Ibuprofen modulates tetrodotoxin-resistant persistent Na+ currents at acidic pH in rat trigeminal ganglion neurons.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to relieve various symptoms such as headache, arthralgia, and dental pain. While the primary mechanism of NSAID-based pain relief is the inhibition of cyclooxygenase-2, several NSAIDs also modulate other molecular targets related to nociceptive transmission such as voltage-gated Na+ channels. In the present study, we examined the effects of NSAIDs on persistent Na+ current (INaP) mediated by tetrodotoxin-resistant (TTX-R) Na+ channels in small-to medium-sized trigeminal ganglion neurons using a whole-cell patch-clamp technique. At clinically relevant concentrations, all propionic acid derivatives tested (ibuprofen, naproxen, fenoprofen, and flurbiprofen) preferentially inhibited the TTX-R INaP. The inhibition was more potent at acidic extracellular pH (pH 6.5) than at normal pH (pH 7.4). Other NSAIDs, such as ketorolac, piroxicam, and aspirin, had a negligible effect on the TTX-R INaP. Ibuprofen both accelerated the onset of inactivation and retarded the recovery from inactivation of TTX-R Na+ channels at acidic extracellular pH. However, all NSAIDs tested in this study had minor effects on voltage-gated K+ currents, as well as hyperpolarization-activated and cyclic nucleotide-gated cation currents, at both acidic and normal extracellular pH. Under current-clamp conditions, ibuprofen decreased the number of action potentials elicited by depolarizing current stimuli at acidic (pH 6.5) extracellular pH. Considering that extracellular pH falls as low as 5.5 in inflamed tissues, TTX-R INaP inhibition could be a mechanism by which ibuprofen and propionic acid derivative NSAIDs modulate inflammatory pain.

Hypoxia stabilizes SETDB1 to maintain genome stability.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Design of hypoxia responsive CRISPR-Cas9 for target gene regulation.

The CRISPR-Cas9 system is a widely used gene-editing tool, offering unprecedented opportunities for treating various diseases. Controlling Cas9/dCas9 activity at specific location and time to avoid undesirable effects is very important. Here, we report a conditionally active CRISPR-Cas9 system that regulates target gene expression upon sensing cellular environmental change. We conjugated the oxygen-sensing transcription activation domain (TAD) of hypoxia-inducing factor (HIF-1α) with the Cas9/dCas9 protein. The Cas9-TAD conjugate significantly increased endogenous target gene cleavage under hypoxic conditions compared with that under normoxic conditions, whereas the dCas9-TAD conjugate upregulated endogenous gene transcription. Furthermore, the conjugate system effectively downregulated the expression of SNAIL, an essential gene in cancer metastasis, and upregulated the expression of the tumour-related genes HNF4 and NEUROD1 under hypoxic conditions. Since hypoxia is closely associated with cancer, the hypoxia-dependent Cas9/dCas9 system is a novel addition to the molecular tool kit that functions in response to cellular signals and has potential application for gene therapeutics.

Anti­tumor properties of FoxO1 in YD­9 oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC) is a tumor with a poor prognosis and a high recurrence rate. Despite its high annual incidence worldwide, appropriate therapeutic strategies have not yet been developed. Consequently, the 5­year survival rate for OSCC is low when advanced stages or recurrence is diagnosed. Forkhead transcriptional factor O1 (FoxO1) is a key mediator for maintaining cellular homeostasis. FoxO1 can function as a tumor suppressor as well as an oncogene depending on the cancer type. Therefore, the precise molecular functions of FoxO1 need to be validated, considering intracellular factors and the extracellular environment. To the best of our knowledge, however, the roles of FoxO1 in OSCC have not yet been defined. The present study examined FoxO1 levels under pathological conditions (oral lichen planus and oral cancer) and selected an appropriate OSCC cell line (YD­9). Crispr/Cas9 was used to generate FoxO1­deficient YD­9 cells in which the protein levels of phospho ERK and phospho STAT3 were upregulated, promoting cancer proliferation and migration. In addition, FoxO1 reduction increased the levels of the cell proliferation markers phospho H3 (Ser10) and PCNA. FoxO1 loss significantly reduced cellular ROS levels and apoptosis in YD­9 cells. Collectively, the present study demonstrated that FoxO1 exerted an anti­tumor effect by suppressing proliferation and migration/invasion but promoting oxidative stress­linked cell death in YD­9 OSCC cells.

CDK9 inhibitors downregulate DKK1 expression to suppress the metastatic potential of HCC cells.

BACKGROUND: Elevated expression of Dickkopf-1 (DKK1) is frequently observed in hepatocellular carcinoma (HCC) patients with poor clinical outcomes. Several reports indicating the functional involvement of DKK1 in HCC progression have suggested DKK1 as a promising therapeutic target for HCC. OBJECTIVE: In this study, to develop an efficient way to target DKK1, we assessed the effect of CDK9 inhibitors on DKK1 expression linked to metastatic movement of HCC. METHODS: The expression of DKK1 in CDK9 inhibitor-treated HCC cells was measured by western blot, ELISA and quantitative real-time reverse transcription PCR. Wound healing assay, migration assay, invasion assay and western blot were examined to evaluate the functional role of DKK1 in CDK9 inhibitors-treated HCC. RESULTS: Inactivation of CDK9 either by a catalytic inhibitor being clinically evaluated or by a specific CDK9 protein degrader largely downregulated DKK1 expression at the transcript and protein levels. In addition, CDK9 inhibitors suppressed the migration and invasion of HCC cells. We observed that ectopic high expression of DKK1 at least partially reversed the defects in metastatic movement of HCC cells mediated by CDK9 inhibitors. We further discovered that the DKK1-nuclear ß-catenin axis associated with the metastatic potential of HCC cells was impaired by CDK9 inhibitors. CONCLUSION: Taken together, our findings suggest that CDK9 inhibitors are potent tools to target DKK1, which can suppress the metastatic progression of HCC.

Discovery of pan-IAP degraders via a CRBN recruiting mechanism.

Inhibitors of apoptosis proteins (IAPs), defined by the presence of baculovirus IAP repeat (BIR) protein domain, are critical regulators of cell survival and cell death processes. Cellular IAP 1/2 (cIAP1/2) and X-linked IAPs (XIAPs) regulate the innate immune signaling pathway through their E3 ubiquitin ligase activity. Peptidomimetics or small-molecule IAP antagonists have been developed to treat various diseases, such as cancer, infection, and inflammation. In this study, we synthesized and characterized IAP-cereblon (CRBN) heterodimerizing proteolysis-targeting chimera (PROTAC), which induces the degradation of cIAP1/2 and XIAP but not CRBN. We demonstrated that this PROTAC inhibits tumor necrosis factor alpha (TNFα)-induced innate immune response and cancer cell migration and invasion, leading to apoptotic cell death. Our study is the first to demonstrate that both cIAPs and XIAP are degradable when applied to the PROTAC strategy.

Structure-activity relationship analysis of novel GSPT1 degraders based on benzotriazinone scaffold and its antitumor effect on xenograft mouse model.

Molecular glue degraders, such as lenalidomide and pomalidomide, bind to cereblon (CRBN) E3 ligase and subsequently recruit neosubstrate proteins, Ikaros (IKZF1) and Aiolos (IKZF3), for the ubiquitination-proteasomal degradation process. In this study, we explored structure-activity relationship analysis for novel GSPT1 degraders utilizing a benzotriazinone scaffold previously discovered as a novel CRBN binder. In particular, we focused on the position of the ureido group on the benzotriazinone scaffold, substituent effect on the phenylureido group, and methyl substitution on the benzylic position of benzotriazinone. As a result, we identified 34f (TD-522), which exhibits strong anti-proliferative effects in both KG-1 (EC50 = 0.5 nM) and TMD-8 (EC50 = 5.2 nM) cell lines. Compound 34f effectively induced GSPT1 degradation with a DC50 of 0.269 nM and Dmax of >95 % at 10 nM concentration in KG-1 cells. An in vivo xenograft study showed that compound 34f effectively suppressed TMD8-driven tumor growth, suggesting a potential role in the development of novel GSPT1 degraders.

Artificial intelligence-based identification of octenidine as a Bcl-xL inhibitor.

Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein-protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells.

Sevoflurane modulation of tetrodotoxin-resistant Na+ channels in small-sized dorsal root ganglion neurons of rats.

OBJECTIVE: Volatile anesthetics are widely used for general anesthesia during surgical operations. Voltage-gated Na+ channels expressed in central neurons are major targets for volatile anesthetics; but it is unclear whether these drugs modulate native tetrodotoxin-resistant (TTX-R) Na+ channels, which are involved in the development and maintenance of inflammatory pain. METHODS: In this study, we examined the effects of sevoflurane on TTX-R Na+ currents (INa) in acutely isolated rat dorsal root ganglion neurons, using a whole-cell patch-clamp technique. RESULTS: Sevoflurane slightly potentiated the peak amplitude of transient TTX-R INa but more potently inhibited slow voltage-ramp-induced persistent INa in a concentration-dependent manner. Sevoflurane (0.86 ± 0.02 mM) (1) slightly shifted the steady-state fast inactivation relationship to hyperpolarizing ranges without affecting the voltage-activation relationship, (2) reduced the extent of use-dependent inhibition of Na+ channels, (3) accelerated the onset of inactivation and (4) delayed the recovery from inactivation of TTX-R Na+ channels. Thus, sevoflurane has diverse effects on TTX-R Na+ channels expressed in nociceptive neurons. CONCLUSIONS: The present results suggest that the inhibition of persistent INa and the modulation of the voltage dependence and inactivation might be, at least in part, responsible for the analgesic effects elicited by sevoflurane.

Antioxidant and Antiproliferative Activity of Finasteride against Glioblastoma Cells.

Glioblastoma is an actively growing and aggressive brain tumor with a high propensity of recurrence. Although the surgical removal of tumor mass is the primary therapeutic option against glioblastoma, supportive pharmacotherapy is highly essential due to incredibly infiltrative characteristic of glioblastoma. Temozolomide, an FDA-approved alkylating agent, has been used as a first-line standard pharmacological approach, but several evident limitations were repeatedly reported. Despite additional therapeutic options suggested, there are no medications that successfully prevent a recurrence of glioblastoma and increase the five-year survival rate. In this study, we tested the possibility that finasteride has the potential to be developed as an anti-glioblastoma drug. Finasteride, an FDA-approved medication for the treatment of benign prostate hyperplasia and androgenic alopecia, is already known to pass through the blood-brain barrier and possess antiproliferative activity of prostate epithelial cells. We showed that finasteride inhibited the maintenance of glioma stem-like cells and repressed the proliferation of glioblastoma. Mechanistically, finasteride lowered intracellular ROS level by upregulating antioxidant genes, which contributed to inefficient ß-catenin accumulation. Downregulated ß-catenin resulted in the reduction in stemness and cell growth in glioblastoma.

Sevoflurane excites nociceptive sensory neurons by inhibiting K+ conductances in rats.

Sevoflurane, which is preferentially used as a day-case anesthetic based on its low blood solubility, acts on the central nervous system and exerts analgesic effects. However, it still remains unknown whether sevoflurane affects the excitability of nociceptive sensory neurons. Therefore, we conducted this study to examine the effects of sevoflurane on the excitability of small-sized dorsal root ganglion (DRG) neurons of rats using the whole-cell patch-clamp technique. In a voltage-clamp condition, sevoflurane elicited the membrane current in a concentration-dependent manner, in which the reversal potential was similar to the equilibrium potential of K+. In a current-clamp condition, sevoflurane directly depolarized the membrane potentials in a concentration-dependent manner. Moreover, at a clinically relevant concentration, sevoflurane decreased the threshold for action potential generation. These findings suggest that sevoflurane acts on the leak K+ channels to increase the excitability of DRG neurons. Sevoflurane increased the half-width of single action potentials, which resulted from the inhibition of voltage-gated K+ currents, including the fast inactivating A-type and non-inactivating delayed rectifier K+ currents. Our study indicates that sevoflurane could exhibit pronociceptive effects on nociceptive sensory neurons by inhibiting K+ conductances.

Ginsenoside F2 induces cellular toxicity to glioblastoma through the impairment of mitochondrial function.

BACKGROUND: Glioblastoma (GBM) is the most aggressive tumor residing within the central nervous system, with extremely poor prognosis. Although the cytotoxic effects of ginsenoside F2 (GF2) on GBM were previously suggested, the precise anti-GBM mechanism of GF2 remains unclear. The aim of this study was to explore the anti-cancer molecular mechanism of GF2 toward human GBM. METHODS: GF2-driven cellular toxicity was confirmed in two different GBM cells, U373 and Hs683. To test mitochondrial impairment driven by GF2, we examined the mitochondrial membrane potential, OCR, and ATP production. An intracellular redox imbalance was identified by measuring the relative ratio of reduced glutathione to oxidized glutathione (GSH/GSSG), glutaredoxin (GLRX) mRNA expression, intracellular NAD+ level, and AMPK phosphorylation status. RESULTS: GF2 increased the percentage of cleaved caspase 3-positive cells and γH2AX signal intensities, confirming that GF2 shows the cytotoxicity against GBM. GO enrichment analysis suggested that the mitochondrial function could be negatively influenced by GF2. GF2 reduced the mitochondrial membrane potential, basal mitochondrial respiratory rate, and ATP production capacity. Our results showed that GF2 downregulated the relative GSH/GSSG, intracellular NAD+ level, and GLRX expression, suggesting that GF2 may alter the intracellular redox balance that led to mitochondrial impairment. CONCLUSION: GF2 reduces mitochondrial membrane potential, inhibits cellular oxygen consumption, activates AMPK signaling, and induces cell death. Our study examined the potential vulnerability of mitochondrial activity in GBM, and this may hold therapeutic promise.

Phosphorylation of REPS1 at Ser709 by RSK attenuates the recycling of transferrin receptor.

RalBP1 associated EPS domain containing 1 (REPS1) is conserved from Drosophila to humans and implicated in the endocytic system. However, an exact role of REPS1 remains largely unknown. Here, we demonstrated that mitogen activated protein kinase kinase (MEK)-p90 ribosomal S6 Kinase (RSK) signaling pathway directly phosphorylated REPS1 at Ser709 upon stimulation by epidermal growth factor (EGF) and amino acid. While REPS2 is known to be involved in the endocytosis of EGF receptor (EGFR), REPS1 knockout (KO) cells did not show any defect in the endocytosis of EGFR. However, in the REPS1 KO cells and the KO cells reconstituted with a non-phosphorylatable REPS1 (REPS1 S709A), the recycling of transferrin receptor (TfR) was attenuated compared to the cells reconstituted with wild type REPS1. Collectively, we suggested that the phosphorylation of REPS1 at S709 by RSK may have a role of the trafficking of TfR. [BMB Reports 2021; 54(5): 272-277].

Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways.

The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized ß-transducin repeat-containing protein 1 (ßTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.

Menthol facilitates excitatory and inhibitory synaptic transmission in rat medullary dorsal horn neurons.

Menthol, which acts as an agonist for transient receptor potential melastatin 8 (TRPM8), has complex effects on nociceptive transmission, including pain relief and hyperalgesia. Here, we addressed the effects of menthol on spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs, respectively) in medullary dorsal horn neurons, using a whole-cell patch-clamp technique. Menthol significantly increased sEPSC frequency, in a concentration-dependent manner, without affecting current amplitudes. The menthol-induced increase in sEPSC frequency could be completely blocked by AMTB, a TRPM8 antagonist, but was not blocked by HC-030031, a transient receptor potential ankyrin 1 (TRPA1) antagonist. Menthol still increased sEPSC frequency in the presence of Cd2+, a general voltage-gated Ca2+ channel blocker, suggesting that voltage-gated Ca2+ channels are not involved in the menthol-induced increase in sEPSC frequency. However, menthol failed to increase sEPSC frequency in the absence of extracellular Ca2+, suggesting that TRPM8 on primary afferent terminals is Ca2+ permeable. On the other hand, menthol also increased sIPSC frequency, without affecting current amplitudes. The menthol-induced increase in sIPSC frequency could be completely blocked by either AMTB or CNQX, an AMPA/KA receptor antagonist, suggesting that the indirect increase in excitability of inhibitory interneurons may lead to the facilitation of spontaneous GABA and/or glycine release. The present results suggested that menthol exerts analgesic effects, via the enhancement of inhibitory synaptic transmission, through central feed-forward neural circuits within the medullary dorsal horn region.

Aryl Sulfonamides Induce Degradation of Aryl Hydrocarbon Receptor Nuclear Translocator through CRL4DCAF15 E3 Ligase.

Aryl hydrocarbon receptor nuclear translocator (ARNT) plays an essential role in maintaining cellular homeostasis in response to environmental stress. Under conditions of hypoxia or xenobiotic exposure, ARNT regulates the subset of genes involved in adaptive responses, by forming heterodimers with hypoxia-inducible transcription factors (HIF1α and HIF2α) or aryl hydrocarbon receptor (AhR). Here, we have shown that ARNT interacts with DDB1 and CUL4-associated factor 15 (DCAF15), and the aryl sulfonamides, indisulam and E7820, induce its proteasomal degradation through Cullin-RING finger ligase 4 containing DCAF15 (CRL4DCAF15) E3 ligase. Moreover, the two known neo-substrates of aryl sulfonamide, RNA-binding motif protein 39 (RBM39) and RNA-binding motif protein 23 (RBM23), are not required for ARNT degradation. In line with this finding, aryl sulfonamides inhibited the transcriptional activities of HIFs and AhR associated with ARNT. Our results collectively support novel regulatory roles of aryl sulfonamides in both hypoxic and xenobiotic responses.

Efficacy and pharmacokinetics evaluation of 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) as an anti-seizure agent.

Epilepsy is a common chronic neurological disease characterized by recurrent epileptic seizures. A seizure is an uncontrolled electrical activity in the brain that can cause different levels of behavior, emotion, and consciousness. One-third of patients fail to receive sufficient seizure control, even though more than fifty FDA-approved anti-seizure drugs (ASDs) are available. In this study, we attempted small molecule screening to identify potential therapeutic agents for the treatment of seizures using seizure-induced animal models. Through behavioral phenotype-based screening, 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) was identified as a prototype. GM-90432 treatment effectively decreased seizure-like behaviors in zebrafish and mice with chemically induced seizures. These results were consistent with decreased neuronal activity through immunohistochemistry for pERK in zebrafish larvae. Additionally, electroencephalogram (EEG) analysis revealed that GM-90432 decreases seizure-specific EEG events in adult zebrafish. Moreover, we revealed the preferential binding of GM-90432 to voltage-gated Na+ channels using a whole-cell patch clamp technique. Through pharmacokinetic analysis, GM-90432 effectively penetrated the blood-brain barrier and was distributed into the brain. Taken together, we suggest that GM-90432 has the potential to be developed into a new ASD candidate.

Quadrella incana (Capparaceae) Leaf Extract Enhances Proliferation and Maintenance of Neural Stem/Progenitor Cells through Upregulating Glycolytic Flux and Redox Potential.

Neural stem/progenitor cells (NSPCs) are self-renewing, multipotent cells located in the embryonic and adult central nervous system (CNS). Extensive preclinical and clinical studies have shed light on the potential of stem cell replacement therapy for various neurodegenerative diseases. The key prerequisite for the success of these clinical applications is the procurement of a sufficient number of high-quality NSPCs. In this study, we explored the biological activity of Quadrella incana leaf in NSPC homeostasis. We showed that the leaf extract of Quadrella incana upregulated NSPC marker and proliferative potential. On the other hand, Quadrella incana leaf suppressed spontaneous unintended NSPC differentiation. Mechanistically, Quadrella incana leaf contributed to the maintenance of NSPCs by upregulating glycolytic flux and redox potential.

Effects of cenobamate (YKP3089), a newly developed anti-epileptic drug, on voltage-gated sodium channels in rat hippocampal CA3 neurons.

New, more effective pharmacologic treatments for epilepsy are needed, as a substantial portion of patients (>30%) are refractory to currently available anti-epileptic drugs. Cenobamate (YKP3089) is an investigational anti-epileptic drug in clinical development. Two completed adequate and well-controlled studies demonstrated a significant reduction in focal seizures with cenobamate in patients with epilepsy. In this study, we characterized the effects of cenobamate on voltage-gated Na+ channels in acutely isolated rat hippocampal CA3 neurons using a whole-cell patch-clamp technique. While cenobamate had little effect on the peak component of transient Na+ current (INaT) induced by brief depolarizing step pulses, it potently inhibited the non-inactivating persistent component of INa (INaP). In addition, cenobamate potently inhibited the current by slow voltage-ramp stimuli. Cenobamate significantly shifted the steady-state fast inactivation relationship toward a hyperpolarizing range, indicating that cenobamate binds to voltage-gated Na+ channels at the inactivated state with a higher affinity. Cenobamate also accelerated the development of inactivation and retarded recovery from inactivation of voltage-gated Na+ channels. In current clamp experiments, cenobamate hyperpolarized membrane potentials in a concentration-dependent manner, and these effects were mediated by inhibiting the INaP. Cenobamate also increased the threshold for generation of action potentials, and decreased the number of action potentials elicited by depolarizing current injection. Given that the INaP plays a pivotal role in the repetitive and/or burst generation of action potentials, the cenobamate-mediated preferential blockade of INaP might contribute to anti-epileptic activity.

Cytoplasmic pro-apoptotic function of the tumor suppressor p73 is mediated through a modified mode of recognition of the anti-apoptotic regulator Bcl-XL.

In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL or BCL2L1). This binding disrupted an interaction between Bcl-XL and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-XL Interestingly, the structure of the Bcl-XL-p73-TAD16 peptide complex revealed a novel mechanism of Bcl-XL recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-XL, comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-XL recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.