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

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.

. Figure 3. DFL20656 activity in rabbit aorta assay. In the rabbit thoracic aorta, DFL20656 at a concentration as high as 3.0E-05 M, did not induce a contraction but inhibited the response desArg9-BK. The following results indicate that DFL20656 behaves as an antagonist at the Bl receptors in this tissue. Data shown are the mean of a triplicate experiment.

Radioligand Binding Assays
The kinetic binding data were calculated as follows: Calculation of Kobs and association half-life values using prism software Y = Ymax × (1 − exp(− 1*Kobs*X)) Kobs is the observed association rate constant in units of M−1 min−1 Ymax is the maximum plateau at equilibrium in the unit of the y axis.
The half-life equals ln(2) divided by Kobs. Calculation of koff (= k2) values using prism software Y= (Y0 − NS)*exp(− K*X) + NS Y0 is the binding at time zero in the units of the Y axis. NS is the non-specific binding at infinite times in the units of the Y axis. Koff is the rate constant in inverse units of the X axis. The half-life equals ln (2)

Pharmacokinetics
Sprague Dawley male rats (body weights 250 g at the time of the treatment) were used in this study. The animals were originally supplied by Harlan, Italy. The animals were housed, in a group of four, in cages suitable for the species, also during dosing and feeding periods. The animals were housed in a single, exclusive room, air conditioned to provide a minimum of 15 air changes/hour. The environmental controls were set to maintain temperature within the range 22°C and relative humidity within the range 50 to 60% with an approximate 12 h light and 12 h dark cycle that is controlled automatically. Food and water were available ad libitum throughout the study. All animals were weighed on the day of the treatment. Clinical signs were monitored at regular intervals throughout the study in order to assess any reaction to treatment. The experiment was carried on in agreement with the Italian Law D. L.vo 4 marzo 2014, n. 26 Blood samples were collected in heparinized eppendorfs (Heparin Vister 5000 U.I/mL), gently mixed and placed immediately on ice; then eppendorfs were centrifuged (3500 × g, at 4 °C for 15 min) and the resulted plasma collected and transferred to uniquely labelled eppendorfs and frozen at -20 °C till the analysis. At the end of the study animals were sacrificed by exsanguination under deep isoflurane anesthesia. After the bolus, blood (approximately 100ul) was sampled from tail vein at the following timepoints: 5, 15, 30, 60, 120 240, 480 min and 24 h. Samples were analyzed on UPLC (Acquity, Waters) coupled with a API 3200 Triple Quadrupole AB Sciex. Non compartmental analysis was applied and the following PK parameters were evaluated for each subject-Maximum plasma concentration (Cmax), Plasma concentration at last timepoint (Clast), Time of maximum plasma concentration (tmax), AUC from time zero to the time of the last quantifiable plasma concentration (AUC0-last), AUC from time zero extrapolated to infinity (AUCinf), Mean Residence Time (MRT), Half-life (T½), Clearance Dose/AUC (Cli), Apparent distribution volume at the steady-state (Vss): Dose*AUMCinf/(AUCinf)2. Concentration data were extrapolated using the software Analyst™ 6.1 (Applied Biosystems); AUCs were calculated by linear trapezoidal rule and a uniform weight was performed as a first general approach. Graphical concentration-time curves are produced after Log transformation. Softwares (PK Solver 2.0, Excel 2007 Microsoft add in).

Rabbit Thoracic Aorta Assay
Rings of rabbit thoracic aorta denuded of endothelium were suspended in 20 mL organ baths filled with an oxygenated (95% O2 and 5% CO2) and pre-warmed (37 °C) physiological salt solution of the following composition (in nM): NaCl 118.0, KCl 4.7, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25 and glucose 11.0 (pH 7.4). The tissues were connected to force transducers for isometric tension recordings. They were stretched to a resting tension of 4 g, washed several times then allowed to equilibrate overnight. The experiments were carried out using semi-automated isolated organ systems possessing eight organ baths, with multichannel data acquisition. The tissues are exposed to a submaximal concentration of the reference agonist Lys-desArg9-BK (0.1 μM) to obtain a control contractile response.
After stabilization of the agonist-induced response, the tissues are exposed to increasing concentrations of the antagonists. The concentrations are added cumulatively and each is left in contact with the tissues until a stable response is obtained or for a maximum of 30 min. The parameter measured is the maximum change in tension induced by each compound concentration. The results are expressed as a percent of the control response to Lys-desArg9-BK.