Affinity Labeling Mu Opioid Receptors With Novel Radioligands

Abstract

1. A series of novel opiate ligands based upon 6alpha-naloxamine have been examined in opioid receptor binding assays. 2. Coupling an ethylamine spacer alone to 6-alpha-naloxamine gave a compound with relatively poor affinity for mu opioid receptors compared to naloxone, although it retained high affinity for kappa1 opioid receptors. Coupling a benzoyl group significantly increased the affinity. The presence at the 4-position of the benzoyl moiety of an amino-(NalAmiBen) or an azido-substituent (NalAziBen) did not significantly effect the affinity at mu receptors. However, iodinating the benzoyl moiety at the 3-position increased the affinity of the derivatives. 3. Two compounds were radiolabeled and evaluated in receptor binding assays. Both radioligands labeled sites in CHO cells stably transfected with the mouse MOR-1 clone. The amino coupound [125I]NalAmiBen and the azido derivative [125I]NalAziBen reversibly bound to membranes from CHO cells transfected with MOR-1 with high affinity in the dark. Exposure of [125I]NalAmiBen to UV did not alter the reversibility of binding, but exposure of [125I]NalAziBen to UV light led to the covalent coupling of the radioligand to the receptor. When run on SDS-PAGE, [125I]NalAziBen binding showed a band at approximately 70-80 kDa. A control corresponding to nonspecific binding failed to reveal any labeling. No bands were observed from membranes labeled with [125I]NalAmiBen.

Generation of novel radiolabeled opiates through site-selective iodination

“…Prior studies suggested benzoic acid substitutions at the 6-position of the opiate scaffold can maintain activity. [12,13] We now report the design and synthesis of three novel, high affinity 125 I-labeled opiates based upon 6-substituted amines of naltrexone ( 6 ), naloxone ( 7 ), and oxymorphone ( 8 ).…”