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PLoS Biology May 2024Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In...
Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.
Topics: Humans; Animals; Proteolysis; Mice; Ubiquitin-Protein Ligases; Oxindoles; Cell Cycle Proteins; Transcription Factors; Cell Line, Tumor; Xenograft Model Antitumor Assays; Mice, Nude; HEK293 Cells; Structure-Activity Relationship; Proteasome Endopeptidase Complex; Azepines; Antineoplastic Agents; Female; Bromodomain Containing Proteins; Receptors, Interleukin-17
PubMed: 38768083
DOI: 10.1371/journal.pbio.3002550 -
International Immunopharmacology Jun 2024Epilepsy is a chronic disabling disease poorly controlled by available antiseizure medications. Oridonin, a bioactive alkaloid with anti-inflammatory properties and...
BACKGROUND
Epilepsy is a chronic disabling disease poorly controlled by available antiseizure medications. Oridonin, a bioactive alkaloid with anti-inflammatory properties and neuroprotective effects, can inhibit the increased excitability of neurons caused by glutamate accumulation at the cellular level. However, whether oridonin affects neuronal excitability and whether it has antiepileptic potential has not been reported in animal models or clinical studies.
METHOD
Pentylenetetrazol was injected into mice to create a model of chronic epilepsy. Seizure severity was assessed using the Racine scale, and the duration and latency of seizures were observed. Abnormal neuronal discharge was detected using electroencephalography, and neuronal excitability was assessed using calcium imaging. Damage to hippocampal neurons was evaluated using Hematoxylin-Eosin and Nissl staining. The expression of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome and other pyroptosis-related proteins was determined using western blotting and immunofluorescence. A neuronal pyroptosis model was established using the supernatant of BV2 cells treated with lipopolysaccharide and adenosine triphosphate to stimulate hippocampal neurons.
RESULTS
Oridonin (1 and 5 mg/kg) reduced neuronal damage, increased the latency of seizures, and shortened the duration of fully kindled seizures in chronic epilepsy model mice. Oridonin decreased abnormal discharge during epileptic episodes and suppressed increased neuronal excitability. In vitro experiments showed that oridonin alleviated pyroptosis in hippocampal HT22 neurons.
CONCLUSION
Oridonin exerts neuroprotective effects by inhibiting pyroptosis through the NLRP3/caspase-1 pathway in chronic epilepsy model mice. It also reduces pyroptosis in hippocampal neurons in vitro, suggesting its potential as a therapy for epilepsy.
Topics: Animals; Diterpenes, Kaurane; NLR Family, Pyrin Domain-Containing 3 Protein; Neuroprotective Agents; Epilepsy; Pyroptosis; Mice; Anticonvulsants; Male; Hippocampus; Neurons; Disease Models, Animal; Pentylenetetrazole; Mice, Inbred C57BL; Inflammasomes; Cell Line; Seizures
PubMed: 38759374
DOI: 10.1016/j.intimp.2024.112247 -
ENeuro May 2024The voltage-gated calcium channel subunit α2δ-2 controls calcium-dependent signaling in neurons, and loss of this subunit causes epilepsy in both mice and humans. To...
The voltage-gated calcium channel subunit α2δ-2 controls calcium-dependent signaling in neurons, and loss of this subunit causes epilepsy in both mice and humans. To determine whether mice without α2δ-2 demonstrate hippocampal activation or histopathological changes associated with seizure activity, we measured expression of the activity-dependent gene c and various histopathological correlates of temporal lobe epilepsy (TLE) in hippocampal tissue from wild-type (WT) and α2δ-2 knock-out ( KO) mice using immunohistochemical staining and confocal microscopy. Both genotypes demonstrated similarly sparse c- and expressions within the hippocampal dentate granule cell layer (GCL) at baseline, consistent with no difference in basal activity of granule cells between genotypes. Surprisingly, when mice were assayed 1 h after handling-associated convulsions, KO mice had fewer c--positive cells but dramatically increased expression in the dentate gyrus compared with WT mice. After administration of a subthreshold pentylenetetrazol dose, however, KO mice dentate had significantly more c- expression compared with WT mice. Other histopathological markers of TLE in these mice, including markers of neurogenesis, glial activation, and mossy fiber sprouting, were similar between WT and KO mice, apart from a small but statistically significant increase in hilar mossy cell density, opposite to what is typically found in mice with TLE. This suggests that the differences in seizure-associated dentate gyrus function in the absence of α2δ-2 protein are likely due to altered functional properties of the network without associated structural changes in the hippocampus at the typical age of seizure onset.
Topics: Animals; Mice, Knockout; Seizures; Hippocampus; Proto-Oncogene Proteins c-fos; Male; Calcium Channels; Mice, Inbred C57BL; Pentylenetetrazole; Mice; Disease Models, Animal; Neurons; Convulsants
PubMed: 38749701
DOI: 10.1523/ENEURO.0486-23.2024 -
Biomedicine & Pharmacotherapy =... Jun 2024Brain apoptosis is one of the main causes of epileptogenesis. The antiapoptotic effect and potential mechanism of Q808, an innovative anticonvulsant chemical, have never...
Brain apoptosis is one of the main causes of epileptogenesis. The antiapoptotic effect and potential mechanism of Q808, an innovative anticonvulsant chemical, have never been reported. In this study, the seizure stage and latency to reach stage 2 of pentylenetetrazol (PTZ) seizure rat model treated with Q808 were investigated. The morphological change and neuronal apoptosis in the hippocampus were detected by hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, respectively. The hippocampal transcriptomic changes were observed using RNA sequencing (RNA-seq). The expression levels of hub genes were verified by quantitative reverse-transcription PCR (qRT-PCR). Results revealed that Q808 could allay the seizure score and prolong the stage 2 latency in seizure rats. The morphological changes of neurons and the number of apoptotic cells in the DG area were diminished by Q808 treatment. RNA-seq analysis revealed eight hub genes, including Map2k3, Nfs1, Chchd4, Hdac6, Siglec5, Slc35d3, Entpd1, and LOC103690108, and nine hub pathways among the control, PTZ, and Q808 groups. Hub gene Nfs1 was involved in the hub pathway sulfur relay system, and Map2k3 was involved in the eight remaining hub pathways, including Amyotrophic lateral sclerosis, Cellular senescence, Fc epsilon RI signaling pathway, GnRH signaling pathway, Influenza A, Rap1 signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway. qRT-PCR confirmed that the mRNA levels of these hub genes were consistent with the RNA-seq results. Our findings might contribute to further studies exploring the new apoptosis mechanism and actions of Q808.
Topics: Animals; Pentylenetetrazole; Hippocampus; Apoptosis; Anticonvulsants; Male; Transcriptome; Epilepsy; Gene Expression Profiling; Rats; Rats, Sprague-Dawley; Disease Models, Animal; Neurons; Seizures
PubMed: 38739991
DOI: 10.1016/j.biopha.2024.116746 -
Molecules (Basel, Switzerland) Apr 2024Two series, "" and "", each consisting of nine chemical compounds, with 2,3-disubstituted quinazolin-4(3H)-one scaffold, were synthesized and evaluated for their...
Two series, "" and "", each consisting of nine chemical compounds, with 2,3-disubstituted quinazolin-4(3H)-one scaffold, were synthesized and evaluated for their anticonvulsant activity. They were investigated as dual potential positive allosteric modulators of the GABA receptor at the benzodiazepine binding site and inhibitors of carbonic anhydrase II. Quinazolin-4(3H)-one derivatives were evaluated in vivo (D = 50, 100, 150 mg/kg, administered intraperitoneally) using the pentylenetetrazole (PTZ)-induced seizure model in mice, with phenobarbital and diazepam, as reference anticonvulsant agents. The in silico studies suggested the compounds act as anticonvulsants by binding on the allosteric site of GABA receptor and not by inhibiting the carbonic anhydrase II, because the ligands-carbonic anhydrase II predicted complexes were unstable in the molecular dynamics simulations. The mechanism targeting GABA receptor was confirmed through the in vivo flumazenil antagonism assay. The pentylenetetrazole experimental anticonvulsant model indicated that the tested compounds, - and -, present a potential anticonvulsant activity. The evaluation, considering the percentage of protection against PTZ, latency until the onset of the first seizure, and reduction in the number of seizures, revealed more favorable results for the "" series, particularly for compound .
Topics: Anticonvulsants; Animals; Mice; Seizures; Receptors, GABA-A; Pentylenetetrazole; Quinazolinones; Molecular Docking Simulation; Male; Structure-Activity Relationship; Molecular Dynamics Simulation; Computer Simulation; Disease Models, Animal; Molecular Structure; Allosteric Site
PubMed: 38731442
DOI: 10.3390/molecules29091951 -
Journal of Ethnopharmacology Sep 2024The use of medicinal plants for central nervous system (CNS)-related ailments, such as epilepsy and anxiety, is prevalent in South Africa. Plants from the Lamiaceae...
ETHNOPHARMACOLOGICAL RELEVANCE
The use of medicinal plants for central nervous system (CNS)-related ailments, such as epilepsy and anxiety, is prevalent in South Africa. Plants from the Lamiaceae family are commonly used for their therapeutic benefits. Leonotis leonurus (L.) R.Br. has been reported in ethnobotanical literature to have anticonvulsant and anxiolytic effects through the inhalation of pyrolysis products obtained by combustion of the aerial parts.
AIM AND OBJECTIVES
To explore the chemical profiles and CNS activity of the smoke extract and isolated constituents of L. leonurus in zebrafish larvae, through anticonvulsive and anxiolytic activity assays.
MATERIALS AND METHODS
The smoke extract of L. leonurus was obtained through the combustion of the aerial parts of the plant using a custom-built smoke recovery apparatus. The chemical profile of the smoke constituents was determined using Ultra-Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS). Targeted compounds were subjected to preparative High-Performance Liquid Chromatography for separation before structure elucidation using Nuclear Magnetic Resonance (NMR). The maximum tolerated concentrations, as well as the anxiolytic activity of the smoke extract were determined in five days post fertilisation zebrafish larvae. Reverse-thigmotaxis and locomotor activity of larvae in the light/dark transition assay were used to determine anxiolytic activity. Zebrafish larvae at six days post fertilisation (dpf) were subjected to several concentrations of the smoke constituents of L. leonurus. The baseline locomotor activity of the larvae was tracked for 30 min, prior to addition of pentylenetetrazole (PTZ) to induce seizure-like behaviour in the larvae, after which the locomotor activity of the larvae was once again tracked for an additional 30 min.
RESULTS
The UPLC-MS profiles of the smoke extract revealed the presence of two main compounds, leoleorin A and leoleorin B, which were targeted and isolated. Upon subjection to NMR spectroscopy for structure elucidation, the compounds were confirmed to be labdane diterpenoids. Both leoleorin A and leoleorin B, and the smoke extract displayed suppression of the PTZ induced seizure-like behaviour in zebrafish larvae. Under light and dark conditions, the smoke extract and compounds displayed potential anxiolytic activity at different concentrations.
CONCLUSION
Our results suggest that the smoke constituents of L. leonurus may exert anticonvulsant and anxiolytic effects which align with the traditional indications and the mode of administration.
Topics: Animals; Zebrafish; Anti-Anxiety Agents; Smoke; Plant Extracts; Anticonvulsants; Seizures; Larva; Lamiaceae; Pentylenetetrazole; Plant Components, Aerial; South Africa; Behavior, Animal
PubMed: 38688356
DOI: 10.1016/j.jep.2024.118271 -
Experimental Neurology Jul 2024Interleukin-1 receptor-associated kinase 4 (IRAK4) plays an important role in immune modulation in various central nervous system disorders. However, IRAK4 has not been...
BACKGROUND
Interleukin-1 receptor-associated kinase 4 (IRAK4) plays an important role in immune modulation in various central nervous system disorders. However, IRAK4 has not been reported in epilepsy models in animal and clinical studies, nor has its involvement in regulating pyroptosis in epilepsy.
METHOD
First, we performed transcriptome sequencing, quantitative real-time polymerase chain reaction, and western blot analysis on the hippocampal tissues of refractory epilepsy patients to measure the mRNA and protein levels of IRAK4 and pyroptosis-related proteins. Second, we successfully established a pentylenetetrazol (PTZ)-induced seizure mouse model. We conducted behavioral tests, electroencephalography, virus injection, and molecular biology experiments to investigate the role of IRAK4 in seizure activity regulation.
RESULTS
IRAK4 is upregulated in the hippocampus of epilepsy patients and PTZ-induced seizure model mice. IRAK4 expression is observed in the hilar neurons of PTZ-induced mice. Knocking down IRAK4 in PTZ-induced mice downregulated pyroptosis-related protein expression and alleviated seizure activity. Overexpressing IRAK4 in naive mice upregulated pyroptosis-related protein expression and increased PTZ-induced abnormal neuronal discharges. IRAK4 and NF-κB were found to bind to each other in patient hippocampal tissue samples. Pyrrolidine dithiocarbamate reversed the pyroptosis-related protein expression increase caused by PTZ. PF-06650833 alleviated seizure activity and inhibited pyroptosis in PTZ-induced seizure mice.
CONCLUSION
IRAK4 plays a key role in the pathological process of epilepsy, and its potential mechanism may be related to pyroptosis mediated by the NF-κB/NLRP3 signaling pathway. PF-06650833 has potential as a therapeutic agent for alleviating epilepsy.
Topics: Animals; Interleukin-1 Receptor-Associated Kinases; Hippocampus; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Mice; Signal Transduction; Humans; NF-kappa B; Male; Seizures; Neurons; Epilepsy; Female; Mice, Inbred C57BL; Adult; Pentylenetetrazole; Young Adult; Adolescent; Child
PubMed: 38685307
DOI: 10.1016/j.expneurol.2024.114794 -
Molecules (Basel, Switzerland) Apr 2024Bromodomain 4 and 9 (BRD4 and BRD9) have been regarded as important targets of drug designs in regard to the treatment of multiple diseases. In our current study,...
Bromodomain 4 and 9 (BRD4 and BRD9) have been regarded as important targets of drug designs in regard to the treatment of multiple diseases. In our current study, molecular dynamics (MD) simulations, deep learning (DL) and binding free energy calculations are integrated to probe the binding modes of three inhibitors (H1B, JQ1 and TVU) to BRD4 and BRD9. The MD trajectory-based DL successfully identify significant functional function domains, such as BC-loop and ZA-loop. The information from the post-processing analysis of MD simulations indicates that inhibitor binding highly influences the structural flexibility and dynamic behavior of BRD4 and BRD9. The results of the MM-GBSA calculations not only suggest that the binding ability of H1B, JQ1 and TVU to BRD9 are stronger than to BRD4, but they also verify that van der Walls interactions are the primary forces responsible for inhibitor binding. The hot spots of BRD4 and BRD9 revealed by residue-based free energy estimation provide target sites of drug design in regard to BRD4 and BRD9. This work is anticipated to provide useful theoretical aids for the development of selective inhibitors over BRD family members.
Topics: Transcription Factors; Molecular Dynamics Simulation; Cell Cycle Proteins; Protein Binding; Humans; Deep Learning; Binding Sites; Thermodynamics; Triazoles; Azepines; Nuclear Proteins; Molecular Docking Simulation; Bromodomain Containing Proteins
PubMed: 38675678
DOI: 10.3390/molecules29081857 -
The Lancet. Gastroenterology &... Jul 2024In patients with Alagille syndrome, cholestasis-associated clinical features can include high serum bile acids and severe pruritus that can necessitate liver... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
In patients with Alagille syndrome, cholestasis-associated clinical features can include high serum bile acids and severe pruritus that can necessitate liver transplantation. We aimed to evaluate the efficacy and safety of the ileal bile acid transporter inhibitor odevixibat versus placebo in patients with Alagille syndrome.
METHODS
The ASSERT study was a phase 3, double-blind, randomised, placebo-controlled trial that enrolled patients at 21 medical centres or hospitals in ten countries (Belgium, France, Germany, Italy, Malaysia, the Netherlands, Poland, Türkiye, the UK, and the USA). Eligible patients had a genetically confirmed diagnosis of Alagille syndrome, a history of significant pruritus, and elevated serum bile acids. Patients were randomly assigned (2:1) to receive oral odevixibat 120 μg/kg per day or placebo for 24 weeks (in a block size of six and stratified by age: <10 years and ≥10 years to <18 years) via a web-based system. Patients, clinicians, study staff, and people analysing the data were masked to treatment allocation. The primary efficacy endpoint was change in caregiver-reported scratching score (on the PRUCISION instrument; range 0-4) from baseline to weeks 21-24. The prespecified key secondary efficacy endpoint was change in serum bile acid concentration from baseline to the average of weeks 20 and 24. Outcomes were analysed in patients who received at least one dose of study drug (the full analysis set for efficacy outcomes and the safety analysis set for safety outcomes). This trial is registered on ClinicalTrials.gov (NCT04674761) and EudraCT (2020-004011-28), and is completed.
FINDINGS
Between Feb 26, 2021, and Sept 9, 2022, 52 patients were randomly assigned to receive odevixibat (n=35) or placebo (n=17), all of whom were included in the analysis sets. The median age was 5·5 years (IQR 3·2 to 8·9). 27 (52%) of 52 patients were male and 25 (48%) were female. The mean scratching score was elevated at baseline in both groups (2·8 [SD 0·5] for odevixibat vs 3·0 [0·6] for placebo). Mean scratching scores at weeks 21-24 were 1·1 (0·9) for odevixibat and 2·2 (1·0) for placebo, representing a least-squares (LS) mean change of -1·7 (95% CI -2·0 to -1·3) for odevixibat and -0·8 (-1·3 to -0·3) for placebo, which was significantly greater for odevixibat than for placebo (difference in LS mean change from baseline -0·9 [95% CI -1·4 to -0·3]; p=0·0024). Odevixibat also resulted in significantly greater reductions in mean serum bile acids from baseline versus placebo (237 μmol/L [SD 115] with odevixibat vs 246 μmol/L [121] with placebo) to the average of weeks 20 and 24 (149 μmol/L [102] vs 271 μmol/L [167]; LS mean change -90 μmol/L [95% CI -133 to -48] with odevixibat vs 22 μmol/L [-35 to 80] with placebo; difference in LS mean change -113 μmol/L [95% CI -179 to -47]; p=0·0012). The most common treatment-emergent adverse events were diarrhoea (ten [29%] of 35 patients in the odevixibat group vs one [6%] of 17 in the placebo group) and pyrexia (eight [23%] vs four [24%]). Seven patients had serious treatment-emergent adverse events during the treatment period: five (14%) in the odevixibat group and two (12%) in the placebo group. No patients discontinued treatment and there were no deaths.
INTERPRETATION
Odevixibat could be an efficacious non-surgical intervention to improve pruritus, reduce serum bile acids, and enhance the standard of care in patients with Alagille syndrome. Longer-term safety and efficacy data of odevixibat in this population are awaited from the ongoing, open-label ASSERT-EXT study.
FUNDING
Albireo Pharma, an Ipsen company.
Topics: Humans; Double-Blind Method; Alagille Syndrome; Male; Female; Child; Adolescent; Pruritus; Treatment Outcome; Bile Acids and Salts; Adult; Child, Preschool; Young Adult; Carrier Proteins; Membrane Glycoproteins; Methylamines; Thiazepines
PubMed: 38670135
DOI: 10.1016/S2468-1253(24)00074-8 -
Cancer Research Communications May 2024The most common oncogenic driver mutations for non-small cell lung cancer (NSCLC) activate EGFR or KRAS. Clinical trials exploring treatments for EGFR- or KRAS-mutated...
UNLABELLED
The most common oncogenic driver mutations for non-small cell lung cancer (NSCLC) activate EGFR or KRAS. Clinical trials exploring treatments for EGFR- or KRAS-mutated (EGFRmut or KRASmut) cancers have focused on small-molecule inhibitors targeting the driver mutations. Typically, these inhibitors perform more effectively based on combination with either chemotherapies, or other targeted therapies. For EGFRmut NSCLC, a combination of inhibitors of EGFR and Aurora-A kinase (AURKA), an oncogene commonly overexpressed in solid tumors, has shown promising activity in clinical trials. Interestingly, a number of recent studies have indicated that EGFR activity supports overall viability of tumors lacking EGFR mutations, and AURKA expression is abundant in KRASmut cell lines. In this study, we have evaluated dual inhibition of EGFR and AURKA in KRASmut NSCLC models. These data demonstrate synergy between the EGFR inhibitor erlotinib and the AURKA inhibitor alisertib in reducing cell viability and clonogenic capacity in vitro, associated with reduced activity of EGFR pathway effectors, accumulation of enhanced aneuploid cell populations, and elevated cell death. Importantly, the erlotinib-alisertib combination also synergistically reduces xenograft growth in vivo. Analysis of signaling pathways demonstrated that the combination of erlotinib and alisertib was more effective than single-agent treatments at reducing activity of EGFR and pathway effectors following either brief or extended administration of the drugs. In sum, this study indicates value of inhibiting EGFR in KRASmut NSCLC, and suggests the specific value of dual inhibition of AURKA and EGFR in these tumors.
SIGNIFICANCE
The introduction of specific KRAS G12C inhibitors to the clinical practice in lung cancer has opened up opportunities that did not exist before. However, G12C alterations are only a subtype of all KRAS mutations observed. Given the high expression of AURKA in KRASmut NSCLC, our study could point to a potential therapeutic option for this subgroup of patients.
Topics: Carcinoma, Non-Small-Cell Lung; Humans; Aurora Kinase A; ErbB Receptors; Proto-Oncogene Proteins p21(ras); Lung Neoplasms; Animals; Erlotinib Hydrochloride; Mutation; Mice; Cell Line, Tumor; Xenograft Model Antitumor Assays; Protein Kinase Inhibitors; Drug Synergism; Pyrimidines; Azepines; Antineoplastic Combined Chemotherapy Protocols
PubMed: 38639476
DOI: 10.1158/2767-9764.CRC-23-0482