Evo312: An Evodiamine Analog and Novel PKCßI Inhibitor with Potent Antitumor Activity in Gemcitabine-Resistant Pancreatic Cancer.

As an obstinate cancer pancreatic cancer (PC) poses a major challenge due to limited treatment options which include resection surgery, radiation therapy, and gemcitabine-based chemotherapy. In cancer cells, protein kinase C ßI (PKCßI) participates in diverse cellular processes, including cell proliferation, invasion, and apoptotic pathways. In the present study, we created a scaffold to develop PKCßI inhibitors using evodiamine-based synthetic molecules. Among the candidate inhibitors, Evo312 exhibited the highest antiproliferative efficacy against PC cells, PANC-1, and acquired gemcitabine-resistant PC cells, PANC-GR. Additionally, Evo312 robustly inhibited PKCßI activity. Mechanistically, Evo312 effectively suppressed the upregulation of PKCßI protein expression, leading to the induction of cell cycle arrest and apoptosis in PANC-GR cells. Furthermore, Evo312 exerted an antitumor activity in a PANC-GR cell-implanted xenograft mouse model. These findings position Evo312 as a promising lead compound for overcoming gemcitabine resistance in PC through novel mechanisms.

Selective inhibition of PKCß2 improves Caveolin-3/eNOS signaling and attenuates lipopolysaccharide-induced injury by inhibiting autophagy in H9C2 cardiomyocytes.

Lipopolysaccharide (LPS)-induced autophagy is involved in sepsis-associated myocardial injury with increased PKCß2 activation. We previously found hyperglycemia-induced PKCß2 activation impaired the expression of caveolin-3 (Cav-3), the dominant isoform to form cardiomyocytes caveolae which modulate eNOS signaling to confer cardioprotection in diabetes. However, little is known about the roles of PKCß2 in autophagy and Cav-3/eNOS signaling in cardiomyocytes during LPS exposure. We hypothesize LPS-induced PKCß2 activation promotes autophagy and impairs Cav-3/eNOS signaling in LPS-treated cardiomyocytes. H9C2 cardiomyocytes were treated with LPS (10 µg/mL) in the presence or absence of PKCß2 inhibitor CGP53353 (CGP, 1 µM) or autophagy inhibitor 3-methyladenine (3-MA, 10 µM). LPS stimulation induced cytotoxicity overtime in H9C2 cardiomyocytes, accompanied with excessive PKCß2 activation. Selective inhibition of PKCß2 with CGP significantly reduced LPS-induced cytotoxicity and autophagy (measured by LC-3II, Beclin-1, p62 and autophagic flux). In addition, CGP significantly attenuated LPS-induced oxidative injury, and improved Cav-3 expression and eNOS activation, similar effects were shown by the treatment of autophagy inhibitor 3-MA. LPS-induced myocardial injury is associated with excessive PKCß2 activation, which contributes to elevated autophagy and impaired Cav-3/eNOS signaling. Selective inhibition of PKCß2 improves Cav-3/eNOS signaling and attenuates LPS-induced injury through inhibiting autophagy in H9C2 cardiomyocytes.

All-trans retinoic acid and protein kinase C α/ß1 inhibitor combined treatment targets cancer stem cells and impairs breast tumor progression.

Breast cancer is the leading cause of cancer death among women worldwide. Blocking a single signaling pathway is often an ineffective therapy, especially in the case of aggressive or drug-resistant tumors. Since we have previously described the mechanism involved in the crosstalk between Retinoic Acid system and protein kinase C (PKC) pathway, the rationale of our study was to evaluate the effect of combining all-trans-retinoic acid (ATRA) with a classical PCK inhibitor (Gö6976) in preclinical settings. Employing hormone-independent mammary cancer models, Gö6976 and ATRA combined treatment induced a synergistic reduction in proliferative potential that correlated with an increased apoptosis and RARs modulation towards an anti-oncogenic profile. Combined treatment also impairs growth, self-renewal and clonogenicity potential of cancer stem cells and reduced tumor growth, metastatic spread and cancer stem cells frequency in vivo. An in-silico analysis of "Kaplan-Meier plotter" database indicated that low PKCα together with high RARα mRNA expression is a favorable prognosis factor for hormone-independent breast cancer patients. Here we demonstrate that a classical PKC inhibitor potentiates ATRA antitumor effects also targeting cancer stem cells growth, self-renewal and frequency.

PKC-ß/Alox5 axis activation promotes Bcr-Abl-independent TKI-resistance in chronic myeloid leukemia.

Bcr-Abl independent resistance to tyrosine kinase inhibitor (TKI) is a crucial factor lead to relapse or acute leukemia transformation in chronic myeloid leukemia (CML). However, its mechanism is still unclear. Herein, we found that of nine common protein kinases C (PKCs), PKC-ß overexpression was significantly related with TKI resistance. Blockage of its expression in CD34+ cells and CML cell lines increased sensitivity to imatinib. Then, eighty-four leukemia related genes were compared between TKI-resistant CML cell lines with PKC-ß silenced or not. Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that Arachidonate 5-lipoxygenase (Alox5) and its relative pathway mainly participated in the resistance induced by PKC-ß overexpression. It's also observed that Alox5 was increased not only in bone marrow biopsy but also in CD34+ cells derived from IM-resistant CML patients. The signaling pathway exploration indicated that ERK1/2 pathway mediates Alox5 upregulation by PKC-ß. Meanwhile, we also proved that Alox5 induces TKI-insensitivity in CML through inactivation of PTEN. In vivo experiment, PKC-ß elective inhibitor LY333531 prolonged survival time in CML-PDX mice model. In conclusion, targeted on PKC-ß overexpression might be a novel therapy mechanism to overcome TKI-resistance in CML.

Curcumin reverses diabetic nephropathy in streptozotocin-induced diabetes in rats by inhibition of PKCß/p66Shc axis and activation of FOXO-3a.

This study investigated if the nephroprotective effect of Curcumin in streptozotocin-induced type 1 diabetes mellitus (DM) in rats involves downregulation/inhibition of p66Shc and examined the underlying mechanisms. Rats were divided into 4 groups (n = 12/group) as control, control + Curcumin (100 mg/kg), T1DM, and T1DM + Curcumin. Curcumin was administered orally to control or diabetic rats for 12 weeks daily. As compared to diabetic rats, Curcumin didn't affect either plasma glucose or insulin levels but significantly reduced serum levels of urea, blood urea nitrogen, and creatinine, and concurrently reduced albumin/protein urea and increased creatinine clearance. It also prevented the damage in renal tubules and mitochondria, mesangial cell expansion, the thickness of the basement membrane. Mechanistically, Curcumin reduced mRNA and protein levels of collagen I/III and transforming growth factor- ß-1 (TGF-ß1), reduced inflammatory cytokines levels, improved markers of mitochondrial function, and suppressed the release of cytochrome-c and the activation of caspase-3. In the kidneys of both control and diabetic rats, Curcumin reduced the levels of reactive oxygen species (ROS), increased mRNA levels of manganese superoxide dismutase (MnSOD) and gamma-glutamyl ligase, increased glutathione (GSH) and protein levels of Bcl-2 and MnSOD, and increased the nuclear levels of nuclear factor2 (Nrf2) and FOXO-3a. Besides, Curcumin reduced the nuclear activity of the nuclear factor-kappa B (NF-κB), downregulated protein kinase CßII (PKCßII), NADPH oxidase, and p66Shc, and decreased the activation of p66Shc. In conclusion, Curcumin prevents kidney damage in diabetic rats by activating Nrf2, inhibiting Nf-κB, suppressing NADPH oxidase, and downregulating/inhibiting PKCßII/p66Shc axis.

Coronavirus disease 2019 (COVID-19), human erythrocytes and the PKC-alpha/-beta inhibitor chelerythrine -possible therapeutic implication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. Until now, diverse drugs have been used for the treatment of COVID-19. These drugs are associated with severe side effects, e.g. induction of erythrocyte death, named eryptosis. This massively affects the oxygen (O2) supply of the organism. Therefore, three elementary aspects should be considered simultaneously: (1) a potential drug should directly attack the virus, (2) eliminate virus-infected host cells and (3) preserve erythrocyte survival and functionality. It is known that PKC-α inhibition enhances the vitality of human erythrocytes, while it dose-dependently activates the apoptosis machinery in nucleated cells. Thus, the use of chelerythrine as a specific PKC-alpha and -beta (PKC-α/-ß) inhibitor should be a promising approach to treat people infected with SARS-CoV-2.

Antiproliferative effects of chalcones on T cell acute lymphoblastic leukemia-derived cells: Role of PKCß.

In this study, a series of 20 chalcone derivatives was synthesized, and their antiproliferative activity was tested against the human T cell acute lymphoblastic leukemia-derived cell line, CCRF-CEM. On the basis of the structural features of the most active compounds, a new library of chalcone derivatives, according to the structure-activity relationship design, was synthesized, and their antiproliferative activity was tested against the same cancer cell line. Furthermore, four of these derivatives (compounds 3, 4, 8, 28), based on lower IC50 values (between 6.1 and 8.9 µM), were selected for further investigation regarding the modulation of the protein expression of RACK1 (receptor for activated C kinase), protein kinase C (PKC)α and PKCß, and their action on the cell cycle level. The cell cycle analysis indicated a block in the G0/G1 phase for all four compounds, with a statistically significant decrease in the percentage of cells in the S phase, with no indication of apoptosis (sub-G0/G1 phase). Compounds 4 and 8 showed a statistically significant reduction in the expression of PKCα and an increase in PKCß, which together with the demonstration of an antiproliferative role of PKCß, as assessed by treating cells with a selective PKCß activator, indicated that the observed antiproliferative effect is likely to be mediated through PKCß induction.

Selective Inhibition of PKCß2 Restores Ischemic Postconditioning-Mediated Cardioprotection by Modulating Autophagy in Diabetic Rats.

Diabetic hearts are more susceptible to myocardial ischemia/reperfusion (I/R) injury and less sensitive to ischemic postconditioning (IPostC), but the underlying mechanisms remain unclear. PKCß2 is preferentially overactivated in diabetic myocardium, in which autophagy status is abnormal. This study determined whether hyperglycemia-induced PKCß2 activation resulted in autophagy abnormality and compromised IPostC cardioprotection in diabetes. We found that diabetic rats showed higher cardiac PKCß2 activation and lower autophagy than control at baseline. However, myocardial I/R further increased PKCß2 activation and promoted autophagy status in diabetic rats. IPostC significantly attenuated postischemic infarct size and CK-MB, accompanied with decreased PKCß2 activation and autophagy in control but not in diabetic rats. Pretreatment with CGP53353, a selective inhibitor of PKCß2, attenuated myocardial I/R-induced infarction and autophagy and restored IPostC-mediated cardioprotection in diabetes. Similarly, CGP53353 could restore hypoxic postconditioning (HPostC) protection against hypoxia reoxygenation- (HR-) induced injury evidenced by decreased LDH release and JC-1 monomeric cells and increased cell viability. These beneficial effects of CGP53353 were reversed by autophagy inducer rapamycin, but could be mimicked by autophagy inhibitor 3-MA. It is concluded that selective inhibition of PKCß2 could attenuate myocardial I/R injury and restore IPostC-mediated cardioprotection possibly through modulating autophagy in diabetes.

PKCß and reactive oxygen species mediate enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia in neonatal rats.

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCß and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCß and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCß inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCß-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCß in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCß activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCß and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.

PKCßII specifically regulates KCNQ1/KCNE1 channel membrane localization.

The slow voltage-gated potassium channel (IKs) is composed of the KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Activation of protein kinase C (PKC) has been linked to cardiac arrhythmias. Although PKC has been shown to be a regulator of a number of cardiac channels, including IKs, little is known about regulation of the channel by specific isoforms of PKC. Here we studied the role of different PKC isoforms on IKs channel membrane localization and function. Our studies focused on PKC isoforms that translocate to the plasma membrane in response to Gq-coupled receptor (GqPCR) stimulation: PKCα, PKCßI, PKCßII and PKCε. Prolonged stimulation of GqPCRs has been shown to decrease IKs membrane expression, but the specific role of each PKC isoform is unclear. Here we show that stimulation of calcium-dependent isoforms of PKC (cPKC) but not PKCε mimic receptor activation. In addition, we show that general PKCß (LY-333531) and PKCßII inhibitors but not PKCα or PKCßI inhibitors blocked the effect of cPKC on the KCNQ1/KCNE1 channel. PKCß inhibitors also blocked GqPCR-mediated decrease in channel membrane expression in cardiomyocytes. Direct activation of PKCßII using constitutively active PKCßII construct mimicked agonist-induced decrease in membrane expression and channel function, while dominant negative PKCßII showed no effect. This suggests that the KCNQ1/KCNE1 channel was not regulated by basal levels of PKCßII activity. Our results indicate that PKCßII is a specific regulator of IKs membrane localization. PKCßII expression and activation are strongly increased in many disease states, including heart disease and diabetes. Thus, our results suggest that PKCßII inhibition may protect against acquired QT prolongation associated with heart disease.

Variegatic acid from the edible mushroom Tylopilus ballouii inhibits TNF-α production and PKCß1 activity in leukemia cells.

Variegatic acid, isolated from Tylopilus ballouii dry fruiting bodies, is an inhibitor of ß-hexosaminidase release and tumor necrosis factor (TNF)-α secretion from rat basophilic leukemia (RBL-2H3) cells, with IC50 values of 10.4 µM and 16.8 µM, respectively. On the other hand, it inhibits PKCß1 activity with an IC50 value of 36.2 µM.

Targetable cellular signaling events mediate vascular pathology in vascular Ehlers-Danlos syndrome.

Vascular Ehlers-Danlos syndrome (vEDS) is an autosomal-dominant connective tissue disorder caused by heterozygous mutations in the COL3A1 gene, which encodes the pro-α 1 chain of collagen III. Loss of structural integrity of the extracellular matrix is believed to drive the signs and symptoms of this condition, including spontaneous arterial dissection and/or rupture, the major cause of mortality. We created 2 mouse models of vEDS that carry heterozygous mutations in Col3a1 that encode glycine substitutions analogous to those found in patients, and we showed that signaling abnormalities in the PLC/IP3/PKC/ERK pathway (phospholipase C/inositol 1,4,5-triphosphate/protein kinase C/extracellular signal-regulated kinase) are major mediators of vascular pathology. Treatment with pharmacologic inhibitors of ERK1/2 or PKCß prevented death due to spontaneous aortic rupture. Additionally, we found that pregnancy- and puberty-associated accentuation of vascular risk, also seen in vEDS patients, was rescued by attenuation of oxytocin and androgen signaling, respectively. Taken together, our results provide evidence that targetable signaling abnormalities contribute to the pathogenesis of vEDS, highlighting unanticipated therapeutic opportunities.

Molecular mechanisms of hydrogen sulfide against uremic accelerated atherosclerosis through cPKCßII/Akt signal pathway.

BACKGROUND: Cardiovascular disease is the most common complication and leading cause of death in maintenance hemodialysis patients. The protection mechanism of hydrogen sulfide (H2S) and the specific role of conventional protein kinase C ßII (cPKCßII)/Akt signaling pathway in the formation of atherosclerosis is still controversial. METHODS: 8-week-old male ApoE-/- mice were treated with 5/6 nephrectomy and high-fat diet to make uremia accelerated atherosclerosis (UAAS) model. Mice were divided into normal control group (control group), sham operation group (sham group), UAAS group, L-cysteine group (UAAS+L-cys group), sodium hydrosulfide group (UAAS+NaHS group), and propargylglycine group (UAAS+PPG group). Western blot was used to detect cPKCßII activation, Akt phosphorylation and endothelial nitric oxide synthase (eNOS) expression in mice aorta. RESULTS: The membrane translocation of cPKCßII in UAAS group was higher than sham group, and L-cys or NaHS injection could suppress the membrane translocation, but PPG treatment resulted in more membrane translocation of cPKCßII (P < 0.05, n = 6 per group). Akt phosphorylation and the eNOS expression in UAAS group was lower than sham group, and L-cys or NaHS injection could suppress the degradation of Akt phosphorylation and the eNOS expression, but PPG treatment resulted in more decrease in the Akt phosphorylation and the eNOS expression (P < 0.05, n = 6 per group). CONCLUSION: Endogenous cystathionine-γ-lyase (CSE)/H2S system protected against the formation of UAAS via cPKCßII/Akt signal pathway. The imbalance of CSE/H2S system may participate in the formation of UAAS by affecting the expression of downstream molecule eNOS, which may be mediated by cPKCßII/Akt signaling pathway.

Toxoplasma gondii induces prolonged host epidermal growth factor receptor signalling to prevent parasite elimination by autophagy: Perspectives for in vivo control of the parasite.

Toxoplasma gondii causes retinitis and encephalitis. Avoiding targeting by autophagosomes is key for its survival because T. gondii cannot withstand lysosomal degradation. During invasion of host cells, T. gondii triggers epidermal growth factor receptor (EGFR) signalling enabling the parasite to avoid initial autophagic targeting. However, autophagy is a constitutive process indicating that the parasite may also use a strategy operative beyond invasion to maintain blockade of autophagic targeting. Finding that such a strategy exists would be important because it could lead to inhibition of host cell signalling as a novel approach to kill the parasite in previously infected cells and treat toxoplasmosis. We report that T. gondii induced prolonged EGFR autophosphorylation. This effect was mediated by PKCα/PKCß âž” Src because T. gondii caused prolonged activation of these molecules and their knockdown or incubation with inhibitors of PKCα/PKCß or Src after host cell invasion impaired sustained EGFR autophosphorylation. Addition of EGFR tyrosine kinase inhibitor (TKI) to previously infected cells led to parasite entrapment by LC3 and LAMP-1 and pathogen killing dependent on the autophagy proteins ULK1 and Beclin 1 as well as lysosomal enzymes. Administration of gefitinib (EGFR TKI) to mice with ocular and cerebral toxoplasmosis resulted in disease control that was dependent on Beclin 1. Thus, T. gondii promotes its survival through sustained EGFR signalling driven by PKCα/ß âž” Src, and inhibition of EGFR controls pre-established toxoplasmosis.

A novel form of glycolytic metabolism-dependent cardioprotection revealed by PKCα and ß inhibition.

KEY POINTS: Acute hyperglycaemia at the time of a heart attack worsens the outcome for the patient. Acute hyperglycaemia is not limited to diabetic patients and can be due to a stress response in non-diabetics. This study suggests that the damaging cardiac effects of hyperglycaemia can be reversed by selective PKC inhibition. If PKCα/ß isoforms are inhibited, then high glucose itself becomes protective against ischaemic damage. Selective PKC inhibition may therefore be a useful therapeutic tool to limit the damage that can occur during a heart attack by stress-induced hyperglycaemia. ABSTRACT: Hyperglycaemia has a powerful association with adverse prognosis for patients with acute coronary syndromes (ACS). Previous work shows that high glucose prevents ischaemic preconditioning and causes electrical and mechanical disruption via protein kinase C α/ß (PKCα/ß) activation. The present study aimed to: (i) determine whether the adverse clinical association of hyperglycaemia in ACS can be replicated in preclinical cellular models of ACS and (ii) determine the importance of PKCα/ß activation to the deleterious effect of glucose. Freshly isolated rat, guinea pig or rabbit cardiomyocytes were exposed to simulated ischaemia after incubation in the presence of normal (5 mm) or high (20 mm) glucose in the absence or presence of small molecule or tat-peptide-linked PKCαß inhibitors. In each of the four conditions, the following hallmarks of cardioprotection were recorded using electrophysiology or fluorescence imaging: cardiomyocyte contraction and survival, action potential stability and time to failure, intracellular calcium and ATP, mitochondrial depolarization, ischaemia-sensitive leak current, and time to Kir 6.2 opening. High glucose alone resulted in decreased cardiomyocyte contraction and survival; however, it also imparted cardioprotection in the presence of PKCα/ß inhibitors. This cardioprotective phenotype displayed improvements in all of the measured parameters and decreased myocardium damage during whole heart coronary ligation experiments. High glucose is deleterious to cellular and whole-heart models of simulated ischaemia, in keeping with the clinical association of hyperglycaemia with an adverse outcome in ACS. PKCαß inhibition revealed high glucose to show a cardioprotective phenotype in this setting. The results of the present study suggest the potential for the therapeutic application of PKCαß inhibition in ACS associated with hyperglycaemia.

The protein kinase Cß-selective inhibitor, enzastaurin, attenuates amphetamine-stimulated locomotor activity and self-administration behaviors in rats.

RATIONALE: Pathological amphetamine (AMPH) use is a serious public health concern with no pharmacological treatment options. Protein kinase Cß (PKCß) has been implicated in the mechanism of action of AMPH, such that inhibition of PKCß attenuates AMPH-stimulated dopamine efflux in vivo. With this in mind, inhibition of PKCß may be a viable therapeutic target for AMPH use disorder. OBJECTIVE: The purpose of this study is to demonstrate that selective pharmacological inhibition of PKCß alters AMPH-stimulated behaviors in rats. METHODS: Rats were administered intracerebroventricular (i.c.v.) injections of the PKCß-selective inhibitor enzastaurin 0.5, 3, 6, or 18 h before evaluating AMPH-stimulated locomotion (0.32-3.2 mg/kg). Rats were trained to make responses for different doses of AMPH infusions or sucrose under a fixed ratio 5 schedule of reinforcement, and the effects of enzastaurin pretreatment 3 or 18 h prior to a self-administration session were determined. Also, the effect of enzastaurin on AMPH-stimulated PKC activity in the ventral striatum was evaluated. RESULTS: A large dose of enzastaurin (1 nmol) decreased AMPH-stimulated locomotor activity 0.5 h following enzastaurin administration. Small doses of enzastaurin (10-30 pmol) attenuated AMPH-stimulated locomotor activity and shifted the AMPH dose-effect curve to the right following an 18-h pretreatment. Rats pretreated with enzastaurin 18 h, but not 3, prior to a self-administration session showed a decrease in the number of responses for AMPH, shifted the ascending limb of the amphetamine dose effect curve, and produced no change in responses for sucrose. AMPH-stimulated PKC activity was decreased following a 0.5- or 18-h pretreatment, but not a 3-h pretreatment of enzastaurin. CONCLUSIONS: These results demonstrate that inhibition of PKCß will decrease AMPH-stimulated behaviors and neurobiological changes and suggest that PKCß is potentially a viable target for AMPH use disorder.

Cross-species genetic screens to identify kinase targets for APP reduction in Alzheimer's disease.

An early hallmark of Alzheimer's disease is the accumulation of amyloid-ß (Aß), inspiring numerous therapeutic strategies targeting this peptide. An alternative approach is to destabilize the amyloid beta precursor protein (APP) from which Aß is derived. We interrogated innate pathways governing APP stability using a siRNA screen for modifiers whose own reduction diminished APP in human cell lines and transgenic Drosophila. As proof of principle, we validated PKCß-a known modifier identified by the screen-in an APP transgenic mouse model. PKCß was genetically targeted using a novel adeno-associated virus shuttle vector to deliver microRNA-adapted shRNA via intracranial injection. In vivo reduction of PKCß initially diminished APP and delayed plaque formation. Despite persistent PKCß suppression, the effect on APP and amyloid diminished over time. Our study advances this approach for mining druggable modifiers of disease-associated proteins, while cautioning that prolonged in vivo validation may be needed to reveal emergent limitations on efficacy.

A selective inhibitor of mitofusin 1-ßIIPKC association improves heart failure outcome in rats.

We previously demonstrated that beta II protein kinase C (ßIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that ßIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. ßIIPKC siRNA or a ßIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-ßIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMßA, a rationally-designed peptide that selectively antagonizes Mfn1-ßIIPKC association. SAMßA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMßA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMßA may be a potential treatment for patients with heart failure.

Synthesis and Characterization of the Selective, Reversible PKCß Inhibitor (9 S)-9-[(Dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, Ruboxistaurin (LY333531).

The demonstrated role of PKCß in  mediating amphetamine-stimulated dopamine efflux, which regulates amphetamine-induced dopamine transporter trafficking and activity, has promoted the research use of the selective, reversible PKCß inhibitor (9 S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, ruboxistaurin. Despite the interest in development of ruboxistaurin as the mesylate monohydrate (Arxxant) for the treatment of diabetic retinopathy, macular edema, and nephoropathy, several crucial details in physicochemical characterization were erroneous or missing. This report describes the synthesis and full characterization of ruboxistaurin free base (as a monohydrate), including X-ray crystallography to confirm the absolute configuration, and of the mesylate salt, isolated as a hydrate containing 1.5 mol of water per mole.

Glycine Protects H9C2 Cardiomyocytes from High Glucose- and Hypoxia/Reoxygenation-Induced Injury via Inhibiting PKCß2 Activation and Improving Mitochondrial Quality.

BACKGROUND: Patients with diabetes are more vulnerable to myocardial ischemia reperfusion injury (IRI), which is involved in PKCß2 activation and mitochondrial dysfunction. Glycine has been documented as a cytoprotective agent to attenuate diabetes-related abnormalities and reduce myocardial IRI, but the underlying mechanisms are still unclear. We determined whether glycine could attenuate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by inhibiting PKCß2 activation and improving mitochondrial quality in cultured H9C2 cells. METHODS: H9C2 cells were either exposed to low glucose (LG) or HG conditions with or without treatment of glycine or CGP53353 (a selective inhibitor of PKCß2) for 48 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). Cell viability, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using corresponding commercial kits. Mitochondrial quality control-related proteins (LC-3II, Mfn-2, and Cyt-C) and PKCß2 activation were detected by Western blot. RESULTS: HG stimulation significantly decreased cell viability and SOD activity and increased LDH release, MDA production, and PKCß2 activation as compared to LG group, all of which changes were further increased by H/R insult. Glycine or CGP53353 treatment significantly reduced the increase of LDH release, MDA production, PKCß2 activation, and Cyt-C expression and the decrease of cell viability, SOD activity, MMP, Mfn-2 expression, and LC-3II/LC-3I ratio induced by HG and H/R stimulation. CONCLUSIONS: Supplementary glycine protects H9C2 cells from HG- and H/R-induced cellular injury by suppressing PKCß2 activation and improving mitochondria quality.