EURON and THEME joint PhD meeting

40
EURON and THEME joint meeting 2011
Conformational strain may underlie biased agonism
of dualsteric ligands at the M 2 receptor
Bock, A. 1 , Müller, A. 2 , Holzgrabe, U. 3 , De Amici, M. 4 , Kostenis, E. 2 , Mohr, K. 1
1 Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn; 2 Molecular, Cellular,
and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn; 3 Institute of Pharmaceutical
Chemistry, University of Würzburg; 4 Dipartimento di Scienze Farmaceutiche ‘Pietro Pratesi’,
Università degli Studi di Milano, Milano, Italy.
G protein-coupled receptors (GPCRs) are seven transmembrane (7TM)-spanning
proteins representing the largest and most ubiquitously expressed type of cell
surface receptors. Many 7TMRs contain at least one allosteric binding site which
is topographically distinct from the orthosteric site recognized by the respective
endogenous messenger compound. The muscarinic M 2 acetylcholine receptor is
an excellent model to study allosteric/orthosteric interactions as the core region
of the allosteric binding site is well characterized.
Recently, bisquaternary allosteric/orthosteric hybrid compounds were designed
consisting of an allosteric inverse agonist fragment linked via an aliphatic
hexamethylene middle chain with an orthosteric high affinity agonist. In
exclusively activating the Gi pathway these dualsteric compounds showed biased
agonism at the M 2 receptor [1] . However, hybrids’ potency was considerably lower
than that of the orthosteric building block alone, suggesting a suboptimal fit of
the hybrids’ building blocks to their corresponding binding sites. Here we show
that middle-chain elongation to octamethylene considerably increases potency
for M 2 receptor-mediated G protein activation as measured by [ 35 S]GTPγS binding
in membranes of hM 2 -CHO cells. In order to check whether middle chain length
affects biased signaling we carried out real-time measurements of dynamic mass
redistribution in hM 2 -CHO cells using the EPIC® system (Corning, New York). Our
findings show that the elongated hybrids regain modest ability for G s activation.
We conclude that a spatial misfit between a dualsteric ligand and its corresponding
orthosteric/allosteric receptor sites might impose a conformational strain on the
receptor protein that underlies biased agonism of hexamethylene-type dualsteric
compounds.
[1] Antony J et al. (2009). Dualsteric GPCR targeting: a novel route to binding and
signaling pathway selectivity. FASEB J 23: 442-450
Support by the DFG is gratefully acknowledged (HO 1368/12-1, MO 821/2-1).
This abstract is published in Naunyn Schmiedebergs Arch Pharmacol. 2011,
March (383).