Irreversible Binding of cis-(+)-3-Methylfentanyl Isothiocyanate to the δ Opioid Receptor and Determination of Its Binding Domain

Abstract

Binding of cis-(؉)-Opiate and opioid drugs, acting on membrane-bound receptors, have been widely used as analgesics. They, however, also produce side effects such as respiratory depression, decreased gastrointestinal motility, sedation, and mood changes (1). At least three types (, ␦, and ) of opioid receptor are present in the nervous system (1). Many structurally diverse opiates and opioid compounds have been synthesized aiming to minimize the side effects and to understand the structure-function relationship. Some drugs act nonselectively on all three types of opioid receptors, yet there are ligands selective for each receptor.Among the agents synthesized are affinity ligands or irreversible ligands. An affinity ligand is thought to bind to the receptor and then form a covalent bond on or near the binding site, resulting in irreversible attachment (2, 3). Affinity ligands have been very useful in the purification of receptors such as the ␦ opioid receptor (4) and in the elucidation of receptor structure. Specific incorporation of radiolabeled affinity ligand into the receptor followed by polyacrylamide gel electrophoresis and fluorography or autoradiography has been used to identify the receptor, to determine molecular mass of the receptor without purification, and to examine the nature of carbohydrate moieties (for example, see Refs. 5 and 6). Because of the covalent nature of the bond between the ligand and the receptor, the site of incorporation, thus part of the binding domain, can be precisely determined by peptide mapping and/or determination of amino acid sequences of labeled fragments (for example, see Ref. 7).SUPERFIT, 1 an isothiocyanate derivative of cis-(ϩ)-3-methylfentanyl (Fig. 1), was synthesized by Burke et al. (8) and found to bind irreversibly to the ␦ receptor. Incubation of NG108-15 cell membranes with SUPERFIT (1-5 nM) followed by extensive washing reduced ␦ binding by 60 -90%. The reduction in ␦ binding was due to a decrease in B max with no change in K d (8). Similar preincubation of rat brain membranes with SUPERFIT (1-5 nM) greatly reduced ␦ receptor binding, without affecting receptor binding. The (ϩ)-enantiomer (SU-PERFIT) was 50 times more potent than its (Ϫ)-enantiomer (9). The high affinity and enantioselectivity of SUPERFIT indicate that the irreversible binding is the result of selective interaction with the ␦ receptor. ␦ opioid receptors have been cloned from several species (10 -16). In addition, and opioid receptors have been cloned (Refs. 17 and 18 and references therein). All three opioid receptors contain seven putative transmembrane helices (TMHs), a common structural motif of a G protein-coupled receptor superfamily. Sequence comparison among the three types of opioid receptors shows substantial divergence in the N-and C-terminal domains as well as extracellular loops, while sequences within TMHs and intracellular loops are very similar. These divergent sequences may contribute to the binding of type-selective ligands.

The region in the μ opioid receptor conferring selectivity for sufentanil over the δ receptor is different from that over the κ receptor

“…This finding is reminiscent of our observation on SUPERFIT [29]. Despite the fact that 8/IX4 had more 8 sequence than 8/IX3, chimera 8/ IX4 had a lower affinity for SUPERFIT than 8 Table 1.…”

“…Chimeras 8/ I.t4 (aa 81 187/~t207-398) and pJ54 (aa ml-206/~188-372) were constructed by exchanging the segments from the N-terminus to the beginning of the second extracellular (e2) loop. Details of generation of these five la/8 chimeras has been described [29].…”

“…Membranes were prepared from CHO cells and COS-1 cells as described previously [29]. Protein contents of membranes were determined by the BCA method of Smith et al [33] with bovine serum albumin as the standard.…”

“…(--)Naloxone (10 ktM) was used to define nonspecific binding. Saturation experiments were performed with various concentrations of [3H]diprenorphine (ranging from 0.05 nM to 5 nM) as described [28,29]. Inhibition of [3H]diprenorphine binding was performed with [3H]diprenorphine at a concentration close to its Kd for each receptor and various concentrations of sufentanil or lofentanil.…”

(-)-7′-Isothiocyanato-11-hydroxy-1′,1′-dimethylheptylhexahydrocannabinol (AM841), a High-Affinity Electrophilic Ligand, Interacts Covalently with a Cysteine in Helix Six and Activates the CB1 Cannabinoid Receptor

“…Moreover, an improvement in affinity of nearly 2-fold was noted for ⌬ 9 -THC with C6.47(355)A, in which the WT cysteine is mutated to alanine, resulting in a more hydrophobic methyl group in this position (Fig. 5 (Zhu et al, 1996;Picone et al, 2002;Tahtaoui et al, 2003). Specific binding of [ 3 H]CP55940 was reduced from 303 Ϯ 58.4 pmol/g (buffer-treated) to 50.33 Ϯ 27.8 pmol/g (Fig.…”