Deliverables
Screening for ORL1 receptor ligands.
A selective and quantitative radio-receptor assay has been set up. This assay is based upon the ability of a compound to compete with equilibrium binding of radiolabelled nociceptin in a crude membrane fraction from recombinant CHO cells expressing the human ORL1 receptor (CHO[hORL1] cells). A miniaturized scintillation proximity-based assay, well adapted to automated high throughput screening is now available.
Screening for ORL1 receptor agonists versus antagonists.
The ORL1 receptor-mediated inhibition of adenylyl cyclase in intact recombinant CHO[hORL1] cells can be used as a functional assay to discriminate agonist and antagonist activities. Alternative functional assays have been set up. One involves the expression of the human ORL1 receptor in a cell line co-expressing apoaequorin-a Ca++ dependent light emitter-and the Galpha16 subunit that couples all G protein-coupled receptors to phospholipase C and intracellular calcium release. Here, receptor activation is recorded as a flash of light. The other is based on the ability of ORL1 receptor agonists to promote fluorescence resonance energy transfer (FRET) in transiently transfected cells co-expressing ORL1 receptors fused to a donor (blue) or acceptor (green) fluorescent protein.
The result provides methods for high throughput screening of chemical libraries in search of novel ORL1 (nociceptin) receptor ligands. These novel ligands, preferably non peptidic agonists and/or antagonists could be chemically optimized into clinically useful drugs for the treatment of such neurological disorders as chronic pain, stress and anxiety, and drug addiction.
By using these methods, partner 1 have discovered that the opiate lofentanil is a potent ORL1 receptor agonist. Partner 1 have therefore advocated the use of the 4-anilino-piperidine molecular skeleton as a lead structural motif toward new ORL1 receptor ligands. Following this lead, several drug companies have now patented 4-anilinopiperidine derivatives as novel non-peptidic ORL1 receptor agonists (Hoffmann-La Roche, Pfizer), and antagonists (Merck/Banyu Pharm, Hoffmann-La Roche). However, the Banyu antagonist excepted, these new ligands show only low to moderate ORL1 versus opioid receptor selectivity. The quest for highly selective non peptidic ORL1 receptor ligands must therefore go on.
The broad pharmacological profile of nociceptin, the endogenous neuropeptide agonist of the ORL1 receptor, points to a plethora of potential therapeutic applications, particularly in the treatment of pain, stress and anxiety. A molecular model of the complex formed between nociceptin and the ORL1 receptor has been built. The modelled complex is in full agreement with the results of a photo-affinity labelling study using a radio-labelled nociceptin derivative containing p-benzoyl-L-phenylalanine. Rational design of selective, high affinity ORL1 receptor agonists and antagonists, can now be achieved using 3-D QSAR methods. These methods permit the spatial definition of ligand binding sites from correlations of the binding and activity properties of lead compounds with their interaction energies in model complexes. New lead compounds can be identified by high throughput screening of natural product and/or combinatorial non peptidic chemical libraries.
IA molecular model of the receptor has been built, comprising the seven-helix fold and the extra- and intra-cellular loops which have been structurally validated using an environmental amino acid propensity table. An extended binding site able to accommodate nociceptin-(1-13), the shortest fully active analogue of nociceptin, has been characterised. The N-terminal FGGF tetrapeptide is proposed to bind in a highly conserved region, comprising two distinct hydrophobic pockets in a cavity formed by helices 3, 5, 6 and 7, capped by the acidic second extracellular (EL2) loop controlling access to the TM elements of the peptide binding site. The nociceptin conformation provides for the interaction of its highly positively charged core (residues 8-13) with the EL2 loop, thought to mediate receptor activation. Other aspects of the model are in accordance with experimental results, such as the tolerance of nociceptin position (serine) 10 to the bulky sidechain groups. Photo-affinity labelling of the ORL1 receptor with radio-iodinated [Bpa10, Tyr14]-nociceptin has identified the photo-reactive region as Thr296-Arg302, indicating that the nociceptin Ser10 sidechain is appropriately orientated in the model, and that the ligand itself is sunk to a correct depth within the seven-helix transmembrane bundle.
To the best of our knowledge, the model is the only one in existence. The atomic coordinates have not been released in the public domain and are therefore patentable.
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