Label-free integrative pharmacology on-target of opioid ligands at the opioid receptor family

Abstract

BackgroundIn vitro pharmacology of ligands is typically assessed using a variety of molecular assays based on predetermined molecular events in living cells. Many ligands including opioid ligands pose the ability to bind more than one receptor, and can also provide distinct operational bias to activate a specific receptor. Generating an integrative overview of the binding and functional selectivity of ligands for a receptor family is a critical but difficult step in drug discovery and development. Here we applied a newly developed label-free integrative pharmacology on-target (iPOT) approach to systematically survey the selectivity of a library of fifty-five opioid ligands against the opioid receptor family. All ligands were interrogated using dynamic mass redistribution (DMR) assays in both recombinant and native cell lines that express specific opioid receptor(s). The cells were modified with a set of probe molecules to manifest the binding and functional selectivity of ligands. DMR profiles were collected and translated to numerical coordinates that was subject to similarity analysis. A specific set of opioid ligands were then selected for quantitative pharmacology determination.ResultsResults showed that among fifty-five opioid ligands examined most ligands displayed agonist activity in at least one opioid receptor expressing cell line under different conditions. Further, many ligands exhibited pathway biased agonism.ConclusionWe demonstrate that the iPOT effectively sorts the ligands into distinct clusters based on their binding and functional selectivity at the opioid receptor family.

Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies

“…DMR has been already applied to study the pharmacological properties of new ligands acting at various GPCRs, such as histamine H 1 , β 2 adrenergic, muscarinic M 3 , purinergic P2Y, formyl peptide, and protease‐activated receptors. Classical opioid and the nociceptin/orphanin FQ peptide (NOP) receptors were also investigated in DMR studies. Additionally, the DMR assay, together with different biochemical tools, has been successfully used for GPCRs signaling deconvolution studies …”

Synthesis and Opioid Activity of Tyr1‐ψ[(Z)CF=CH]‐Gly2 and Tyr1‐ψ[(S)/(R)‐CF3CH‐NH]‐Gly2 Leu‐enkephalin Fluorinated Peptidomimetics

“…Analogues 6 – 8 were also screened at the KOPR. Conflicting reports indicate that: 1) compound 5 does not bind to the KOPR, but that 2) 5 has partial agonist activity . However, using the functional assay described above, 5 demonstrated full KOPR agonist activity with an EC 50 value of 80±20 n m , 1000‐fold less potent than at the DOPR.…”

“…[59] Analogues 6-8 werea lso screened at the KOPR.C onflicting reports indicate that:1 )compound 5 does not bind to the KOPR, [60] but that 2) 5 has partial agonist activity. [61] However,u sing the functional assay described above, 5 demonstrated full KOPR agonista ctivity with an EC 50 value of 80 AE 20 nm,1 000-fold less potent than at the DOPR. This result highlights the differences amongst screening meth-ods and assays.I nc ontrastt o5,f luoroalkene 6 and trifluoroethylamine derivatives, (S)-7 and (R)-8 had no activity at KOPR up to concentrationsof10mm.…”

Label-free drug discovery

“…Owing to its integrative nature in measurement (Fang et al, 2006), label-free cell phenotypic profiling, by de facto , is not ideal for directly assessing biased agonism (Morse et al, 2013). However, several label-free approaches may be useful to manifest biased agonism.…”