Proteostasis Regulators Restore Function of Epilepsy-Associated GABAA Receptors.

Proteostasis deficiency in mutated ion channels leads to a variety of ion channel diseases that are caused by excessive endoplasmic reticulum-associated degradation (ERAD) and inefficient membrane trafficking. We investigated proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors, the primary mediators of neuronal inhibition in the mammalian central nervous system. We screened a structurally diverse, Food and Drug Administration-approved drug library and identified dinoprost (DNP) and dihydroergocristine (DHEC) as highly efficacious enhancers of surface expression of four epilepsy-causing trafficking-deficient mutant receptors. Furthermore, DNP and DHEC restore whole-cell and synaptic currents by incorporating mutated subunits into functional receptors. Mechanistic studies revealed that both drugs reduce subunit degradation by attenuating the Grp94/Hrd1/Sel1L/VCP-mediated ERAD pathway and enhance the subunit folding by promoting subunit interactions with major GABAA receptors-interacting chaperones, BiP and calnexin. In summary, we report that DNP and DHEC remodel the endoplasmic reticulum proteostasis network to restore the functional surface expression of mutant GABAA receptors.

In Vitro Effect and Mechanism of Action of Ergot Alkaloid Dihydroergocristine in Chemoresistant Prostate Cancer Cells.

BACKGROUND/AIM: Chemoresistance is a major obstacle in the treatment of prostate cancer (PCa). It is imperative to develop novel strategies for overcoming chemoresistance and improving clinical outcomes. We evaluated the in vitro activity and mechanism of action of dihydroergocristine (DHECS), an ergot alkaloid approved for the treatment of dementia, in PCa cells. MATERIALS AND METHODS: The in vitro effects of DHECS on PCa cell cycle and viability were determined by flow cytometry and colorimetric assay. The effects of DHECS on PCa cell signaling were evaluated by quantitative PCR, western blot analysis and reporter assay. RESULTS: DHECS was effective in inducing cell cycle arrest and apoptosis in human PCa cells. Of particular interest, DHECS demonstrated high potency against chemoresistant PCa cells. At the molecular level, DHECS affected multiple factors implicated in the regulation of cancer cell cycle and programmed cell death, including p53, mouse double minute 2 homolog (MDM2), retinoblastoma protein (RB), p21, E2F transcription factor 1 (E2F1), survivin, myeloid cell leukemia 1 (Mcl-1) and poly ADP ribose polymerase (PARP). Furthermore, DHECS may function through dopamine receptor-mediated effects on 5'-AMP-activated protein kinase (AMPK) and nuclear factor kappa B (NF-ĸB). CONCLUSION: DHECS has the potential to be repurposed as a novel anticancer agent for the management of chemoresistant PCa.

The FDA-approved natural product dihydroergocristine reduces the production of the Alzheimer's disease amyloid-ß peptides.

Known γ-secretase inhibitors or modulators display an undesirable pharmacokinetic profile and toxicity and have therefore not been successful in clinical trials for Alzheimer's disease (AD). So far, no compounds from natural products have been identified as direct inhibitors of γ-secretase. To search for bioactive molecules that can reduce the amount of amyloid-beta peptides (Aß) and that have better pharmacokinetics and an improved safety profile, we completed a screen of ~400 natural products by using cell-based and cell-free γ-secretase activity assays. We identified dihydroergocristine (DHEC), a component of an FDA- (Food and Drug Administration)-approved drug, to be a direct inhibitor of γ-secretase. Micromolar concentrations of DHEC substantially reduced Aß levels in different cell types, including a cell line derived from an AD patient. Structure-activity relationship studies implied that the key moiety for inhibiting γ-secretase is the cyclized tripeptide moiety of DHEC. A Surface Plasmon Resonance assay showed that DHEC binds directly to γ-secretase and Nicastrin, with equilibrium dissociation constants (Kd) of 25.7 nM and 9.8 µM, respectively. This study offers DHEC not only as a new chemical moiety for selectively modulating the activity of γ-secretase but also a candidate for drug repositioning in Alzheimer's disease.

Roles of adenosine and serotonin receptors on the antinociception of sildenafil in the spinal cord of rats.

PURPOSE: The phosphodiesterase 5 inhibitor sildenafil has antinociceptive effects, mediated by an increase in cGMP. This study examined the role of spinal adenosine and serotonin receptors played in the antinociceptive effects of intrathecal sildenafil. MATERIALS AND METHODS: Intrathecal catheters were inserted into the subarachnoid space of Sprague-Dawley male rats as a drug delivery device. Pain was induced by injecting formalin into the plantar surface of rats and observing nociceptive behavior (flinching response) for 60 minutes. Then, the effects of intrathecal adenosine and serotonin receptor antagonists on the antinociceptive activity of intrathecal sildenafil were examined. RESULTS: Intrathecal sildenafil suppressed the flinching response in a dose-dependent manner during phases 1 and 2 in the formalin test. Both CGS 15943 and dihydroergocristine decreased the antinociceptive effects of sildenafil during phases 1 and 2 in the formalin test. CONCLUSION: Intrathecal sildenafil effectively attenuated the pain evoked by formalin injection. Both adenosine and serotonin receptors may be involved in the antinociceptive action of sildenafil at the spinal level.

Roles of Adenosine and Serotonin Receptors on the Antinociception of Sildenafil in the Spinal Cord of Rats

PURPOSE: The phosphodiesterase 5 inhibitor sildenafil has antinociceptive effects, mediated by an increase in cGMP. This study examined the role of spinal adenosine and serotonin receptors played in the antinociceptive effects of intrathecal sildenafil. MATERIALS AND METHODS: Intrathecal catheters were inserted into the subarachnoid space of Sprague-Dawley male rats as a drug delivery device. Pain was induced by injecting formalin into the plantar surface of rats and observing nociceptive behavior (flinching response) for 60 mininutes. Then, the effects of intrathecal adenosine and serotonin receptor antagonists on the antinociceptive activity of intrathecal sildenafil were examined. RESULTS: Intrathecal sildenafil suppressed the flinching response in a dose-dependent manner during phases 1 and 2 in the formalin test. Both CGS 15943 and dihydroergocristine decreased the antinociceptive effects of sildenafil during phases 1 and 2 in the formalin test. CONCLUSION: Intrathecal sildenafil effectively attenuated the pain evoked by formalin injection. Both adenosine and serotonin receptors may be involved in the antinociceptive action of sildenafil at the spinal level.