Structure-Activity Relationship and Bioactivity Studies of Neurotrophic trans-Banglene

Neurotrophic small molecule natural products are functional analogs of signaling proteins called neurotrophins, which cause a pro-growth, pro-survival, or pro-differentiation response in neuronal cells. While these phenotypic responses are desirable to combat neurodegenerative disease progression, the pharmacokinetic properties of neurotrophins present challenges to their administration. Therefore, neurotrophic small molecules such as the cis- and trans-banglenes offer attractive alternatives. We describe the synthesis and testing of banglene derivatives and establish a structure-activity response for the banglene family. We demonstrate that (–) trans-banglene is the primarily active enantiomer, and that select modifications on the cyclohexene ring of trans-banglene do not significantly impair its bioactivity. Finally, we demonstrate that (–) trans-banglene potentiation of NGF induced neuritogenesis is unaffected by the presence of these Erk1/2, Akt and Pkc inhibitors. Our structure-activity results also suggest that (–) trans-banglene neurotrophic activity and its potentiation of NGF activity might be distinct unassociated processes.

Structure-Activity-Relationship and Bioactivity of Neurotrophic trans-Banglene

Neurotrophic small molecule natural products are functional analogs of signaling proteins called neurotrophins, which cause a pro-growth, pro-survival, or pro-differentiation response in neuronal cells. While these phenotypic responses are desirable to combat neurodegenerative disease progression, the pharmacokinetic properties of neurotrophins present challenges to their administration. Therefore, neurotrophic small molecules such as the cis- and trans-banglenes offer attractive alternatives. We describe the synthesis and testing of banglene derivatives and establish a structure-activity response for the banglene family. We demonstrate that (–) trans-banglene is the primarily active enantiomer, and that select modifications on the cyclohexene ring of trans-banglene do not significantly impair its bioactivity. Finally, we demonstrate that (–) trans-banglene potentiation of NGF induced neuritogenesis is unaffected by the presence of these Erk1/2, Akt and Pkc inhibitors. Our structure-activity results also suggest that (–) trans-banglene neurotrophic activity and its potentiation of NGF activity might be distinct unassociated processes.

Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials

Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinase, is classed into three subfamilies based on their structural and activation characteristics: conventional or classic PKC isozymes (cPKCs; α, βI, βII, and γ), novel or non-classic PKC isozymes (nPKCs; δ, ε, η, and θ), and atypical PKC isozymes (aPKCs; ζ, ι, and λ). PKC inhibitors and activators are used to understand PKC-mediated intracellular signaling pathways and for the diagnosis and treatment of various PKC-associated diseases, such as cancers, neurological diseases, cardiovascular diseases, and infections. Many clinical trials of PKC inhibitors in cancers showed no significant clinical benefits, meaning that there is a limitation to design a cancer therapeutic strategy targeting PKC alone. This review will focus on the activators and inhibitors of PKC and their applications in clinical trials.

Berberine Reverses Nitroglycerin Tolerance Through Suppressing PKCα Activity in Vascular Smooth Muscle Cells

Purpose The aim of the present study was to evaluate the effects of berberine on nitroglycerin (NTG) tolerance and explore the underlying mechanism involved. Methods NTG tolerance was induced by pre-exposure of Sprague-Dawley rat aortas to NTG in vitro or by pretreating Sprague-Dawley rats with NTG patch in vivo. The aortas were pre-treated with berberine or PKC inhibitors for different durations of time before induction of NTG tolerance. NTG-induced vasorelaxations was measured on wire myograph. Primary vascular smooth cells (VSMCs) were used to dissect the underlying mechanism of berberine-induced inhibition of NTG tolerance. Results NTG tolerance induced by either prior exposure of rat aortas to NTG in vitro or pretreatment with NTG patch in vivo was reversed by co-treatment with berberine, as well as the inhibitors of PKC and PKCα. The mechanistic study revealed that PKCα participated in the development of NTG tolerance as NTG increased the activity of PKCα with enriched PKCα membrane localization and elevated phosphorylation of PKCα in VSMCs, which was reversed by berberine or PKCα inhibitors. Conclusion The present study is probably the first demonstration that berberine reverses NTG tolerance through inhibiting PKCα activity in VSMCs and PKCα is an important contributor to the development of NTG tolerance. These new findings suggest that berberine could become a promising drug for prevention of NTG tolerance and that targeting PKCα in VSMCs is likely to be a potential therapeutic target for reversal of NTG tolerance in blood vessels.

Protein Kinase C Downregulation upon Rapamycin Treatment Attenuates Neuroinflammation and Mitochondrial Disease

Mitochondrial dysfunction causes many poorly understood diseases, such as Leigh Syndrome, that are often caused by dysfunctions in proteins involved in the electron transport chain. My lab previously reported mTOR is pathologically involved in the neurodegenerative phenotype and premature death of mice missing the Complex I subunit Ndufs4 (Ndufs4-/- mice). We discovered treatment with rapamycin extends lifespan, reduces neuroinflammation, and attenuates the neurodegenerative phenotype in these mice, although the mechanisms remain unclear. Rapamycin-treated Ndufs4-/- mice exhibited decreased activation of the mTORC1 pathway. It also deactivated the mTORC2 pathway. We observed that phosphorylation of the canonical protein kinase C (PKC) isoforms (PKC-α, -β, and -γ) decreased more than any other kinases, leading us to hypothesize its deactivation contributes to the observed lifespan extension. To test this, we treated Ndufs4-/- mice with three different PKC inhibitors: the pan-PKC inhibitors GO6983 and GF109203X, and the PKC-β specific inhibitor ruboxistaurin. Similar to rapamycin, all three drugs were able to significantly delay the onset of neurological symptoms (i.e. clasping) and increase survival. We also observed that PKC-β inhibition reduced skin inflammation to suppress the hair loss phenotype displayed by Ndufs4-/- mice at weaning. We further discovered PKC-β inhibition reduces neuroinflammation by deactivating the NF-kB inflammatory pathway. These results suggest that mTORC2 may play a critical role in the etiology of mitochondrial diseases such as Leigh Syndrome.

Yoda1 and phosphatidylserine exposure in red cells from patients with sickle cell anaemia

Phosphatidylserine (PS) exposure is increased in red cells from sickle cell anaemia (SCA) patients. Externalised PS is prothrombotic and attractive to phagocytes and activated endothelial cells and thus contributes to the anaemic and ischaemic complications of SCA. The mechanism of PS exposure remains uncertain but it can follow increased intracellular Ca2+ concentration ([Ca2+]i). Normally, [Ca2+]i is maintained at very low levels but in sickle cells, Ca2+ permeability is increased, especially following deoxygenation and sickling, mediated by a pathway sometimes called Psickle. The molecular identity of Psickle is also unclear but recent work has implicated the mechanosensitive channel, PIEZO1. We used Yoda1, an PIEZO1 agonist, to investigate its role in sickle cells. Yoda1 caused an increase in [Ca2+]i and PS exposure, which was inhibited by its antagonist Dooku1 and the PIEZO1 inhibitor GsMTx4, consistent with functional PIEZO1. However, PS exposure did not necessitate an increase in [Ca2+]i. Two PKC inhibitors were also tested, chelerytherine chloride and calphostin C. Both reduced PS exposure whilst chelerytherine chloride also reduced Yoda1-induced increases in [Ca2+]i. Findings are therefore consistent with the presence of PIEZO1 in sickle cells, able to mediate Ca2+ entry but that PKC was also involved in both Ca2+ entry and PS exposure.

The important role of connexin 43 in subarachnoid hemorrhage-induced cerebral vasospasm

Background Gap junctions are involved in the development of cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). However, the specific roles and regulatory functions of related connexin isoforms remain unknown. The aim of this study was to investigate the importance of connexin 43 (Cx43) in CVS and determine whether Cx43 alterations are modulated via the protein kinase C (PKC) signaling transduction pathway. Methods Oxyhemoglobin (OxyHb)-induced smooth muscle cells of basilar arterial and second-injection model in rat were used as CVS models in vitro and in vivo. In addition, dye transfer assays were used for gap junction-mediated intercellular communication (GJIC) observation in vitro and delayed cerebral ischemia (DCI) was observed in vivo by perfusion-weighted imaging (PWI) and intravital fluorescence microscopy. Results Increase in Cx43 mediated the development of SAH-induced CVS was found in both in vitro and in vivo CVS models. Enhanced GJIC was observed in vitro CVS model, this effect and increased Cx43 were reversed by preincubation with specific PKC inhibitors (chelerythrine or GF 109203X). DCI was observed in vivo on day 7 after SAH. However, DCI was attenuated by pretreatment with Cx43 siRNA or PKC inhibitors, and the increased Cx43 expression in vivo was also reversed by Cx43 siRNA or PKC inhibitors. Conclusions These data provide strong evidence that Cx43 plays an important role in CVS and indicate that changes in Cx43 expression may be mediated by the PKC pathway. The current findings suggest that Cx43 and the PKC pathway are novel targets for developing treatments for SAH-induced CVS.

Protein kinase C inhibitors override ZEB1-induced chemoresistance in HCC

Epithelial–mesenchymal transition (EMT) is a process by which tumour cells lose epithelial characteristics, become mesenchymal and highly motile. EMT pathways also induce stem cell features and resistance to apoptosis. Identifying and targeting this pool of tumour cells is a major challenge. Protein kinase C (PKC) inhibition has been shown to eliminate breast cancer stem cells but has never been assessed in hepatocellular cancer (HCC). We investigated ZEB family of EMT inducer expression as a biomarker for metastatic HCC and evaluated the efficacy of PKC inhibitors for HCC treatment. We showed that ZEB1 positivity predicted patient survival in multiple cohorts and also validated as an independent biomarker of HCC metastasis. ZEB1-expressing HCC cell lines became resistant to conventional chemotherapeutic agents and were enriched in CD44high/CD24low cell population. ZEB1- or TGFβ-induced EMT increased PKCα abundance. Probing public databases ascertained a positive association of ZEB1 and PKCα expression in human HCC tumours. Inhibition of PKCα activity by small molecule inhibitors or by PKCA knockdown reduced viability of mesenchymal HCC cells in vitro and in vivo. Our results suggest that ZEB1 expression predicts survival and metastatic potential of HCC. Chemoresistant/mesenchymal HCC cells become addicted to PKC pathway and display sensitivity to PKC inhibitors such as UCN-01. Stratifying patients according to ZEB1 and combining UCN-01 with conventional chemotherapy may be an advantageous chemotherapeutic strategy.