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Anti-hypertensive Mechanisms of Cyclic Depsipeptide Inhibitor Ligands for G q/11 Class G Proteins

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Anti-hypertensive Mechanisms of Cyclic Depsipeptide Inhibitor Ligands for G q/11 Class G Proteins

Matthew M Meleka et al. Pharmacol Res.

Abstract

Augmented vasoconstriction is a hallmark of hypertension and is mediated partly by hyper-stimulation of G protein couple receptors (GPCRs) and downstream signaling components. Although GPCR blockade is a key component of current anti-hypertensive strategies, whether hypertension is better managed by directly targeting G proteins has not been thoroughly investigated. Here, we tested whether inhibiting Gq/11 proteins in vivo and ex vivo using natural cyclic depsipeptide, FR900359 (FR) from the ornamental plant, Ardisia crenata, and YM-254890 (YM) from Chromobacterium sp. QS3666, or it's synthetic analog, WU-07047 (WU), was sufficient to reverse hypertension in mice. All three inhibitors blocked G protein-dependent vasoconstriction, but to our surprise YM and WU and not FR inhibited K+-induced Ca2+ transients and vasoconstriction of intact vessels. However, each inhibitor blocked whole-cell L-type Ca2+ channel current in vascular smooth muscle cells. Subcutaneous injection of FR or YM (0.3 mg/kg, s.c.) in normotensive and hypertensive mice elicited bradycardia and marked blood pressure decrease, which was more severe and long lasting after the injection of FR relative to YM (FRt1/2 ≅ 12 h vs. YMt1/2 ≅ 4 h). In deoxycorticosterone acetate (DOCA)-salt hypertension mice, chronic injection of FR (0.3 mg/kg, s.c., daily for seven days) reversed hypertension (vehicle SBP: 149 ± 5 vs. FR SBP: 117 ± 7 mmHg), without any effect on heart rate. Our results together support the hypothesis that increased LTCC and Gq/11 activity is involved in the pathogenesis of hypertension, and that dual targeting of both proteins can reverse hypertension and associated cardiovascular disorders.

Keywords: Arginine vasopressin (CID: 644077); Bay K8644 (CID: 2303; Blood pressure; Calcium signaling; Cyclic depsipeptides; Endothelin-1 (CID: 16212950); FR900359 (CID: 14101198); G proteins; G(q/11) inhibitor ligands; L-type calcium channel; Nifedipine (CID: 63011); Phenylephrine (CID: 5284443); U46619 (CID: 5311493); WU-07047 (PMID: 25875152); YM-254890 (CID: 9919454).

Conflict of interest statement

Conflict of interest: The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
Inhibition of Gq-coupled GPCR-induced vasoconstriction of small mesenteric arteries by cyclic depsipeptide Gq/11 inhibitor ligands FR900359 (FR), YM-254890 (YM), and WU-07047 (WU). Vasoconstrictor responses to phenylephrine (PE) are expressed as mean percent decrease in diameter (n=3 animals [2 vessels/animal] per group). A – C, Concentration-dependent blockade of PE-induced vasoconstriction by FR, YM, and WU. D, E, F, Effects of the highest concentrations of FR (1 μM), YM (1 μM), and WU (50 μM) on contractile responses to the thromboxane analogue, U-46619, arginine vasopressin (AVP), and endoethelin-1 (ET-1), respectively. Values are mean ± s.e.m. **P< 0,01 vs. control; *, **P< 0.05, 0.01 WU vs. control; #, ##P< 0.05, 0.01 YM vs. control; ++P< 0.01 FR vs. control
Figure 2.
Figure 2.
Effects of Gq/11 inhibitor ligands on G protein-independent, high-potassium and L-type calcium channel (LTCC)-mediated Ca2+ transients and vasoconstriction. Contractile responses were elicited by abluminal application of PSS containing increasing concentrations of potassium to cause membrane depolarization, or the LTCC activator Bay K 8644 (Bay K), opening LTCC and facilitating the influx of Ca2+ into vascular smooth muscle. A-C, High potassium-induced Ca2+ transients in small mesenteric arteries in the absence or presence of FR (1 μM), YM (1 μM), or WU (50 μM). Ca2+ transients were measured as changes in fluorescence intensity of the Ca2+ binding dye, Fluo-4 that was preloaded in the vessel prior to incubation with an inhibitor or the application of high potassium PSS. Values are expressed as percent change in fluorescence from baseline. Vasoconstrictor responses are expressed as mean percent decrease in diameter (n=6 animals [2 vessels/animal] per group). D – F, High potassium-induced vasoconstriction in the absence and presence of FR (1 μM), YM (1 μM), or WU (50 μM). YM and WU but not FR inhibit high potassium-evoked contractile responses. G, Calcium-induced vasoconstriction in the absence and presence of the LTCC opener, Bay K8644. Both low and high concentrations of Bay K have similar facilitative effects on Ca2+-induced vasoconstriction. H, I, Effects of YM (1 μM) and FR (1 μM) on Ca2+-induced, Bay K-facilitated vasoconstriction. YM but not FR blocks LTCC-mediated vasoconstriction. All values are mean ± s.e.m. *,**P<0.05, 0,01 vs. control.
Figure 3.
Figure 3.
Structural comparison of the binding modes of amlodipine (A, green), YM-254890 (B, pink), FR 900359 (C, yellow), and WU-07047 (D, orange) bound within the 5KMD dihydropyridine binding site of bacterial CaVAb, viewed from the side of the pore module. A, 5KMD x-ray crystal structure with amlodipine bound within the bacterial homotetrameric model CaV channel, CaVAb. B – D, docked complexes of YM, FR and WU compounds, respectively, bound within the amlodipine binding site of CaVAb. Comparison of the binding modes reveal that the phenyl ring of amlodipine, YM and FR orients in the same direction towards amino acid residue Y168. YM and FR make more extensive hydrophobic interactions within the pocket and have the same binding modes. The binding mode of WU is distinct from all others with the phenyl ring pointing down towards the hydrophobic amino acid residue F203. The CaV channel is oriented such that the pore opening is on the top. The structure orientation is similar to what is shown in Figure 1e/f of the reference paper (doi:10.1038/nature19102), which reports details on the 5KMD crystal structure. Residues T138, Y195, I199, P200, F203 are part of the S6 helix as previously reported in the paper. Residues E165, F167, F171 and Y168 are part of the P helix. E, ribbon and stick structure of CavAb with superimposed poses of docked Gq/11 inhibitor ligands in side view in 5KMD protein.
Figure 4.
Figure 4.
Inhibition of A7r5 vascular smooth muscle L-type calcium (LCa) current by Gq/11 inhibitor ligands. A, left panel, representative tracings of whole-cell currents (top left panel) recorded with a 500 ms step-pulse from a holding membrane potential of −80 mV to testing potential from −60 mV to +40 V at a pulse frequency of 0.1 Hz (bottom left panel). The A7r5 cells were pre-incubated with vehicle (0.02% DMSO) for 10–60 min prior to establishing whole-cell patch; right panel, whole cell LCa tracings in the presence of the 1,4 dihydropyridine, nifedipine. The cells were pre-incubated with nifedipine (1 μM) for 15 min prior to the recording of LCa. B, Conductance – voltage curves of LTCC derived from peak Lca - voltage relationships in the presence of Gq/11 inhibitors. Prior to LCa current (Ica-L) recordings, the cells were incubated with vehicle (control), YM (1 μM), FR (1 μM), or WU (1 or 50 μM) for 10–60 min. For control and each compound, an average of 10 cells were recorded. All values are mean ± s.e.m. *P<0.05, vs. control.
Figure 5.
Figure 5.
Summary of the acute effects of systemic administration of FR and YM on overnight systolic blood pressure (SBP) and heart rate (HR) of conscious normotensive mice. Each dose of the inhibitors was administered subcutaneously as a bolus at the same time (5 pm, indicated by the dashed line) of the day, after 3 hr of baseline recording. Hemodynamic parameters were continuously recorded for at least 24 hr. A, FR (n=7) elicits depressor responses only at medium and high doses. B, YM (n=7) elicits depressor responses at all doses. Note the blockade of nighttime rise in SBP by the lowest dose of YM. C, Comparison of the rate of SBP recovery from the nadir following the injection of FR and YM, using the same data as in A and B. SBP returns to control levels 7 and 20 hr after reaching the nadir following the injection of YM and FR, respectively. D, FR causes transient decrease in HR within 2 hr after injection of the highest dose. E, Both the medium and high dose of YM cause a rapid decrease in HR within 1 hr after injection. F, HR slowly returns to baseline after FR injection whereas it rapidly returns to control levels after YM injection. All values are mean ± s.e.m.
Figure 6.
Figure 6.
Blood pressure and heart rate responses to systemic administration of Gq/11 inhibitors FR and YM in normotensive mice. A, B, maximal depressor response to increasing doses of FR and YM, respectively. Both inhibitors caused a dose-dependent decrease in SBP following a bolus subcutaneous (s.c.) injection in conscious mice. C, D, maximal bradycardic response to increasing doses of FR and YM, respectively. Only the highest dose of FR caused a significant decrease in HR, whereas the medium and high dose YM significantly decrease HR following a bolus s.c. injection. All values are mean ± s.e.m. **P<0.01 vs. control; ##P<0.01, 0.05 vs. 0.1 mg/kg of FR or YM; ++P<0.01, 0.1 vs. 0.3 mg/kg of FR.
Figure 7.
Figure 7.
Changes in mean arterial pressure (MAP) and heart rate (HR) in response to intravenous injection of FR900359 (FR) or YM-254890 (YM), or increasing doses of the alpha-adrenergic receptor agonist, phenylephrine (PE), and the nitric oxide donor, sodium nitroprusside (SNP) in the absence or presence of FR or YM in anesthetized mice. A, B, representative time-course tracings of changes in MAP and HR following a bolus administration (0.1 mg/kg, i.v.) of FR (blue tracings) or YM (orange tracings). The arrow indicates the time of drug injection. C, D, maximum changes in BP and HR in response to increasing doses (1.5, 3, 6, 12, 24 μg/kg, i.v.) of phenylephrine (PE) and sodium nitroprusside (SNP), respectively, before (closed squares) or after intravenous injection of 0.1 mg/kg FR (white triangles) or YM (inverted gray triangles). All values are mean ± s.e.m. **P< 0.01 vs. control
Figure 8.
Figure 8.
Summary of the acute effects of FR and YM on overnight systolic blood pressure (SBP) and heart rate of conscious hypertensive mice. The mice were made hypertensive by placing them on L-NAME in their drinking water (0.5 g/L) for 10 days. Each dose of FR or YM was administered subcutaneously as a bolus at the same time (5 pm, indicated by the dashed line) of the day, after 3 hr of baseline recording. Hemodynamic parameters were continuously recorded for at least 24 hr. A, B, both FR (n=4) and YM (n=4) elicited depressor responses only at medium and high doses. Note the lack of effect of the lowest dose of YM on nighttime SBP. C, a comparison of the rate of SBP recovery from the nadir following the injection of FR and YM, using the same data as in A and B. Note that a higher dose of YM was required to reduce SBP to the same nadir as 0.3 mg/kg of FR in hypertensive state. SBP returned to control levels 5 hr after YM injection; in contrast, SBP reached control levels 16 hr after FR injection. D, E, F, both FR and YM did not affect HR at any dose in hypertensive mice. All values are mean ± s.e.m.
Figure 9.
Figure 9.
Effects of FR900359 on systolic blood pressure (SBP) and heart rate (HR) of conscious, deoxycorticosterone acetate (DOCA)-salt hypertensive male and female mice. A, C, 24-hour SBP (A) and HR (C) responses to a single injection of FR (0.1 or 0.3 mg/kg, s.c.). FR was administered subcutaneously as a bolus at the same time (5 pm, indicated by the dashed line) of the day, after 3 hr of baseline recording. Hemodynamic parameters were continuously recorded for at least 24 hr. B, D, SBP and HR levels before and after DOCA implantation, during daily injection (shaded area) of vehicle (0.01% DMSO in 0.9% saline) or FR (0.3 mg/kg, s.c.), and during post-injection period. All values are mean ± s.e.m. *,**P<0.05, 0,01 vs. control.

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