(D)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Ialpha

Abstract

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Ialpha inhibitors with K(i) values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Ialpha and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.

Figure 1. Stereochemistry and IC₅₀ profiles of the DT-2 family of PKG inhibitors

(A) (L)- and (D)- amino acid sequences of DT-2, ri-(D)-DT-2 and (D)-DT-2 are shown in capital and small letter formats. (B) DT-2 and (D)-DT-2 differ in the side chain orientations relative to the plane of the peptide backbones. (C) The IC₅₀ curves using the substrate TQAKRKKSLAMA [19].

Figure 2

Dixon-plot analyses of (A) W45, a competitive precursor of DT-2, (B) DT-2, (C) ri-(D)-DT-2 and (D) (D)-DT-2. The velocities of three substrate concentrations, 4 μM (■), 8 μM (▲) and 16 μM (▼) TQAKRKKSLAMA were analyzed under varying inhibitor concentrations. (E) Re-plots of the initial 1/v vs. [I] slopes taken from A, B, C, and D.

Figure 3

Schild-plot analyses of (A) W45 and (B) (D)-DT-2. Michaelis-Menten plots were determined for the PKG substrate TQAKRKKSLAMA in the presence of various inhibitor concentrations. The so obtained K_m,app and V_max values were used for further analysis (see Figure 5).

Figure 4. Proposed inhibitory mechanism for (D)-DT-2

(A) Hyperbolic mixed type inhibition model showing the catalytically productive inhibitor complex ESI as part of the non-competitive component (grey) of the model. Determination of (B) βV_max and (C) αK_i was accomplished by plotting V_max or K_m against *I+. The equations used to derive α and β factors are shown in Methods.

Figure 5. Resistance to proteolysis and cellular uptake of (D)-DT-2 in intact vessels

(A) Trypsin pre-incubated (D)-DT-2 inhibits PKG. PKG activity (white) was assayed for DT-2 (gray) and (D)-DT-2 (black) the presence and absence of trypsin. (B) In vitro internalization of Fluo-(D)-DT-2. Mouse mesenteric arteries were incubated with 2 μM (1 nmole) Fluo-(D)-DT-2 for 30 minutes. Live confocal fluorescence imaging was performed without fixation. Scale bar = 50 μm

Figure 6. Kinetic profiles of Fluo-(D)-DT-2 uptake in intact cerebral arteries

In vitro staining of Fluo-(D)-DT-2 in cerebral arteries after incubation with Fluo-(D)-DT-2 at (A) 10 min, (B) 30 minutes and (C) 60 minutes. Nuclear stains (DRAQ5) emphasizing nuclear exclusion or nuclear staining for Fluo-(D)-DT-2 at (D) 10 minutes incubation and (E) 60 minutes incubation.

Figure 7. Effects of PKG inhibitors on 8-Br-cGMP induced dilation of mesenteric resistance arteries

Vessels were superfused with (A) saline), (B) DT-2, or (C) (D)-DT-2 and pressurized to 80 mm Hg. Following development of tone, increasing concentrations of 8-Br-cGMP were titrated. At the end of each experiment vessel viability was tested using high K⁺ (maximal constriction) and Ca²⁺ fresh PSS (maxima dilation. (D) The bar graph summarizes DT-2 and (D)-DT-2 mediated constrictions relative to maximal relaxation observed in Ca²⁺ free PSS.

Figure 8. In vivo uptake of Fluo-(D)-DT-2

C57BL/6 mice were injected with 20 μl, 1mM peptide (20 nmoles) by tail vein injection and left in the circulation for 30 minutes. Live confocal imaging was performed on small resistance arteries immediately after harvest. A: mesenteric artery, B: femoral artery, C: posterior cerebral artery. D: injection of Fluo-W45, a PKG specific inhibitor sequence without tat-translocation sequence showed no uptake in arteries from mesentery. Arrows depict vessel orientation. Scale bar= 100 μm

Figure 9. DT-2 and (D)-DT-2 increase blood pressure

Mean arterial blood pressures (MAP) was recorded from alert C57BL6xSV129 hybrid background mice implanted with a telemetric DataScience transponder system after i.p. injection of 256 nmoles DT-2 (A, n=8), 134 nmoles (D)-DT-2 (B, n=9) or saline (C, n=8) for a total of 70 minutes. D; Statistical analysis was performed on three time intervals (a) 10–20 minutes, (b) 30–40 minutes and (c) 10–70 min after injection. DT-2 increased MAP significantly only in the first time interval. Blood pressure returned to normal after 20–40 minutes. In contrast, (D)-DT-2 increased blood pressure significantly by 15–25 mmHg for all three intervals.

Figure 10. In vivo treatment with (D)-DT2 suppresses vasodilation induced by 8-Br-cGMP in vitro

(A) A mesenteric arteriole isolated from an untreated mouse relaxes to near maximal diameter in response to 8-Br-cGMP. (B) The 8-Br-cGMP response is greatly reduced in an arteriole isolated from a treated (10 nmoles (D)-DT-2, i.p.) mouse. (C) 8-Br-cGMP induces an 80% dilation in arteries from untreated mice, but only a 12% dilation in arteries from mice treated with (D)-DT2 in vivo. It should be noted that only very small mesenteric arteries (30–50 μm starting diameter) developed sufficient pressure induced tone to reveal the in vitro vasodilator response to 8-Br-cGMP.