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

Department of Pharmacology, University of Vermont, College of Medicine, HSRF 330, 149 Beaumont Avenue, Burlington, VT 05405, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 12/2009; 1804(3):524-32. DOI: 10.1016/j.bbapap.2009.12.004
Source: PubMed


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.

    • "Cancer-Selective Targeting of the NF-kB Pathway followed by secondary screening and optimization of the resulting hits, yielded two acetylated L-tetrapeptides of similar structure, namely, Ac-LTP1 and Ac-LTP2, which disrupted the GADD45b/MKK7 complex, in vitro, with remarkable halfmaximal inhibitory concentration (IC 50 ) values in the subnanomolar range (Figure 3A; Figure S3B and Table S2), in line with the top-end potencies of other hits isolated from similar peptide library screens (Houghten et al., 1999). Next, we examined whether these two L-tetrapeptides retained in vitro potency upon synthesis in the D configuration, a strategy used successfully in some cases to render small peptides resistant to proteolysis (Zhou et al., 2002; Nickl et al., 2010). Strikingly, as shown in Figures 3A and 3B, the D-enantiomers of Ac-LTP1 and Ac-LTP2 (termed Ac-DTP1 and Ac-DTP2, respectively) displayed no loss of activity in vitro and, unlike their L counterparts, were highly stable in human serum, even after prolonged incubation. "

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    • "Anterior cerebral arteries from male Sprague-Dawley rat and third order mesenteric arteries from male mice (~100 μm starting diameter) were isolated and cannulated in physiological saline solution (PSS; 119 mM NaCl, 4.7 mM KCl, 24 mM NaHCO3, 1.2 mM KH2PO4, 0.03 mM EDTA, 1.2 mM MgSO4, 1.6 mM CaCl2, 10.6 mM glucose, pH 7.4 at 4°C) as previously described in a DMT organ culture myograph (Danish Myo Technology A/S). Warmed (37°C) and gassed (20% O2/5% CO2/75% N2) PSS was circulated through the myograph chamber and arterial diameters were measured using edge detection software (VediView, DMT; Nickl et al., 2010). Intraluminal pressure was progressively increased in 20 mmHg steps using a pressure manometer to allow for the development of myogenic tone (usually between 60 and 80 mmHg) before each experiment. "
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    ABSTRACT: Nitric oxide (NO) is a potent dilator of vascular smooth muscle (VSM) by modulating intracellular cGMP ([cGMP](i)) through the binding and activation of receptor guanylyl cylases (sGC). The kinetic relationship of NO and sGC, as well as the subsequent regulation of [cGMP](i) and its effects on blood vessel vasodilation, is largely unknown. In isolated VSM cells exposed to both pulsed and clamped NO we observed transient and sustained increases in [cGMP](i), with sub-nanomolar sensitivity to NO (EC(50) = 0.28 nM). Through the use of pharmacological inhibitors of sGC, PDE5, and PKG, a comprehensive VSM-specific modeling algorithm was constructed to elucidate the concerted activity profiles of sGC, PDE5, phosphorylated PDE5, and PDE1 in the maintenance of [cGMP](i). In small pressure-constricted arteries of the resistance vasculature we again observed both transient and sustained relaxations upon delivery of pulsed and clamped NO, while maintaining a similarly high sensitivity to NO (EC(50) = 0.42 nM). Our results propose an intricate dependency of the messengers and enzymes involved in cGMP homeostasis, and vasodilation in VSM. Particularly, the high sensitivity of sGC to NO in primary tissue indicates how small changes in the concentrations of NO, irrespective of the form of NO delivery, can have significant effects on the dynamic regulation of vascular tone.
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    • "The resulting increased molecular weight often accompanied by increased polarity of compounds leads to complications related to cellular uptake and proteolytic stability. Nevertheless , several more or less selective peptide-based inhibitors have been recently developed and successfully used for the regulation of physiological processes in cells and tissues [Dostmann et al. 2002; Lawrence 2005; Shiga et al. 2009; Bogoyevitch et al. 2010; Ekokoski et al. 2010; Nickl et al. 2010]. Bisubstrate-analogue (bisubstrate) inhibitors are compounds that simultaneously associate with the ATP-and protein/peptide substrate-binding sites of the enzyme. "
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    ABSTRACT: Proteiinkinaaside (PK-de) poolt katalüüsitav valkude fosforüleerimine on üks tähtsamaid valkude aktiivsuse reguleerimise mehhanisme, mis võimaldab rakkudel reageerida väliskeskkonna muutustele ning suunata elutegevuse tagamiseks olulisi rakusiseseid protsesse. PK-de funktsioneerimishäiretega (eelkõige nende üleekspressiooni ning anomaalselt kõrge aktiivsususega) on seotud mitmed haigused, sealhulgas vähkkasvajad, suhkruhaigus ja Alzheimeri tõbi. Seetõttu on PK-de uurimine ja PK-de aktiivsust blokeerivate inhibiitorite arendamine muutunud oluliseks nii teadusasutustes kui ka ravimitööstustes. Käesoleva töö raames töötati välja fluorestentsi mõõtmisel põhinevad meetodid kinaaside aktiivsuse määramiseks ning kinaaside inhibiitorite iseloomustamiseks, mille rakendatavust demonstreeriti nii kinaase sisaldavates lahustes kui ka elusrakkudes. Esimene arendatud meetod põhineb fluorestsentsmärgistatud cAMP-sõltuva proteiinkinaasi (PKA) substraadi (5-TAMRA-kemptiid) lahutamisel fosforüleeritud produktist planaarkromatograafiliselt ning fosforüleerimisreaktsiooni komponentide kvantifitseerimisel fluorestsentsskänneriga. Antud meetodit rakendati edukalt inhibiitorite, sealhulgas ARC-tüüpi inhibiitorite, iseloomustamiseks. Teine meetod on fluorestentsanisotroopia mõõtmisel põhinev sidumismeetod, mis kasutab fluorestsentsvärviga märgistatud adenosiini analoogi ja arginiini-rikka peptiidi konjugaate, ARC-Photo sonde. Uudseid fluorestsentssonde kasutati kinaaside PKAc ja ROCKII inhibiitorite tuvastamiseks ning sidumisomaduste määramiseks. Tõestati ARC-Photo sondide bisubstraatne iseloom ning nende sobivus kinaaside aktiivsuse määramiseks ning rakendatavus sensorina cAMP kontsentratsiooni mõõtmisel. Järgmiseks sammuks oli sidumismeetodi ülekandmine elusrakkudesse, kuna just elusraku keskkond võimaldab adekvaatselt hinnata inhibiitorite omadusi ning proteiinkinaaside inhibeerimisest tingitud füsioloogilisi efekte. Näidati, et ARC-Photo sondid on võimelised läbima raku plasmamembraani. Sealjuures sõltus sondide võime rakku tungida ja nende lokalisatsioon rakus arginiinide arvust ARC-i peptiidses osas, ühendi kontsentratsioonist ning fluorestsentsvärvi keemilisest loomusest. ARC-Photo sondide struktuuride optimeerimine võimaldas välja töötada Försteri resonantsienergia ülekande (FRET) efektiivsuse mõõtmisel põhineva meetodi PKA aktiivsuse jälgimiseks elusrakkudes. Protein kinases (PKs) play a key role in the regulation of protein functions in living cells. Many heavy human diseases (including cancer, diabetes, and Alzheimer’s disease) have been linked to the aberrant PK signalling, which has made PKs important drug targets. This has caused an increasing need for the elaboration and improvement of analytical methods for high-throughput screening and characterization of new compounds. This thesis describes the progress with the development of fluorescence-based assays for protein kinase research, starting with a biochemical kinetic method and getting at a binding assay applicable for monitoring of protein kinase activity in living cells. A kinetic assay based on the chromatographic separation of the fluorescently labelled substrate peptide TAMRA-Kemptide from its phosphorylated counterpart and quantification of the phosphorylation extent ratiometrically with a fluorescence imager was worked out. The assay was successfully applied for the characterization of the inhibitory properties of inhibitors of cAMP-dependent protein kinase (PKA). The fluorescent probe ARC-583 was developed and its bisubstrate character demonstrated. The probe was applied for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of PKs in an assay with fluorescence polarization readout. High affinity of ARC-583 (KD = 0.48 nM towards PKA) enabled its application for the characterization of inhibitors with nanomolar and micromolar potency. The effect of structural elements of ARC-Photo probes on the cellular internalization efficiency and intracellular localization was studied. It was demonstrated that the cellular uptake and localization of the labelled inhibitors were influenced by several factors such as the number of arginine residues in the peptide moiety, the concentration of extracellularly applied compounds and the origin of the fluorescent dye of the ARC-Photo probe. The ability ARC-Photo probes to bind with high affinity to the free catalytic subunit of PKA was used to construct a cellular sensor for monitoring PKA activity in living cells. Väitekirja elektrooniline versioon ei sisalda publikatsioone.
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