Agonist-selective activation of individual G-proteins by muscarinic receptors

Abstract

Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.

Figure 1

Structures of novel agonists. Top, published compounds JR-6 (hit compound 7A) and its chlorine derivate PN-152 (7D). Left, novel analogues with the THP ring were replaced by quinuclidine (JB-8A) or morpholine (JB-12-1 and JB-12-2). Right, novel analogues with methyl ester were added to position 5 of the THP ring (JB-13-1 and JB-13-2).

Figure 2

Agonist-induced activation of individual types of Gαi/o on M2 receptor. Y-axis, functional responses measured as agonist-induced GTPγ[³⁵S]binding to membranes from Sf9 cells expressing a particular combination of M2 receptor with a given Gαi/o (indicated in the legend) are expressed as a fold increase over the basal level (in the absence of agonist). X-axis, the molar concentration of agonists is expressed as a logarithm. Data are means ± S.E.M from representative experiments performed in quadruplicates.

Figure 3

Signaling bias among individual Gi/o proteins activated by M2. (a) spider plots show relative intrinsic activity RAi at individual Gαi/o types for agonists (indicated in legend). Intrinsic activities of individual agonists relative to reference agonist carbachol (RAi) calculated according to Eq. (7) from the measurement of GTPγ[³⁵S]binding (Table S2) are plotted as ratios to RAi at GαoA for a given agonist. (Values of reference agonist carbachol are equal to 1). The thickness of the line represents confidence intervals of calculated RAi values. (b) bias plot among individual types of Gi/o G-protein. Values are expressed as log(2) of the bias factor, where the bias factor is calculated according to Eq. (8). Values > 1 (more than two-fold preference) were taken as relevant and displayed in a red frame. Values > 7 mean no response at the Gα subunit in the left column. Data of RAi and bias factors are summarized in SI (Tables S2 and S3, respectively).

Figure 4

Schematic representation of the BRET assay for real-time optical imaging of G protein activity. Agonist-induced activation of a GPCR leads to the dissociation of Gα-GTP and Venus-Gβγ subunits. The released Venus-Gβγ then interacts with the Gβγ effector mimetic masGRK3ct-Nluc to produce the BRET signal.

Figure 5

Agonist-induced activation of individual Gα subunits via the M2 receptor. Time-courses of the representative experiments of agonists-induced real-time monitoring of activation individual Gα subunits (indicated in the legend) induced by reference agonist CBC (100 μM) (a) THP-based compounds JB-13-1, JB-12-2 (1 mM), JR-6, JB-8A and PN-152 (100 μM) (c) are plotted in the x, y graphs (X-axis, time in seconds; Y-axis, amplitudes of Gα activation expressed as ΔBRET ratio normalized to % of the maximal response of the reference agonist CBC at GαoA). Traces are running averages over nine consecutive determinations. Bar plots show means ± SD of observed maximal amplitudes and activation rate constants Kobs, expressed in s-1, obtained by fitting Eq. (9) to the data from three independent experiments performed in triplicates. *P < 0.05 significantly different from the maximal response at GoαA (set to 100) induced by CBC according to one-sample t-test; #P < 0.05 significantly different from the response of given agonist at GoA, according to one-way ANOVA followed by Dunnet’s multiple comparison test or t-test as appropriate. Data of maximum amplitudes and activation rate constants of reference agonist CBC are also expressed in the polar plot (b), where amplitudes are expressed as % of maximal response at GαoA and activation rate constants as logarithms of the observed rate per minute. Line thickness represents the SD of three independent experiments performed in triplicates. Data are summarized in SI Table S4.

Figure 6

Comparison of agonist-induced activation of individual Gq/11 α subunits via the M1 and M3 receptors. Time-courses of the representative experiments of agonists-induced real-time monitoring of activation individual Gα subunits (indicated in the legend) induced by reference agonist CBC (100 μM) and THP-based compounds, JB-12-2, JB-13-1 (1 mM), JR6, PN-152 and JB-8A (100 μM) are plotted in the x, y graphs (X-axis, time in seconds; Y-axis, amplitudes of Gα activation expressed as ΔBRET ratio normalized to % of the maximal response of the reference agonist CBC at Gαq). Traces are running averages over nine consecutive determinations [(A) on the left]. Bar plots [(A) on the right] show means ± SD of observed maximal amplitudes and activation rate constants Kobs, expressed in s-1, obtained by fitting Eq. (9) to the data from at least three independent experiments performed in triplicates. *P < 0.05 significantly different from the maximal response at Gαq (set to 100) induced by CBC according to one-sample t-test; #P < 0.05 significantly different from the response of given agonist at Gq, according to one-way ANOVA followed by Dunnet’s multiple comparison test or t-test as appropriate. Data of maximum amplitudes and activation rate constants of individual agonists are also expressed in the polar plot (B), where amplitudes are expressed as % of maximal response at Gαq and activation rate constants as logarithms of the observed rate per minute. Line thickness represents the SD of at least three independent experiments performed in triplicates. Data are summarized in SI Table S5.

Figure 7

Binding of JR-6 and JB-13-2 in the orthosteric binding site of M₂ receptor. The model of the M₂ receptor in an active conformation (4MQS) was used. Top: reversed orientation of R- enantiomers of compounds JR-6 (left) and JB-13-2 (right) is shown. Orange arrows indicate the position of the THP. The hydrogen bond between N404 at TM6 and the carbonyl oxygen of JB-13-2 is shown as red dots. Bottom: schematic of detailed JR-6 (left) and JB-13-2 (right) interactions with the protein residues. Interactions that occur more than 30% of the simulation time of molecular dynamics trajectory are shown.