Structure-affinity and structure-residence time relationships of macrocyclic Gαq protein inhibitors

Abstract

The macrocyclic depsipeptides YM-254890 (YM) and FR900359 (FR) are potent inhibitors of Gα_q/11 proteins. They are important pharmacological tools and have potential as therapeutic drugs. The hydrogenated, tritium-labeled YM and FR derivatives display largely different residence times despite similar structures. In the present study we established a competition-association binding assay to determine the dissociation kinetics of unlabeled G_q protein inhibitors. Structure-affinity and structure-residence time relationships were analyzed. Small structural modifications had a large impact on residence time. YM and FR exhibited 4- to 10-fold higher residence times than their hydrogenated derivatives. While FR showed pseudo-irreversible binding, YM displayed much faster dissociation from its target. The isopropyl anchor present in FR and some derivatives was essential for slow dissociation. These data provide a basis for future drug design toward modulating residence times of macrocyclic G_q protein inhibitors, which has been recognized as a crucial determinant for therapeutic outcome.

Keywords: Biochemistry; Biological sciences; Pharmacology.

Graphical abstract

Figure 1

Chemical structures, affinity values (pK_i or pK_D), and residence times (τ, in min) of FR, YM, and all characterized derivatives and analogs; n.d., not determined due to low affinity. The labeling position of the radioligands was highlighted in green

Figure 2

Binding experiments of [³H]PSB-15900 to Gα_q proteins, expressed in HEK293 cell membranes (A) Saturation binding of [³H]PSB-15900 to HEK293-Gα_q membranes (25 μg of protein). The following values were determined: pK_D = 8.19 ± 0.16, B_max = 27.5 ± 2.9 pmol/mg. (B) Association kinetics of [³H]PSB-15900 (5 nM) to HEK293-Gα_q membrane preparations. The observed association rate k_obs was 0.080 ± 0.006 min-1, resulting in an association half-life (ln 2/kobs) of 8.71 ± 0.60 min. (C) Dissociation kinetics of [³H]PSB-15900 to HEK293-Gα_q membrane preparations. The dissociation rate k_off was determined as 0.0078 ± 0.0005 min⁻¹ resulting in a dissociation half-life (ln 2/k_off) of 91.0 ± 5.4 min and a residence time (1/k_off) of 131 ± 8 min. All data points represent means ± SD of three separate experiments, performed in duplicates.

Figure 3

Characterization of FR and YM derivatives in radioligand-binding assays versus [³H]PSB-15900 Competition-binding experiments of (A) FR and its derivatives, (B) YM and its derivatives YM-1, YM-3, YM-7, and YM-8, (C) YM and its derivatives YM-9 to YM-12, and (D) YM and its derivatives YM-13, YM-14, YM-15, and YM-18 versus [³H]PSB-15900. (E) pK_i values of FR, YM, and all investigated derivatives as determined in competition-binding assays. For affinity values see Table 1. Significance levels were obtained from a one-way ANOVA with multiple comparisons (FR derivatives were compared to FR, YM derivatives were compared to YM) using Dunnett’s post-hoc test; p > 0.05 not significant (n.s.), p < 0.05 ∗; p < 0.01 ∗∗; p < 0.001 ∗∗∗. Data is represented as mean ± SD of three separate experiments, performed in duplicates. (F) Correlation of affinity data from competition-binding assays with functional data from previously performed IP₁ assays; slope = 0.75, R² = 0.89,^20–22 an ideal line (slope = 1) is plotted in light gray for comparison.

Figure 4

Establishing competition-association binding assays (A) Competition-association binding assays with the FR-derived radioligand [³H]PSB-15900 at HEK293-Gα_q membrane preparations (25 μg of protein per vial). (B–D) Competition-association binding assays of [³H]PSB-15900 versus the indicated competitor (B. FR, C. YM, D. YM-18) at 1-, 3-, and 10-fold of their IC₅₀ value. The inhibitor-free association curve is displayed in light blue. (E) The correlation between competitor concentration of FR (red), YM (blue), and YM-18 (gray) and log k_off as determined by the “Kinetics of competitive binding”-fit in Prism 8.4.0. All data are as means ± SD from 3 to 4 independent experiments performed in duplicates.

Figure 5

Competition-association assays for the determination of the residence time of Gα_q inhibitors The curve of the indicated compound is shown in black, the curves of competitor-free [³H]PSB-15900 association are displayed in blue, and the curves of either FR (for FR-derived compounds) or YM (for YM-derived compounds) is displayed in gray for reference. A-D. Competition-association curves of FR derivatives (A. FR, B. FR-1, C. FR-2, D. FR-3/4). E-J. Competition-association curves of YM derivatives and analogs (E. YM, F. YM-10, G. YM-12, H. YM-13, I. YM-14, J. YM-18). All data are means ± SD of 3–4 independent experiments performed in duplicates.

Figure 6

Binding site of macrocyclic Gα_q protein inhibitors and inhibitor-protein interactions (A) Binding site of YM (magenta) at the heterotrimeric Gα_qβ₁γ₂ protein (Gα_q cyan, Gβ₁ light gray, Gγ₂ dark gray) as determined by X-ray crystallography (PDB: 3AH8). Guanosine diphosphate is shown in yellow. A close-up illustrating the binding pose of YM, highlighting the amino acids Arg60 and Glu191, at the membrane is displayed at the right. (B) 2D-ligand interaction diagram of YM at the binding pocket of the Gα_q protein, generated by Molecular Operating Environment (Chemical Computing Group, Cambridge, UK). See also Figures S1–S4 for ligand-binding site interactions of YM and FR derivatives.

Figure 7

Correlations between affinity (pK_i) and kinetic rate constants obtained by competition-association binding assays Correlations (A) between affinity (pK_i) and association rate (log k_on), and (B) between affinity (pK_i) and dissociation rate (log k_off). The 95%-confidence interval is shown as dotted lines in (B).