Binding Mode Exploration of B1 Receptor Antagonists' by the Use of Molecular Dynamics and Docking Simulation-How Different Target Engagement Can Determine Different Biological Effects

Abstract

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules' mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC₅₀, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.

Keywords: allosteric inhibitors; biased signaling; bradykinin 1; neuropathic pain.

Figure 1

Chemical structures of DFL20656 (on the left) and Merck compound 14 (on the right).

Figure 2

3D representation of DFL20656 binding the B1 receptor pocket. DFL20656 is represented in orange sticks, protein and binding site residues (colored in slate) are represented as cartoon and lines respectively, whereas hydrogen bond, aromatic hydrogen bond and pi-pi interactions are reported in yellow, cyan and green dashes respectively. In the figure are shown the interaction of the carbonyl group of amidic region and methylphenyl group of DFL20656, with N114^3.29 and W93^2.60 residues, respectively. Labels are reported with Ballesteros–Weinstein numbering scheme.

Figure 3

3D representation of Merck compound 14 binding the B1 receptor pocket. Merck compound 14 is reported in cyan sticks, protein and binding site residues (colored in slate) are represented as cartoon and lines respectively, whereas hydrogen bond and pi-pi interactions are reported in yellow and green dashes respectively. In the figure are shown the interaction of the carbonyl moiety of methyl ester group with N298^7.39 and the pi-pi interaction between the Merck compound and residue Y266^6.51. Labels are reported with Ballesteros–Weinstein numbering scheme.

Figure 4

Effect of single point mutations on compound activity. [Leu⁸]-Lys-desArg⁹-BK activity is affected only by D291A, which is known to be a key residue for orthosteric cavity (A). DFL20656 and Merck compounds are affected by mutations inside the minor pocket but with a different pattern, to note that both are unaffected by the D291A mutation. DFL20656 presents a single key interaction with the N114A (B). The Merck compound 14 is not affected by N114A mutation by engaging the minor pocket with a different pattern of interactions, in fact its activity is reduced by N298A, Y266A and Q295A (C). The data represent the mean of six experimental replicates. All data were presented as the mean ± SEM. Bonferroni-Dunn post hoc test was used. p < 0.05 was considered representative of significant differences.

Figure 5

Selected compound activity in the intracellular calcium mobilization assay. Dose-response curves were generated to evaluate compound activity and to determine the IC₅₀ both in the transfectant CHO-B1 (up) and in the IMR90 human cell line (down) physiologically expressing the B1 receptor. The orthosteric peptidic inhibitor [Leu⁸]-Lys-desArg⁹-BK (A,D), the Merck compound 14 (B,E) and Dompé DFL20656 (C,F) inhibited calcium release in CHO-B1 cells with pIC₅₀ of 9.16 ± 0.05, 9.42 ± 0.07 and 7.75 ± 0.16 respectively. The inhibition of Ca²⁺ signaling was also observed in the IMR90 cell model with pIC₅₀ values of 7.0, 6.4 and 5.8 respectively. The data represent the mean ± SEM of six experimental replicates.

Figure 6

Analysis of B1 expressing cells: IMR90 cells were treated with selected inhibitors for 30′ at [2000 nM], subsequently the peptidic B1 agonist, Lys-desArg⁹-BK, was added and incubated for an additional 30′. Steady-state IMR90 (control) presented a distribution of B1 expressing cells (in black) and B1 negative cells (in grey). When antagonists were added the distribution changed. In particular, the peptidic antagonist [Leu⁸]-Lys-desArg⁹-BK, stabilized the presence of B1 upon the membrane while Dompé DFL20656 increased the percentage of B1-negative cells thus promoting B1 receptor internalization. The data represent the mean of 5 replicates. All data were presented as the mean ± SEM. Bonferroni-Dunn post hoc test was used. p < 0.05 was considered representative of significant differences. **** p < 0.0001.

Figure 7

Antiallodynic effect of DFL20656 and Merck compound 14 after intravenous administration in a model of neuropathic pain. The efficacy of DFL20656 and the Merck compound were evaluated in a CCI rat model. Both mechanical allodynia (A) and thermal hyperalgesia (B) were evaluated. The DFL20656 showed a significant effect in reducing mechanical allodynia up to 8 h following intravenous administration, while Merck compound was active up to 4 h. For thermal hyperalgesia DFL20656 was active up to 12 h following administration, while Merck compound did not display significant efficacy after 4 h. The data represent the mean of 10 rats per experimental group. All data were presented as the mean ± SEM. Bonferroni-Dunn post hoc test was used. p < 0.05 was considered representative of significant differences. *** p < 0.001, ** p < 0.01 and * p < 0.05 vs CTR group.