Novel Ligands and assays for ociceptin/orphanin FQ and classical opioid receptors


  • Davide Malfacini


The aim of the present study was twofold: pharmacologically characterize novel ligands and set-up and validate novel in vitro assays for nociceptin/orphanin FQ (N/OFQ) peptide (NOP) and classical opioid receptors. NOP and opioid receptors are 7TM receptors coupled with inhibitory G proteins; receptor activation leads to the inhibition of cAMP formation and calcium currents, and opening of potassium channels. Via these cellular inhibitory mechanisms, the N/OFQ – NOP receptor and classical opioid systems regulate a variety of biological functions both in the central nervous system and in the periphery. The calcium mobilization assay has been and still is broadly used as primary screening for novel molecules in academic and industrial in vitro pharmacology laboratories. The use of chimeric G proteins allows to extend the calcium mobilization assay to virtually all types of G protein coupled receptors. This approach was previously used in our laboratories for characterizing NOP receptor ligands. In the frame of the present study, the calcium mobilization assay has been extended and validated for classical opioid receptors using a panel of standard opioid receptor agonists and antagonist. This test was used for investigating the pharmacological profile of novel opioid ligands including a series of morphine and oxymorphone analogues and novel cyclic endomorphin-2 derivatives. Calcium mobilization studies together with classical in vitro assays such as receptor binding, [35S]GTPγS binding and bioassays with isolated organs were applied to novel NOP receptor ligands including i) 3 different series of spiroxatrine derivatives; ii) the antagonist NiK-21273; iii) [X5]N/OFQ(1-13)-NH2 derivatives; iv) three tetrabranched derivatives of N/OFQ generated with an innovative chemical approach named peptide welding technology. Recent data demonstrated that biased agonists, i.e. receptor ligands able to select which signaling pathways become activated upon binding to the receptor, may display advantages over unbiased ligands. In particular, in the field of opioids, G-protein (vs arrestin) biased agonists for the mu receptor displayed an increased therapeutic index associated to reduced tolerance liability. No data are yet available about biased agonism in the NOP receptor field. Therefore a novel bioluminescence resonance energy transfer (BRET) based assay was set-up for the NOP receptor. This method that allows to study both NOP/G-protein and NOP/β-arrestin interactions has been validated using a large panel of NOP ligands encompassing full and partial agonist as well as antagonist activity. The comparison of data achieved investigating NOP/G-protein and NOP/β-arrestin interaction allowed us to perform the very first study of biased agonism in the NOP receptor field. In summary the studies performed in the frame of my PhD project extend our knowledge on the pharmacological profile of NOP and classical opioid receptors, provided to the scientific community novel compounds, pharmacologically characterized in detail, to be used as research tools and possibly as drug prototypes, and made available novel pharmacological assays useful for selecting fully innovative drugs such NOP receptor biased agonists.






Macroarea MED-BIO - Farmacologia e Oncologia Molecolare