Dmt1]DALDA, we have prepared [3H][Dmt1]DALDA and compared its binding properties with that of [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Kinetic studies revealed rapid association of [3H][Dmt1]DALDA when incubated with mouse brain membranes (K+1 = 0.155 nM(-1) min(-1)). Dissociation of [3H][Dmt1]DALDA was also rapid (K(-1) = 0.032 min(-1)) and indicated binding to a single site. [3H][Dmt1]DALDA binds with very high affinity to human mu opioid receptor (hMOR) (Kd = 0.199 nM), and Kd and Bmax were reduced by sodium but not Gpp(NH)p [guanosine 5'-(beta,gamma-imido)triphosphate]. Similar Kd values were obtained in brain and spinal cord tissues and SH-SY5Y cells. The hMOR:hDOR (human delta opioid receptor) selectivity of [Dmt1]DALDA ( approximately 10,000) is 8-fold higher than DAMGO. However, [Dmt1]DALDA is less selective than DAMGO against hKOR (human kappa opioid receptor) (26-versus 180-fold). The Ki values for a number of opioid ligands were generally higher when determined by competitive displacement binding against [3H][Dmt1]DALDA compared with [3H]DAMGO, with the exception of Dmt1-substituted peptide analogs. All Dmt1 analogs showed much higher affinity for the mu receptor than corresponding Tyr1 analogs. [35S]GTPgammaS (guanosine 5'-O -(3-[35S]thio)triphosphate) binding showed that [Dmt1]DALDA and DAMGO are full agonists at hMOR and hDOR but are only partial agonists at hKOR. The very high affinity and selectivity of [3H][Dmt1]DALDA for the mu receptor, together with its very low nonspecific binding (10-15%) and metabolic stability, make [3H][Dmt1]DALDA an ideal radioligand for labeling mu receptors.