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

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 Iα inhibitors with Ki 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 Iα 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.

Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2

“…by guest on May 11, 2018 http://circres.ahajournals.org/ Downloaded from required for BAY 60-7550 to prevent norepinephrine-induced nuclear translocation of NFAT. In contrast, treatment of hypertrophic myocytes with sildenafil significantly blocked nuclear translocation of both wild-type and triple-mutant NFAT-GFP (Figure 7D), as expected given that the antihypertrophic effect mediated by sildenafil is independent of PKA phosphorylation (Figure 2 and Nickl et al 27 ). Inhibition of PDE4 with rolipram did not affect the nuclear translocation of either wild-type or triple-mutant NFAT ( Figure 7D).…”

“…To establish whether PKA or protein kinase G mediates the antihypertrophic effect of PDE2 inhibition, we measured cell surface area and NFAT-GFP nuclear translocation in norepinephrine-treated NRVMs in the presence of selective kinase inhibitors. The selective protein kinase G inhibitor DT-2 27 did not affect the ability of BAY 60-7550 to significantly reduce hypertrophy (Figure 4A and 4B). As a control, we measured the effect of DT-2 on the antihypertrophic effect of selective inhibition of the cyclic GMP-specific PDE5 with sildenafil (10 nmol/L), previously reported to counteract hypertrophy via protein kinase G activity.…”

Pressure-induced oxidative activation of PKG enables vasoregulation by Ca 2+ sparks and BK channels

“…S1B). Notably, however, the synthetic oligopeptide DT-2, a direct inhibitor of PKG that does not distinguish between oxidant- and cGMP-activated PKG10,11, constricted pressurized (80 mmHg) mesenteric arteries from wild-type mice (fig. S1, B and C), but did not affect arteries from PKG[C42S] KI mice (fig.…”

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

“…, Hidaka’s inhibitors [14,15], KT5823 [16]), and peptidic inhibitors that presumably bind to the protein/peptide substrate pocket of PKGIα ( e.g. , TQAKRKKALAMA [17], LRK 5 H, DT-2 and its analogues [18,19], etc . [20,21]).…”

“…[20,21]). Within this set, the best characteristics from the aspect of inhibition potential ( K i =0.8 nM in the presence of cGMP [19]) and selectivity towards PKG1α versus PKA, are exposed by D-DT-2 (although D-DT-2 has not been fully characterized in terms of broad selectivity profiling).…”