Baker, Jillian G.

Mistry, Shailesh N.

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Mistry, Shailesh N. and Baker, Jillian G. (2016) The composite influence of the imidazolone moiety of CGP 12177 on its affinity and efficacy at the two conformations of the human β1-adrenoceptor. In: British Pharmacological Society - 6th Focused Meeting on Cell Signalling, Spring 2016, 18-19 Apr 2016, Leicester, UK. Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/38644/1/vol14issue1abst012p.pdfCopyright and reuse: The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions.This article is made available under the University of Nottingham End User licence and may be reused according to the conditions of the licence.  For more details see: http://eprints.nottingham.ac.uk/end_user_agreement.pdfA note on versions: The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the repository url above for details on accessing the published version and note that access may require a subscription.For more information, please contact eprints@nottingham.ac.uk  Proceedings of the British Pharmacological Society at http://www.pA2online.org/abstracts/Vol14Issue1abst012P.pdf   The composite influence of the imidazolone moiety of CGP 12177 on its affinity and efficacy at the two conformations of the human β1-adrenoceptor  There are at least two active conformations of the β1-adrenoceptor (AR): a high affinity conformation (HAC), where cimaterol is readily inhibited by antagonists (including CGP 12177); a secondary low affinity conformation (LAC) where CGP 12177 stimulates agonist responses that are relatively resistant to antagonism (1). Thus, CGP 12177 is a neutral antagonist of the HAC, but at higher concentrations, activates agonist responses via the secondary LAC. This study investigated the role of each component of the imidazolone moiety of CGP 12177 (carbonyl group at position 2 and NH groups in positions 1 and 3 of the aromatic core) towards this unusual pharmacological finding at the human β1-AR.   CGP 12177 analogues (rac-1g, 3-5) were synthesised and 3H-CGP 12177 whole cell binding and CRE-SPAP reporter gene assay were examined in cells stably expressing the human β1-AR as previously described (2).    The affinity at the HAC was investigated using 3H-CGP 12177 binding. The affinity of 3H-CGP 12177 was 0.27±0.02nM n=7 (log KD = -9.48), determined from saturation binding. The analogue affinities (log KD) were rac-1g: -9.47±0.08 n=10, 3: -9.23±0.06 n=9, 4: -8.08±0.06 n=6 and 5: -7.75±0.11 n=6.  CRE-SPAP studies demonstrated that cimaterol responses (log EC50 -8.55±0.06, 69.9±2.5% isoprenaline maximum (isop max), n=11) were readily inhibited by CGP20712A (log KD -9.27±0.07, n=13) whereas responses to CGP12177 (log EC50 -8.51±0.10, 84.7±6.0% isop max) required higher concentrations of CGP20712A (log KD for CGP20712A = -7.14±0.10, n=12), thus demonstrating the presence of a HAC and LAC of the β1-AR. The agonist responses to rac-1g (log EC501 -8.94±0.10, log EC502 = -6.80±0.23, 57.8±4.7% site 1, 86.3±7.5% isop max, n=8) and 3 (log EC501 = -8.86±0.09, log EC502 -6.45±0.15, 51.0±3.2% site 1, 69.9±10.0% isop max, n=8) were best described by two-component responses, suggesting agonism at both conformations (2). Compounds 4 (log EC50 -6.40±0.10, 59.1±6.0% isop max n=9) and 5 (log EC50 -6.64±0.14, 73.7±5.7% isop max n=7) stimulated responses best described by a single component sigmoidal dose response. CGP12177, 4 and 5 inhibited the cimaterol responses as partial agonists, to yield log KD values of -9.43±0.12, n=4, -8.45±0.15, n=5 and -8.09±0.12 n=5, respectively.  Thus, the affinity (KD) of CGP12177, 4 and 5 (measured in both binding and CRE-SPAP assays) is at odds with the concentration required to stimulate agonist responses (EC50).  Furthermore, responses to 4 and 5 were antagonised by CGP20712A to yield log KD values of -7.35±0.10, n=10 and -7.31±0.10 n=6, in keeping with LAC interaction.  Thus the carbonyl moiety of CGP 12177 has little effect on its affinity or efficacy for the HAC.  In contrast, each imidazolone NH group is important for either affinity or efficacy at the HAC. Molecules with the NH in corresponding 3-position of CGP 12177 have lower affinity, and low/no HAC efficacy but do stimulate agonist responses at the LAC of the human β1-AR.  (1) Kaumann and Molenaar (2008). Pharmacol. Ther. 118: 303–336. (2) Baker et al. (2003). Mol. Pharmacol. 63: 1312–1321. HNNHOO NHOHNHOO NHOHNHO NHOH HNOO NHOH HNO NHOHCGP 12177 rac-1g 3 4 5123

The composite influence of the imidazolone moiety of CGP 12177 on its affinity and efficacy at the two conformations of the human β1-adrenoceptor

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