PKCβ Inhibitor Research Articles

Role of Protein Kinase C in Metabolic Regulation of Coronary Endothelial Small Conductance Calcium-Activated Potassium Channels.

Small conductance calcium-activated potassium (SK) channels are largely responsible for endothelium-dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes-induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12-myristate 13-acetate, PKC inhibitors, or endothelial PKCα/PKCβ knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKCβ inhibitor LY333531. NADH (30-300 μmol/L) or PKC activator phorbol 12-myristate 13-acetate (30-300 nmol/L) reduced endothelial SK current density, whereas the selective PKCᵦ inhibitor LY333531 significantly reversed the NADH-induced SK channel inhibition. PKCβ small interfering RNA, but not PKCα small interfering RNA, significantly prevented the NADH- and phorbol 12-myristate 13-acetate-induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKCβ expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKCβ inhibitor LY333531. NADH-induced inhibition of endothelial SK channel function is mediated via PKCβ. These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.

The PKC-β Inhibitor MS-553 Displays Preclinical Efficacy in BTK Inhibitor Resistant Chronic Lymphocytic Leukemia

Background: Treatment of chronic lymphocytic leukemia (CLL) has been revolutionized by usage of targeted therapies against the B-cell receptor (BCR) signaling cascade, specifically Bruton's Tyrosine Kinase (BTK). However, resistance to BTK inhibition occurs through acquisition of various mutations in BTK or its immediate downstream signaling partner, PLCγ2. Clinical outcomes for these patients are poor, emphasizing the need for alternative therapeutics. In the presence of these mutations, BCR signaling remains intact, suggesting that targeting molecules downstream of BTK may be effective. Protein kinase C-β (PKCβ) is a downstream component of the BCR pathway that has been shown to be over-expressed in CLL. Moreover, PKCβ is essential to the development of CLL in the Eμ-TCL1 mouse model and its expression in stromal cells is required for the survival of leukemic B-cells (Holler et al. 2009). This suggests that signaling through PKCβ, in both CLL cells and cells in the microenvironment, is essential to promote leukemogenesis. MS-553 is a potent, ATP competitive, reversible inhibitor of multiple PKC isoforms including PKCβ, with the potential to overcome BTKi mediated resistance. Methods: Primary CLL cells were isolated by negative selection and treated with up to 5 μM MS-553. Cell viability was assessed via Annexin V/PI with and without HS-5 stromal cell co-culture. BCR and canonical WNT signaling changes were evaluated by change in target protein phosphorylation or total protein by immunoblot following drugging. Changes in downstream NFκB and WNT signaling were interrogated by RT-qPCR. Using genetically modified TMD8 cells that express only C481S or T474I mutated BTK, we tested MS-553's effect on cell-viability and BCR signaling. In vivo survival studies were performed using the Eμ-MTCP1 (Walker et al. 2021) adoptive transfer mouse model, an aggressive model of CLL, with subsequent randomization into vehicle or MS-553 treatment arms. Results: Following 72 hours of drugging, 5 μM MS-553 displayed modest cytotoxicity against primary CLL cells both alone (50%, p<0.0001, n=8) and in the presence of HS-5 stromal cells (25%, p=0.0008, n=8). In primary CLL cells harboring the C481S BTK mutation, we observed retained cytotoxicity following 72 hours of drugging (29%, p=0.00029, n=6). At 24 hours, 5 μM MS-553 inhibited downstream BCR and WNT signaling in primary CLL cells, demonstrated by reduced phosphorylation of PKC-β (43%, p=0.0024, n=15) and several of its downstream targets including pGSK3β (45%, p<0.0001, n=15), pIκBα (66%, p<0.0001, n=15), and pERK (17%, p=0.0301, n=15), and reduced total protein of β-Catenin (82%, p<0.0001, n=9), Cyclin D2 (71%, p=0.0011, n=9), and cMyc (85%, p<0.0001, n=9) compared to vehicle treated, stimulated samples. In both treatment naïve and C481S patient samples, mRNA expression of NFκB and WNT pathway targets BCL-2, OCT-2, BFL-1, MYC, CD40, Cyclin D1, and Cyclin D2 were all decreased after 48 hours of treatment with 1 or 5 μM MS-553. MS-553 also showed efficacy in the presence of BTK mutations: viability in TMD8 cells was reduced following 72-hour drugging with MS-553 regardless of BTK mutational status (WT: 22%, p=0.0006, n=3; C481S BTK: 18%, p=0.0032, n=3; T474I BTK: 19%, p=0.0015, n=3). Inflammatory cytokine production is inhibited by MS-553 through its ability to decrease CCL3 and CCL4 cytokine expression in WT and mutant BTK-harboring TMD8 cells (p<0.0001, n=3) (Figure 1A). To validate inhibition of BCR signaling in the C481S BTK setting, we treated primary CLL cells harboring this mutation and observed a reduction in both CCL3 and CCL4 production (61%, p=0.001229, n=5). As proof of concept, we examined MS-553's efficacy in vivo in the Eμ-MTCP1 mouse engraftment model, and found a significant reduction in disease progression and increase in survival (median 117 days) when compared to vehicle control (median 56 days, p=0.0003) (Figure 1B). Conclusion: Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL including that with BTK mutations conferring BTKi resistance. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) (Blachly et al. 2022).

Ginsenoside Rg1 attenuates diabetic vascular endothelial dysfunction by inhibiting the calpain-1/ROS/PKC-β axis

AimsVascular endothelial dysfunction (VED) is the onset event of cardiovascular complications in type 2 diabetes mellitus. Ginsenoside Rg1 (Rg1) can improve the cardiovascular system, but its mechanism in diabetic vascular endothelial dysfunction has received little attention. Main methodsMale calpain-1-knockout and wild-type C57BL/6 J mice were intraperitoneally injected with streptozotocin and treated with Rg1 (10 and 20 mg/kg) for 8 weeks. Human aortic endothelial cells (HAECs) were incubated with high glucose (HG) and were pretreated with Rg1 (10, 20 μM), MDL-28170 (calpain-1 inhibitor), LY-333531 (PKC-β inhibitor), NAC (ROS inhibitor) and calpain-1 overexpression. Then, factors related to mitochondrial dysfunction, oxidative stress and VED were measured. Key findingsThe administration of Rg1 and calpain-1 knockout ameliorated diabetic mitochondrial dysfunction, oxidative stress and VED and inhibited the calpain-1/ROS/PKC-β axis. LY-333531 and NAC treatment restored destructive endothelium-dependent vasodilation in mice with diabetes, while pyrogallol (ROS agonist), PMA (PKC-β agonist) or L-NAME (eNOS inhibitor) treatment abrogated the protective effect of Rg1 against diabetic endothelial dysfunction. The administration of Rg1, MDL-28170, LY-333531 and NAC improved mitochondrial dysfunction, oxidative stress and VED, whereas the overexpression of calpain-1 amplified mitochondrial dysfunction, oxidative stress and VED and further upregulated the expression of PKC-β in HAECs exposed to HG. Overexpression of calpain-1 abrogated the protective effect of Rg1 against HG-induced oxidative stress and VED. SignificanceThese findings reveal that Rg1 can protect against VED by suppressing the calpain-1/ROS/PKC-β axis and alleviating the development of mitochondrial dysfunction and oxidative stress.

Identification of protein kinase C beta as a therapeutic target for neuroendocrine prostate cancer and development of a nanoparticle-based therapeutic strategy

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of malignant prostate cancer for which there is no effective treatment. Although protein kinases are the most attractive therapeutic targets for cancer treatment, there are no clear kinase-targeted drugs available for NEPC treatment. Herein, we showed that protein kinase C beta (PKCβ) is highly expressed in NEPC and can be transcriptionally activated by c-Myc through the E-box elements present in its promoter region. Furthermore, PKCβ can activate PIM1 kinase by phosphorylating Ser51 and Ser54, which in turn stabilizes and enhances c-Myc function. Three formed a PIM1-c-Myc-PKCβ positive feedback loop to promote the progression of NEPC. Enzastaurin, a specific inhibitor of PKCβ, significantly ameliorated NEPC. However, enzastaurin causes thrombus and other side effects in several clinical trials of other diseases, which greatly limits its clinical application. To address these problems, we developed a functional polymer nanoparticle modified with SSTR2-targeted octreotide and a thrombin-binding aptamer to deliver enzastaurin for NEPC treatment. The tailor-designed nanomedicine efficiently retards tumor growth and the incidence of coagulation abnormalities in vitro and in vivo. Collectively, our findings highlight the key role of PKCβ in NEPC and provide a promising nanoparticle-based therapeutic strategy for the treatment of NEPC.

Initial Results from a Phase 1/2 Dose Escalation and Expansion Study Evaluating MS-553, a Novel and Selective PKCβ Inhibitor, in Patients with CLL/SLL

Introduction: Abnormal B-cell receptor (BCR) signaling is a key factor in the pathogenesis of CLL, including in patients who have developed resistance to Bruton tyrosine kinase (BTK) inhibitors. MS-553 is a potent, highly selective, oral, non-covalent inhibitor of protein kinase C (PKC) β, an essential signaling molecule immediately downstream of BTK and PLCγ2 in the BCR pathway. As PKCβ is downstream of both BTK and PLCγ2, inhibition of PKCβ has the potential to treat acquired resistance mutations from either protein (the most common mechanisms of BTKi resistance), unlike second generation BTKi. We report early results from the Phase 1/2 dose escalation and expansion study in patients with CLL/SLL treated with MS-553 (NCT03492125). Methods: The primary objective of the study is to evaluate the safety and tolerability of oral daily continuous MS-553 as monotherapy (Cohort A) in relapsed and refractory patients with CLL/SLL, and in combination with acalabrutinib (Cohort B), or with venetoclax plus an anti-CD20 antibody (Cohort C) in earlier lines of therapy. Secondary objectives include assessment of the pharmacokinetic profile, pharmacodynamic activity, and evidence of anti-tumor activity following MS-553 treatment. Dose expansion is underway in cohort A. Dose escalation to determine the recommended Phase 2 dose in the combination cohorts has begun using a conventional 3+3 design. Treatment emergent adverse events (TEAEs) were assessed per NCI CTCAE v.5.0. Tumor responses were evaluated according to IWCLL 2008 guidelines. Results: As of June 20, 2022, a total of 45 patients (median age 69; range 38-82) have been treated with at least one dose of therapy. The median number of prior therapies was 4 (range 1-12) for Cohort A (n= 37), 2 (range 0-2) for Cohort B (n=4) and 0.5 (range 0-2) for Cohort C (n=4). Overall, 72% were IGHV unmutated; 44% had del(17p) or TP53 mutations. A total of 76% had previously been treated with at least one or more BTKi. For Cohort A, 92% had previously received BTKi, 51% had received venetoclax, and 41% had received both BTKi and venetoclax. To date, one dose limiting toxicity (Grade 3 Acute Kidney Injury) has been observed in the highest dose group, but the MTD has not been reached. The majority of the drug-related TEAEs were grade 1 or 2. The most common (>10%) were nausea (36%); diarrhea (29%); fatigue (18%); vomiting, anemia, and abdominal pain (13% each); and anorexia (11%). Drug-related TEAEs ≥ grade 3 include neutropenia (n=2) and one case each of nausea, diarrhea, fatigue, weight loss, lymphocyte increase, hyponatremia, acute kidney injury, and generalized muscle weakness. No drug-related atrial fibrillation or bleeding has been reported. For efficacy evaluable patients in Cohort A, 50% (10/20) of patients exhibited a partial response and 50% stable disease with the median duration of therapy of 6.0 and 4.1 months respectively. In cohort B and C, all patients responded (B, 4/4 PRs; C, 2/3 PRs and 1/3 CR) and all remained on therapy at time of data cut. Of note, responses occurred in several ultra-high-risk patients such as those with BTK/PLCG2/TP53 triple mutations, with relapse after BTKi and venetoclax treatment, and having relapsed after both covalent and non-covalent BTKi. For the combination Cohorts B and C, all participants have shown partial or complete responses and are continuing therapy as of the data lock. While still in the dose escalation phase of the study, the median time on therapy for Cohort B is 5.6 months and for Cohort C is 6 months. Conclusion: MS-553 has been generally well tolerated with initial anti-tumor activity seen in this heavily pretreated population. Half of the evaluable patients have shown partial responses. The combination cohorts have shown good initial safety and tolerability. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MS-553, a Selective PKC-ß Inhibitor Inhibits BCR Signaling and Synergizes with Venetoclax for the Treatment of High Risk CLL

Inhibitors of Bruton's tyrosine kinase (BTK) have been transformative in treating patients with chronic lymphocytic leukemia (CLL) due to their ability to inhibit B-Cell Receptor (BCR) signaling. However, despite durable remissions in many patients, complete responses are uncommon and acquired mutations in BTK and its downstream target phospholipase Cγ2 (PLCγ2) lead to relapses and poor survival. In parallel, genomic high-risk features such as aberrations in TP53 represent an independent risk factor that favor progression on BTK inhibitors (BTKi). TP53 aberrations comprise of deletions in chromosome 17 (del17p) which result in the loss of one allele of TP53 and point mutations in the TP53 gene. CLL patients with del17p acquire clonal mutations that affect the other p53 allele in 82% of cases and largely express only mutant TP53 protein. Therefore, there is an urgent need to identify therapies for patients who progress on BTKi. We have observed over-expression of protein kinase C-β (PKC-β) in primary CLL patient samples including in those that have mutations in TP53. PKC-β is a downstream of the BCR and functions downstream of BTK. PKC-β is required for CLL cell survival and proliferation and therefore can be a potential target for patients with mutations in p53 that do not respond to other targeted therapies. We hypothesized that targeting PKC-β will inhibit BCR signaling downstream of BTK and may offer an effective therapeutic strategy for patients who relapse on ibrutinib. Mechanistically, we found that CLL cells (n=6 patients) in contact with stromal support upregulated their levels of PKC-β. Pre-exposure of stromal cells to MS-553, a selective PKC-β inhibitor prevented the stromal cell contact-induced increase in PKC-β within the CLL cells establishing that stromal contact was an important source of PKC-β in CLL cells. We have shown in primary CLL patient samples that exposure to MS-553 decreases activation of PKC-β and its downstream effectors GSK3-β and ERK in a dose-dependent manner regardless of mutations in p53 both in the presence and absence of stromal support. In parallel, MS-553 also decreased the levels of BCL-xL, a key pro-survival BCL2 family protein via downregulation of β-catenin. Using dynamic BH3 profiling we show that CLL samples (both WT and mutant TP53) exposed to MS-553 (5µM, 24h) increase their dependence on BCL-2 and BCL-xL for survival. This finding suggested that MS-553 could synergize with the BCL-2 inhibitor venetoclax to eliminate CLL cells. Correspondingly, cells exposed to a combination of MS-553 and venetoclax released more cytochrome c (Cyt c), used as a measure of mitochondrial dysfunction, compared to each single agent alone and led to a synergistic increase in apoptosis as measured by Annexin V positivity. These data provide strong rationale for an ongoing Phase I/II trial that evaluates MS-553 as a single agent and in combination with targeted agents such as venetoclax. Early interim data indicate that MS-553 is safe, tolerable and effective both as a single agent and in combination with venetoclax.

Pharmacophore modeling, docking and molecular dynamics simulation for identification of novel human protein kinase C beta (PKCβ) inhibitors.

Protein kinase Cβ (PKCβ) is considered as an attractive molecular target for the treatment of COVID-19-related acute respiratory distress syndrome (ARDS). Several classes of inhibitors have been already identified. In this article, we developed and validated ligand-based PKCβ pharmacophore models based on the chemical structures of the known inhibitors. The most accurate pharmacophore model, which correctly predicted more than 70% active compounds of test set, included three aromatic pharmacophore features without vectors, one hydrogen bond acceptor pharmacophore feature, one hydrophobic pharmacophore feature and 158 excluded volumes. This pharmacophore model was used for virtual screening of compound collection in order to identify novel potent PKCβ inhibitors. Also, molecular docking of compound collection was performed and 28 compounds which were selected simultaneously by two approaches as top-scored were proposed for further biological research. Supplementary InformationThe online version contains supplementary material available at 10.1007/s11224-022-02075-y.

Abstract P125: Long-term Exposure Of Angiotensin II Enhanced Vascular Contraction Via Activation Of Protein Kinase C Beta

Protein kinase C (PKC) β is linked to lymphoma and diabetes complications including diabetic retinopathy, nephropathy and neuropathy. Also, recent studies revealed that PKCβ activation also accelerates atherosclerotic plaque formation in diabetes. These findings strongly indicate that PKCβ is a potential target for the treatment of diabetic vascular complications. Although PKCβ is associated with diabetic vascular dysfunction, role of PKCβ in hypertension remains unclear. Since hypertension is one of most important risk factor for vascular diseases including atherosclerosis, we here examined whether PKCβ is involved in angiotensin II-induced vascular dysfunction. Treatment with angiotensin II (100 nM) for 30 min strongly increased phosphorylation at the PKCβ activation site at S660 in aorta from Wistar rat (n=4). Treatment of rat aorta with angiotensin II (100 nM) for 24 hr increased contractile responses to serotonin (5-HT, Maximum contraction, control vs. Angiotensin II, 92.2±20.0 vs 142.1±20.5%, P<0.05, n=6) or endothelin-1 (ET-1, 157.4±30.6 vs 214.1±24.3%, P<0.05, n=8). In contrast, angiotensin II did not affect vascular contractile response to noradrenaline (n=4). The enhanced contraction induced by angiotensin II was blocked by CGP53353 (1 μM), a PKCβ inhibitor (5-HT, Angiotensin II vs. CGP53353+Angiotensin II,197.8±27.8 vs. 167.4±17.6%, P<0.05, n=4-5). Pretreatment with LY333531 (1 μM), another PKCβ inhibitor, also prevented angiotensin II-induced vascular dysfunction (P<0.05, n=3-4). Next, we generated PKCβ knockout rat using rGONAD. Consistent with the results using PKCβ inhibitors, angiotensin II-induced enhanced contractile responses to 5-HT (122.6±15.6 vs 111.0±16.3%, n=4) and ET-1 (137.9±39.9 vs 139.2±30.1%, n=4) were dismissed in PKCβ knockout rat. We also confirmed that phorbol12-myristate13-acetate (0.05-5 μg/ml), a PKC activator, enhanced vascular contractile responses to 5-HT and ET-1 in dose-dependent manner (P<0.05, n=4). We conclude that long exposure of angiotensin II (24 hr) causes increase in vascular contractile responses to 5-HT and ET-1 and the augmented contraction are due to activation of PKCβ. Our result suggests that PKCβ might be associated with angiotensin II-related vascular dysfunction.

PKCβ Inhibition Promotes TXNIP Degradation to Ameliorate Pancreatic β-Cell Dysfunction

Introduction: Pancreatic β-cell dysfunction is largely regulated by TXNIP accumulation, we have previously disclosed the role of PKA in TXNIP degradation during β-cell dysfunction. However, whether other kinases (PKCs) still regulate TXNIP is unclear, which is beneficial to alleviate β-cell dysfunction. Methods: Thapsigargin (ER stress inducer) was used to induce β-cell dysfunction. PKC’s inhibitors were screened by Western blotting indicated by TXNIP. Also RT-qPCR and Co-immunoprecipitation were applied for evaluating the β-cell improvement ability of PKC’s inhibitors, and the insulin secretion ability was evaluated by glucose-stimulated insulin secretion assay. Results: PKC’s pan-inhibitor, Ro31-8220, decreased β-cell apoptosis and improved insulin secretion under ER stress or high glucose (HG) conditions. Further studies showed that Ro31-8220 reduced ER stress or HG-induced TXNIP levels. On the other side, PKCβ activation or overexpression could reverse the effect of Ro31-8220 on TXNIP. Also, PKCβ selective inhibitor, ruboxistaurin, induced TXNIP degradation as significantly as Ro31-8220 did. Conclusion: This study reveals the regulating mechanism of PKCβ inhibitor on TXNIP degradation to improve β-cell dysfunction. These data indicated PKCβ inhibitor is a promising agent for ameliorating β-cell dysfunction through TXNIP.

Protein kinase C-β distinctly regulates blood-brain barrier-forming capacity of Brain Microvascular endothelial cells and outgrowth endothelial cells

Outgrowth endothelial cells (OECs) provide an endogenous repair mechanism and thus maintain endothelial barrier integrity. As inhibition of protein kinase C-β (PKC-β) activity has been shown to attenuate endothelial damage in various pathological conditions including hyperglycaemia and ischaemic injury, the present study comparatively assessed the effect of LY333531, a PKC-β inhibitor, on the cerebral barrier integrity formed by OECs or human brain microvascular endothelial cells (HBMECs). To this end, an in vitro model of human BBB established by co-culture of astrocytes and pericytes with either OECs or HBMECs was exposed to 4 h of oxygen-glucose deprivation with/out LY333531 (0.05 µM). The inhibition of PKC-β protected the integrity and function of the BBB formed by HBMECs, as evidenced by increases in transendothelial electrical resistance and decreases in sodium fluorescein flux. It also attenuated ischaemia-evoked actin cytoskeleton remodelling, oxidative stress, and apoptosis in HBMECs. In contrast, treatments with LY333531 exacerbated the deleterious effect of ischaemia on the integrity and function of BBB formed by OECs while augmenting the levels of oxidative stress, apoptosis, and cytoskeletal reorganisation in OECs. Interestingly, the magnitude of damage in all aforementioned parameters, notably oxidative stress, was lower with low dose of LY333531 (0.01 µM). It is therefore possible that the therapeutic concentration of LY333531 (0.05 µM) may neutralise the activity of NADPH oxidase and thus trigger a negative feedback mechanism which in turn exacerbate the detrimental effects of ischaemic injury. In conclusion, targeting PKC-β signalling pathway in ischaemic settings requires close attention while using OECs as cellular therapeutic.

Preclinical Efficacy of INST3399, a Synthetic Novel PKCbeta Inhibitor, for the Treatment of Obesity and Fatty Liver Disease

ObjectivesThe prevalence of obesity has increased over the last few decades reaching epidemic proportions. Clearly, public health efforts and anti obesity agents presently in use are inadequate for curbing the obesity epidemic. Currently existing FDA-approved anti-obesity drugs are specific receptor-targeted drugs that focus on reducing energy intake. Energy intake reduction is equivalent to calorie-restricted dieting and is prone to weight cycling because it decreases muscle mass, leading to reduced metabolic function and energy expenditure. Hence, long-term weight loss is difficult to achieve. In consideration of the aforementioned hurdles, a promising anti-obesity drug should consist of multi-functional effects leading to energy expenditure. We previously demonstrated that mice with systemic PKCβ deficiency displayed many of the features one would desire in individuals treated with an ideal anti-obesity drug; elevated metabolic rate, which contribute to protection from diet-induced obesity, hepatic steatosis, and insulin-resistance (Hepatology 49:1525,2009; J. Lipid Res. 286:22,795,2011). We recently reported that depletion of hepatocyte-specific PKCβ recapitulates many of the beneficial features of systemic PKCbeta deficient mice (Mol. Metab. 44: e101133,2021; JCI Insight 6: e149023,2021). These results provide compelling evidence that PKCβ is an important therapeutic target for the treatment of obesity, as well as its related metabolic complications.MethodsOur aim is to evaluate therapeutic effects of a novel PKCβ inhibitor, INST3399 to treat obesity and fatty liver disease in a high-fat diet-induced obese C57BL/6J mouse model.ResultsINST3399 is a novel PKCβ inhibitor, which inhibits this kinase by a mechanism different from a well-known alternative ruboxistaurin. INST3399 inhibits PKCβ in the basal state, about >5-times more potent than ruboxistaurin and exhibit more than >100-fold selectivity for PKCβ versus PKCα. Our data demonstrate that INST3399 is safe and able to decrease the degree of obesity and fatty liver disease.ConclusionsINST3399 is a promising novel drug candidate for the pharmacological treatment of obesity and associated comorbidities. Moreover, INST3399 could further evolve as a novel treatment to manage other metabolic conditions with excessive PKCβ expression.Funding SourcesNIH HL138198; Instacare Funds.

Thrombolytic tPA-induced hemorrhagic transformation of ischemic stroke is mediated by PKCβ phosphorylation of occludin

AbstractThe current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurologic outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy, and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase Cβ (PKCβ) phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We show that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased phosphorylation of occludin serine 490 (S490) in the ischemic penumbra in a tPA-dependent manner, as tPA−/− mice were significantly protected from MCAO-induced occludin phosphorylation. Intraventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCβ-dependent manner. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCβ, reduced MCAO-induced permeability and improved functional outcome. Furthermore, inhibiting PKCβ after MCAO prevented ICH associated with delayed thrombolysis. These results show that PKCβ phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCβ inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.

A Novel PKCβ Inhibitor More Potent than Ruboxistaurin: Potential Therapeutic Tool for Obesity and Fatty Liver Disease

The ongoing epidemic of obesity and its associated comorbidities has increased the demand for novel therapeutics targeted toward modulating appetite and/or energy metabolism. The search for clinically useful drugs has thus far met with limited success, and therefore the identification of novel potential targets remains of great interest. We previously showed that mice with systemic PKCβ depletion displayed many of the features one would desire in individuals treated with an ideal antiobesity drug; increase in lean body mass and elevated metabolic rate contributing to prevention of diet‐induced adiposity, hepatic steatosis, and insulin resistance (Hepatology 49:1525, 2009; J. Lipid Res. 286:22795, 2011). Even depletion of hepatocyte‐specific PKCβ recapitulated many of the bebeficial features of whole‐body knockout mice (Mol. Metab. 44:e101133, 2021; JCI Insight 6:e149023, 2021). Our results provide compelling evidence that PKCβ is an important therapeutic target for treatment of obesity and related metabolic complications. An alternative strategy to genetic manipulation of PKCβ expression is the chronic use of selective inhibitors to resolve obesity, or reverse obesity, or both. About 25 years ago, it was reported that a small, orally available molecule, called ruboxistaurin, (also known as LY333,531) reported to potently and selectively inhibited PKCβ. This inhibitor displayed kinetics consistent with direct competition for ATP binding to PKCβ. This selectivity fueled several randomized clinical trials of ruboxstaurin to resolve chronic diabetic microvascular complications such as neuropathy, retinopathy, and macular oedema. Although the mechanism through which hyperglycemia results in vascular dysfunction has not been well established, but the in vivo and in vitro preclinical evidence suggested that hyperglycemia‐induced activation of PKCβ might underlie the development of these microvascular diseases into chronic diabetic complications. To the best of our knowledge, primary outcomes were never achieved in any of the trials and ruboxistaurin has not been approved from the U. S. Food and Drug Administration. Considering that targeting PKCβ outside of the conserved ATP site may be an effective strategy to achieve greater selectivity and potency, we hereby describe screening of already known and newly synthesized bisindolylmaleimides and their derivatives to evaluate whether these chemicals inhibit PKCβ with similar or higher potency than ruboxistaurin. These chemicals were also used to evaluate inhibition of PKCβ vs PKCα. We identified INST3399 as the most potent inhibitor of PKCβ in the basal state, being about 5‐times more potent than ruboxistaurin and more than 100‐fold selectivity for PKCβ versus PKCα. INST3399 is also several orders of magnitude more selective for inhibition of PKCβ in comparison to other ATP dependent kinases. INST3399 appears to inhibit PKCβ by a mechanism different from ruboxistaurin. As expected, INST3399 prevented diet‐induced obesity and hepatic steatosis. This has clear implications for the treatment of metabolic conditions with excessive PKCβ activity. Further studies are in progress to assess the therapeutic efficacy of this compound in reversing obesity.

PKCβ inhibitor blocked angiotensin II-induced vascular smooth muscle proliferation and oxidative stress

PKCβ inhibitor blocked angiotensin II-induced vascular smooth muscle proliferation and oxidative stress

Abstract 10213: Acute Protein Kinase C Beta Inhibition Preserves Coronary Endothelial Function After Cardioplegic Hypoxia/Reoxygenation

Introduction: Cardioplegic ischemia and reperfusion (CP-I/R) or CP-hypoxia and reoxygenation (CP-H/R) are associated with protein kinase C (PKC) overexpression/activation and coronary endothelial dysfunction. Hypothesis: This study aimed to determine whether acute inhibition of PKC could protect against CP-H/R-induced coronary endothelial dysfunction. Methods: Isolated small coronary arteries from mice (n = 6/group, male and female) were subjected to hyperkalemic, cardioplegic hypoxia (1 hour) and reoxygenation (1 hour) with Krebs buffer. The vessels undergoing CP-H/R were divided into 2 groups: those treated with the selective PKCβ inhibitor Ruboxistaurin (RBX, 50 nM, CP-H/R + RBX group) 5 minutes before and during CP-H/R, and those that received no treatment. Sham control vessels were continuously perfused with oxygenated Krebs buffer without CP-H/R and drug treatment. Vascular reactivity was measured by vessel myography. At the end of 1-hour re-oxygenation, all vessels were pretreated with endothelin-1; then, responses to the endothelium-dependent, NO-mediated vasodilator adenosine-diphosphate (ADP) and the endothelium-dependent/SK channel activator NS309 were examined. Results: CP-H/R caused a significant decrease in the coronary relaxation response to the endothelium-dependent, NO-mediated vasodilator ADP (10 - 4 M, * P =0.006) and the SK channel activator NS309 (10 -5 M, * P =0.0001) as compared with the Sham control group. Treatment with the selective PKCβ inhibitor RBX significantly increased the recovery of coronary relaxation responses to ADP ( # P = 0.031) and NS309 ( # P =0.004) compared with the untreated CP-H/R group (Figure). Conclusion: Acute inhibition of PKCβ significantly improved the recovery of coronary endothelial function after a period of CP-H/R injury in the mouse coronary microvasculature, suggesting that acute PKCβ inhibition may be a novel approach for protecting coronary endothelial function during CP-H/R.

A Predictive Endothelial-Leukemia Pre-Clinical Platform to Uncover Drug Vulnerabilities for Personalized Treatments

Background. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few innovative treatment options. This is also contributed by the lack of models capable of capturing the complexity of the tumor and its microenvironment.Aims. To identify patient-specific vulnerabilities and novel therapeutic strategies in T-ALL and interrogate the mechanisms of the crosstalk between leukemic and stromal elements.Methods. We established a drug-testing platform using patient-derived-tumor-xenografts (PDTX) and a mixed-culture approach using E4ORF1-transduced endothelial cells (ECs) (Seandel M et al, PNAS 2008) to overcome host-mediated chemoresistance. We performed functional experiments using total and single-cell RNA sequencing.Results. First, we established a battery of 22 T-ALL PDTX models that matched both phenotypically (immune-histochemistry, flow cytometry) and genotypically (TCR rearrangement, transcriptome) with the primary patients' samples. We then challenged these models (n=14 samples belonging to different PDTX and serial passages within each model) with a library of compounds (n=433) targeting redundant proteins (n=634). Unsupervised clustering and Principal Component Analysis (PCA) demonstrated two clusters of T-ALL samples based on differential drug susceptibility. We could at least partially correlate these differences to specific transcriptomic signatures predictive of drug response (Figure 1A).We then defined a group of pan-active compounds across all models (n=40), which we validated using an independent screening with/without ECs (Figure 1B). We found that ECs counteracted the activity of selected compounds (i.e. TSA, THZ1 and MLN2238). By PCA, we observed distinct response profiles based on different T-ALL models. We vectorized the EC-rescue and found that the direction was the same across all 3 models tested, indicating that it relied on similar mechanisms regardless of model identity. Based on the known role of IGF1-IGFR1 as a supportive EC-rescue axis (Medyouf H et al, J Exp Med 2011), we performed the same screening with/without recombinant IGFBP-7 (500 ng/mL), a decoy IGF1 molecule. Remarkably, IGFBP-7 completely or partially abrogated the EC-mediated rescue of selected compounds [enzastaurin (PKC-β inhibitor), SC144 (GP130 inhibitor), CHIR124 (Chk1 inhibitor) and YM155 (Survivin inhibitor)] (Figure 1B). Drugs not rescued by ECs (n=30) were considered positive hits and 5 of them (ruxolitinib, tofacitinib, panobinostat, bortezomib, irinotecan) ultimately proved to be effective in vivo in randomized pre-clinical trials either alone or in combination (Figure 1C). Our stepwise endothelial-leukemia platform led to the discovery of “public” and “private” vulnerabilities and the proof-of-principle of prediction-guided in vivo pre-clinical trials. We propose a list of compounds that could be readily translated into T-ALL clinical trials (Figure 1D). We finally proved the validity of our platform using other disease models (i.e. B and T-lymphoma PDTXs).Mechanistically, at single-cell resolution, in vitro interacting T-ALL cells and ECs underwent reciprocal transcriptome changes, with T-ALL shifting towards stemness/undifferentiation and ECs towards tumor-ECs (TECs) phenotypes. Furthermore, in vitro EC-educated T-ALL cells mimicked distinct T-ALL subsets of the leukemic spleen of corresponding PDTX mice (Figure 1E).Conclusions. These data demonstrate that our EC-T-ALL culture system simulates in vivo conditions, offering a robust platform to study drug response, leukemia-host interactions and cell plasticity. This approach will improve the pre-clinical predictability of novel drugs/combinations for T-ALL, as well as for other hematologic malignancies, and propel the development of patient-tailored treatments. [Display omitted] DisclosuresMelnick: Janssen Pharmaceuticals: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Epizyme: Consultancy; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Elemento: AstraZeneca: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Owkin: Consultancy, Other: Current equity holder; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Janssen: Research Funding. Chiaretti: amgen: Consultancy; pfizer: Consultancy; novartis: Consultancy; Incyte: Consultancy. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

Regulation of epithelial sodium channel activity by SARS-CoV-1 and SARS-CoV-2 proteins

Severe acute respiratory syndrome (SARS) coronavirus (CoV) 2 (SARS-CoV-2), which causes the coronavirus disease 2019, encodes several proteins whose roles are poorly understood. We tested their ability either to directly form plasma membrane ion channels or to change functions of two mammalian plasma membrane ion channels, the epithelial sodium channel (ENaC) and the α3β4 nicotinic acetylcholine receptor. In mRNA-injected Xenopus oocytes, none of nine SARS-CoV-2 proteins or two SARS-CoV-1 proteins produced conductances, nor did co-injection of several combinations. Immunoblots for ORF8, spike (S), and envelope (E) proteins revealed that the proteins are expressed at appropriate molecular weights. In experiments on coexpression with ENaC, three tested SARS proteins (SARS-CoV-1 E, SARS-CoV-2 E, and SARS-CoV-2 S) markedly decrease ENaC currents. SARS-CoV-1 S protein decreases ENaC currents modestly. Coexpressing the E proteins but not the S proteins with α3β4 nicotinic acetylcholine receptors significantly reduces acetylcholine-induced currents. ENaC inhibition does not occur if the SARS-CoV protein mRNAs are injected 24 h after the ENaC mRNAs, suggesting that SARS-CoV proteins affect early step(s) in functional expression of channel proteins. Consistent with the hypothesis that the SARS-CoV-2 S protein-induced ENaC inhibition involves competition for available protease, mutating the furin cleavage site in SARS-CoV-2 S protein partially relieves inhibition of ENaC currents. Extending previous suggestions that SARS proteins affect ENaC currents via protein kinase C (PKC) activation, PKC activation via phorbol 12-myristate 13-acetate decreases ENaC and α3β4 activity. Phorbol 12-myristate 13-acetate application reduced membrane capacitance ∼5%, presumably via increased endocytosis, but this decrease is much smaller than the SARS proteins’ effects on conductances. Also, incubating oocytes in Gö-6976, a PKCα and PKCβ inhibitor, did not alter E or S protein-induced channel inhibition. We conclude that SARS-CoV-1 and SARS-CoV-2 proteins alter the function of human plasma membrane channels, via incompletely understood mechanisms. These interactions may play a role in the coronavirus 2019 pathophysiology.

Signalling pathway of U46619-induced vascular smooth muscle contraction in mouse coronary artery.

Thromboxane A2 (TXA2 ) participates in many pathophysiological processes of coronary artery disease. However, its mechanism of TXA2 -induced contraction in the coronary artery remains to be clarified. A multi myograph system was used to measure the isometric tension of the mouse coronary arteries and identify the effect and pathway of TXA2 analogues U46619. Confocal laser scanning microscopy was used to measure the intracellular calcium concentration ([Ca2+ ]i ) in mouse coronary artery smooth muscle cells. Results from the experiment had shown that contraction in coronary artery was generated by U46619 in a concentration-dependent manner, which was completely abolished by a specific TXA2 receptor blocker, GR32191. PI-PLC inhibitors U73122 and D609 and Rho-Kinase inhibitor Y-27632 can block the U46619 elicited coronary artery contraction in a dose-dependent manner. Then, the vasoconstriction response to U46619 was obviously inhibited by two pan-PKC inhibitors chelerythrine or Gӧ6983, and a selective PKCδ inhibitor rottlerin, but was not blocked by a selective PKCζ inhibitor PKC-PS or a selective PKCβ inhibitor hispidin. Meanwhile, the PKC activator PDBu-induced vasoconstriction was significantly inhibited by 1μmol/L nifedipine, then mostly inhibited by 100μmol/L 2-APB and 10μmol/L Y27632. We further found that the response to U46619 was inhibited, respectively, by three calcium channel blockers nifedipine, SKF96356 or 2-APB in a concentration-dependent manner. Although Store-operated Ca2+ (SOC) channels generated the increase of [Ca2+ ]i in mouse coronary artery smooth muscle cells, SOC channels did not contribute to the vasoconstriction in mouse coronary arteries. Caffeine-induced sarcoplasmic reticulum (SR) Ca2+ release could obviously induce coronal vasoconstriction. In addition, NPPB, a cell membrane Ca2+ activated C1- channel blocker, could obviously inhibit the U46619-induced vasoconstriction. The U46619-induced mouse coronary artery contraction was involved in the increase in [Ca2+ ]i mediated by Cav1.2, TRPC channels and SR release through the activation of G-protein-coupled TP receptors and the kinases signalling pathway in TP downstream proteins, while SOC channels did not participate in the vasoconstriction.

Targeting Protein Kinase C in Glioblastoma Treatment.

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor and is associated with a poor prognosis. Despite the use of combined treatment approaches, recurrence is almost inevitable and survival longer than 14 or 15 months after diagnosis is low. It is therefore necessary to identify new therapeutic targets to fight GBM progression and recurrence. Some publications have pointed out the role of glioma stem cells (GSCs) as the origin of GBM. These cells, with characteristics of neural stem cells (NSC) present in physiological neurogenic niches, have been proposed as being responsible for the high resistance of GBM to current treatments such as temozolomide (TMZ). The protein Kinase C (PKC) family members play an essential role in transducing signals related with cell cycle entrance, differentiation and apoptosis in NSC and participate in distinct signaling cascades that determine NSC and GSC dynamics. Thus, PKC could be a suitable druggable target to treat recurrent GBM. Clinical trials have tested the efficacy of PKCβ inhibitors, and preclinical studies have focused on other PKC isozymes. Here, we discuss the idea that other PKC isozymes may also be involved in GBM progression and that the development of a new generation of effective drugs should consider the balance between the activation of different PKC subtypes.

Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry

Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmi...