Selective Phosphorylation Inhibitor of Delta Protein Kinase C–Pyruvate Dehydrogenase Kinase Protein–Protein Interactions: Application for Myocardial Injury <i>in Vivo</i>

Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK’s docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an <i>in vitro</i> <i>K</i>_d of ∼50 nM and reducing cardiac injury IC₅₀ of ∼5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors

Additional file 1: of Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites

Phylogenetic Tree of the 20 species for ALDH2 and εPKC coevolution comparison. Species in green letters are those with a homology of εPKC. Species in red letters are those without a homology of εPKC. (PDF 73 kb

Additional file 2: of Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites

The table shows the 10 species with εPKC (left panel), the 10 species without εPKC (right panel) and their amino acid residues at the three human ALDH2 phosphorylation sites, T185, S279 and T412. (PDF 313 kb

Impaired protein quality control in left ventricular remodeling and heart failure in humans.

<p><b>A</b>. ATP-dependent and -independent proteasomal activity, <b>B</b>. oxidized protein levels (determined by Western blot) and <b>C</b>. polyubiquitinated protein levels (determined by Western blot) in biopsied hearts from aortic stenosis-induced left ventricular remodeling (LVR) patients (green bars), ischemic cardiomyopathy-induced heart failure patients (red bars) and autopsied non-failing human hearts (white bars). <b>D</b>. Negative correlation between proteasomal function and oxidized protein accumulation in failing (aortic stenosis-LVR and ischemic-HF) and non-failing heart samples. <b>E</b>. Total PKC levels in failing hearts compared to non-failing hearts and <b>F</b>. Representative blots of PKCβII and PKCα proteins in total and Triton-soluble fraction (particulate fraction) in biopsied hearts from aortic stenosis-induced left ventricular remodeling patients (n = 6, green bars) and ischemic cardiomyopathy-induced heart failure patients (n = 3, red bars) compared to autopsied non-failing human hearts (n = 6, trace). Total and Triton-soluble fractions were normalized against GAPDH and Gαo, respectively. Error bars indicate SEM. *, p<0.05 compared to control (non-failing heart). §, p<0.05 compared to aortic stenosis-LVR patients.</p

Sustained PKCβII inhibition re-establishes protein quality control and improves cardiac function in myocardial infarction-induced model of heart failure.

<p><b>A</b>. Schematic panel of PKCβII treatment in the post-MI heart failure model, representative blots of PKCβII total level and translocation to particulate fraction, and PKCβII activity from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group). <b>B</b>. 20S proteasome subunits (α5/7, β1, β5 and β7) were precipitated from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group), and then probed with PKCβII, PKCε and anti-serine and threonine phosphorylation antibodies. Equal sample loading was verified using α5/7, β1, β5 and β7 proteasome subunits antibody. <b>C</b>. ATP-dependent and -independent cardiac proteasomal activity. <b>D</b>. Representative blots of proteasome 20S, α-β-crystallin, HSP27, caspase-3, cleaved caspase-3 and GAPDH in heart samples from 22 week-old rats (10 wks after MI surgery) (n = 6 per group). Data quantification and statistical details are in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033175#pone-0033175-g004" target="_blank">Fig. 4</a>. <b>E</b>. Oxidized protein levels, <b>F</b>. polyubiquitinated protein levels and <b>G</b>. soluble oligomer accumulation in heart samples from control (sham, white bars), TAT-treated (gray bars) and βIIV5-3-treated (green bars) heart failure rats as determined by Western blot (E, F) and slot-blot analysis (G). <b>H</b>. Average fractional shortening data from each group at 16 weeks and 22 weeks. All biochemical analyses were performed in the ventricular remote area. Error bars indicate SEM. *, p<0.05 compared to control (sham) rats. §, p<0.05 compared to βIIV5-3-treated heart failure rats.</p

Scheme depicting a possible mechanism of PKCβII-mediated PQC disruption during heart failure establishment.

<p>Scheme depicting a possible mechanism of PKCβII-mediated PQC disruption during heart failure establishment.</p

Development of Selective Inhibitors for Aldehyde Dehydrogenases Based on Substituted Indole-2,3-diones

Aldehyde dehydrogenases (ALDH) participate in multiple metabolic pathways and have been indicated to play a role in several cancerous disease states. Our laboratory is interested in developing novel and selective ALDH inhibitors. We looked to further work recently published by developing a class of isoenzyme-selective inhibitors using similar indole-2,3-diones that exhibit differential inhibition of ALDH1A1, ALDH2, and ALDH3A1. Kinetic and X-ray crystallography data suggest that these inhibitors are competitive against aldehyde binding, forming direct interactions with active-site cysteine residues. The selectivity is precise in that these compounds appear to interact directly with the catalytic nucleophile, Cys243, in ALDH3A1 but not in ALDH2. In ALDH2, the 3-keto group is surrounded by the adjacent Cys301/303. Surprisingly, the orientation of the interaction changes depending on the nature of the substitutions on the basic indole ring structure and correlates well with the observed structure–activity relationships for each ALDH isoenzyme

PKCβII activation inhibits proteasomal activity <i>in vitro</i> and disrupts protein quality control in cultured neonatal cardiomyocytes.

<p><b>A</b>. Proteolytic activity (upper panel) and phosphorylation of purified proteasome 20S (middle and lower panels) by different recombinant PKC isozymes (n = 3 per group). Purified 20S proteasome (1 ug) was individually incubated with recombinant PKCα, PKCβI, PKCβII or PKCε (50 ng) at 37°C for 30 minutes. Proteasome phosphorylation was evaluated using serine/threonine phosphorylation antibody (1∶1000) and [γ³²P] ATP incorporation. Histone phosphorylation was used to check the effectiveness of different PKC isozymes. Error bars indicate SEM. *, p<0.05 compared to other groups. <b>B</b>. Oxidized protein levels (upper panel) and ATP-dependent proteasomal activity (lower panel) in cultured neonatal cardiomyocytes. Cells were stimulated with PMA (a non-specific PKC activator) and the effect of PKC isozyme-specific peptide inhibitors (αV5-3, an αPKC-specific inhibitor; βIV5-3, a βIPKC-specific inhibitor; βIIV5-3, a βIIPKC-specific inhibitor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033175#pone.0033175-Stebbins1" target="_blank">[16]</a>; and εV1-2, an εPKC-specific inhibitor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033175#pone.0033175-Gray1" target="_blank">[46]</a>; or epoxomicin were determined. Error bars indicate SEM. *, p<0.05 compared to control-TAT and βIIV5-3-treated groups. Representative blots of oxidized proteins and GAPDH. Oxidized proteins were normalized against GAPDH. <b>C</b>. Oxidized protein levels (upper panel) and ATP-dependent proteasomal activity (lower panel) in cultured neonatal cardiomyocytes. Cultured neonatal cardiomyocytes were PKCβ or PKCα down-regulated (siRNA) plus/minus βIIV5-3 and challenged with phorbol ester (PMA). NC, negative control for siRNA. Representative blots of PKCβII and PKCα showed siRNA effectiveness. Oxidized proteins were normalized against GAPDH. Error bars indicate SEM. *, p<0.05 compared to control (non-treated cells), PKCβ down-regulated treated cells and PKCα down-regulated treated cells plus βIIV5-3.</p

Protein quality control disruption and PKCβII activation during progression of heart failure in a post-myocardial infarction (MI) model of HF in rats.

<p><b>A</b>. MI-induced HF protocol (10 weeks follow up) and example of heart morphology and echo data before (left) and 4 weeks after myocardial infarction (right) in rats. <b>B</b>. Cardiac fractional shortening, <b>C</b>. Oxidized protein levels, <b>D</b>. Soluble oligomer level and <b>E</b>. Proteasomal activity during the progression of cardiac dysfunction induced by myocardial infarction. *, p<0.05 compared to week 0 (before surgery). §, p<0.05 compared to week 1 after surgery. ‡, p<0.05 compared to week 2 after surgery. <b>F</b>. Concordance between fractional shortening and proteasomal activity, and <b>G</b>. Concordance between accumulation of misfolded cardiac proteins and oxidized proteins at weeks 0, 1, 2, 4 and 10 after myocardial infarction surgery in rats, <b>H</b>. Cardiac fractional shortening, proteasomal activity, oxidized and misfolded protein levels during the progression of cardiac dysfunction induced by myocardial infarction. <b>I</b>. The levels of total PKC isozymes (white bars) and translocated active PKCs (gray bars; Triton-soluble proteins or the particulate fraction/total fraction) at 10 weeks after myocardial infarction relative to control, age-matched rats (n = 6). Total proteins and Triton-soluble proteins of the particulate fraction were normalized against GAPDH and Gαo, respectively. Representative blots are of PKCβII total level and translocation to particulate fraction. All biochemical analyses were performed in the ventricular remote area. Error bars indicate s.e.m. *, p<0.05 compared to control-TAT. <b>J</b>. PKCβII activity at 10 weeks after myocardial infarction relative to control age-matched rats (n = 3). *, p<0.05 compared to control.</p

PKCβII inhibition repairs protein quality control in hypertension-induced heart failure model in rats.

<p><b>A</b>. Schematic panel of sustained PKCβI, βII or ε inhibition in hypertension-induced model of heart failure in rats. Representative blots of PKCβII total level and translocation to particulate fraction. <b>B</b>. ATP-dependent and -independent cardiac proteasomal activity, <b>C</b>. Oxidized proteins (as determined by Western blot) and <b>D</b>. cardiac soluble oligomer accumulation (as determined by slot blot) in heart samples from 17 week-old normotensive rats (white bar), TAT-treated (gray bar), βIV5-3-treated (gray bar), βIIV5-3-treated (green bar) and εV1-2-treated (gray bar) hypertensive rats. <b>E</b>. Average fractional shortening data from each group at the age of 17 weeks-old. <b>F</b>. βIIV5-3 improved survival of rats with hypertension-induced heart failure. Error bars indicate SEM. *, p<0.05 compared to control (sham) rats. §, p<0.05 compared to βIIV5-3-treated heart failure rats. Data from Fig. b–d were analyzed by one-way analysis of variance (ANOVA) with <i>post-hoc</i> testing by Tukey. Survival was analyzed by the standard Kaplan-Meier analysis with log-rank test.</p