Anterior cingulate cortex-related functional hyperconnectivity underlies sensory hypersensitivity in Grin2b-mutant mice.

Sensory abnormalities are observed in ~90% of individuals with autism spectrum disorders (ASD), but the underlying mechanisms are poorly understood. GluN2B, an NMDA receptor subunit that regulates long-term depression and circuit refinement during brain development, has been strongly implicated in ASD, but whether GRIN2B mutations lead to sensory abnormalities remains unclear. Here, we report that Grin2b-mutant mice show behavioral sensory hypersensitivity and brain hyperconnectivity associated with the anterior cingulate cortex (ACC). Grin2b-mutant mice with a patient-derived C456Y mutation (Grin2bC456Y/+) show sensory hypersensitivity to mechanical, thermal, and electrical stimuli through supraspinal mechanisms. c-fos and functional magnetic resonance imaging indicate that the ACC is hyperactive and hyperconnected with other brain regions under baseline and stimulation conditions. ACC pyramidal neurons show increased excitatory synaptic transmission. Chemogenetic inhibition of ACC pyramidal neurons normalizes ACC hyperconnectivity and sensory hypersensitivity. These results suggest that GluN2B critically regulates ASD-related cortical connectivity and sensory brain functions.

Inhibition of endocytic uptake of severe acute respiratory syndrome coronavirus 2 and endo-lysosomal acidification by diphenoxylate.

Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.

Discovery of (E)-2-(hydroxyimino)-N-(2 ((4methylpentyl)amino)ethyl)acetamide (KR-27425) as a non-pyridinium oxime reactivator of paraoxon-inhibited acetylcholinesterase.

This study aimed to explore non-pyridinium oxime acetylcholinesterase (AChE) reactivators that could hold the potential to overcome the limitations of the currently available compounds used in the clinic to treat the neurologic manifestations induced by intoxication with organophosphorus agents. Fifteen compounds with various non-pyridinium oxime moieties were evaluated for AChE activity at different concentrations, including aldoximes, ketoximes, and α-ketoaldoximes. The therapeutic potential of the oxime compounds was evaluated by assessing their ability to reactivate AChE inhibited by paraoxon. Among the tested compounds, α-Ketoaldoxime derivative 13 showed the highest reactivation (%) reaching 67 % and 60 % AChE reactivation when evaluated against OP-inhibited electric eel AChE at concentrations of 1,000 and 100 µM, respectively. Compound 13 showed a comparable reactivation ability of AChE (60 %) compared to that of pralidoxime (56 %) at concentrations of 100 µM. Molecular docking simulation of the most active compounds 12 and 13 was conducted to predict the binding mode of the reactivation of electric eel AChE. As a result, a non-pyridinium oxime moiety 13, is a potential reactivator of OP-inhibited AChE and is taken as a lead compound for the development of novel AChE reactivators with enhanced capacity to freely cross the blood-brain barrier.

Differential effects of two phosphodiesterase 4 inhibitors against lipopolysaccharide-induced neuroinflammation in mice.

BACKGROUND: Several phosphodiesterase 4 (PDE4) inhibitors have emerged as potential therapeutics for central nervous system (CNS) diseases. This study investigated the pharmacological effects of two selective PDE4 inhibitors, roflumilast and zatolmilast, against lipopolysaccharide-induced neuroinflammation. RESULTS: In BV-2 cells, the PDE4 inhibitor roflumilast reduced the production of nitric oxide and tumor necrosis factor-α (TNF-α) by inhibiting NF-κB phosphorylation. Moreover, mice administered roflumilast had significantly reduced TNF-α, interleukin-1ß (IL-1ß), and IL-6 levels in plasma and brain tissues. By contrast, zatolmilast, a PDE4D inhibitor, showed no anti-neuroinflammatory effects in vitro or in vivo. Next, in vitro and in vivo pharmacokinetic studies of these compounds in the brain were performed. The apparent permeability coefficients of 3 µM roflumilast and zatolmilast were high (> 23 × 10-6 cm/s) and moderate (3.72-7.18 × 10-6 cm/s), respectively, and increased in a concentration-dependent manner in the MDR1-MDCK monolayer. The efflux ratios were < 1.92, suggesting that these compounds are not P-glycoprotein substrates. Following oral administration, both roflumilast and zatolmilast were slowly absorbed and eliminated, with time-to-peak drug concentrations of 2-2.3 h and terminal half-lives of 7-20 h. Assessment of their brain dispositions revealed the unbound brain-to-plasma partition coefficients of roflumilast and zatolmilast to be 0.17 and 0.18, respectively. CONCLUSIONS: These findings suggest that roflumilast, but not zatolmilast, has the potential for use as a therapeutic agent against neuroinflammatory diseases.

PredPS: Attention-based graph neural network for predicting stability of compounds in human plasma.

Stability of compounds in the human plasma is crucial for maintaining sufficient systemic drug exposure and considered an essential factor in the early stages of drug discovery and development. The rapid degradation of compounds in the plasma can result in poor in vivo efficacy. Currently, there are no open-source software programs for predicting human plasma stability. In this study, we developed an attention-based graph neural network, PredPS to predict the plasma stability of compounds in human plasma using in-house and open-source datasets. The PredPS outperformed the two machine learning and two deep learning algorithms that were used for comparison indicating its stability-predicting efficiency. PredPS achieved an area under the receiver operating characteristic curve of 90.1%, accuracy of 83.5%, sensitivity of 82.3%, and specificity of 84.6% when evaluated using 5-fold cross-validation. In the early stages of drug discovery, PredPS could be a helpful method for predicting the human plasma stability of compounds. Saving time and money can be accomplished by adopting an in silico-based plasma stability prediction model at the high-throughput screening stage. The source code for PredPS is available at https://bitbucket.org/krict-ai/predps and the PredPS web server is available at https://predps.netlify.app.

Integrative Analyses Reveal the Anticancer Mechanisms and Sensitivity Markers of the Next-Generation Hypomethylating Agent NTX-301.

Epigenetic dysregulation characterized by aberrant DNA hypermethylation is a hallmark of cancer, and it can be targeted by hypomethylating agents (HMAs). Recently, we described the superior therapeutic efficacy of a novel HMA, namely, NTX-301, when used as a monotherapy and in combination with venetoclax in the treatment of acute myeloid leukemia. Following a previous study, we further explored the therapeutic properties of NTX-301 based on experimental investigations and integrative data analyses. Comprehensive sensitivity profiling revealed that NTX-301 primarily exerted anticancer effects against blood cancers and exhibited improved potency against a wide range of solid cancers. Subsequent assays showed that the superior efficacy of NTX-301 depended on its strong effects on cell cycle arrest, apoptosis, and differentiation. Due to its superior efficacy, low doses of NTX-301 achieved sufficiently substantial tumor regression in vivo. Multiomics analyses revealed the mechanisms of action (MoAs) of NTX-301 and linked these MoAs to markers of sensitivity to NTX-301 and to the demethylation activity of NTX-301 with high concordance. In conclusion, our findings provide a rationale for currently ongoing clinical trials of NTX-301 and will help guide the development of novel therapeutic options for cancer patients.

Pharmacokinetics and Tissue Distribution of 13C-Labeled Succinic Acid in Mice.

Succinic acid is widely used as a food additive, and its effects on sepsis, cancer, ataxia, and obesity were recently reported. Dietary drug exposure studies have been conducted to evaluate the in vivo efficacy of succinic acid, but limited pharmacokinetic information is available. Therefore, this study evaluated the pharmacokinetic profiles and tissue distribution of succinic acid following a single intravenous or oral dose. A surrogate analyte, succinic acid-13C4 (13C4SA), was administrated to distinguish endogenous and exogenous succinic acid. The concentration of 13C4SA was determined by a validated analytical method using mass spectrometry. After a 10 mg/kg intravenous dose, non-compartmental pharmacokinetic analysis in plasma illustrated that the clearance, volume of distribution, and terminal half-life of 13C4SA were 4574.5 mL/h/kg, 520.8 mL/kg, and 0.56 h, respectively. Oral 13C4SA was absorbed and distributed quickly (bioavailability, 1.5%) at a dose of 100 mg/kg. In addition, 13C4SA exposure was the highest in the liver, followed by brown adipose tissue, white adipose tissue, and the kidneys. This is the first report on the pharmacokinetics of succinic acid after a single dose in mice, and these results could provide a foundation for selecting dosing regimens for efficacy studies.

Anti-neuroinflammatory Effects and Brain Pharmacokinetic Properties of Selonsertib, an Apoptosis signal-regulating Kinase 1 Inhibitor, in mice.

Selonsertib is a first-in-class apoptosis signal-regulating kinase 1 (ASK1) inhibitor in clinical trials for treating NASH and diabetic kidney disease due to its anti-inflammatory and anti-fibrotic activities. In the present study, we investigated the anti-neuroinflammatory effects and brain pharmacokinetic properties of selonsertib. It inhibited inflammatory cytokines and NO production by suppressing phosphorylated ASK1 in the LPS-stimulated microglial cell line, BV2 cells. Consistent with the in vitro results, selonsertib attenuated plasma and brain TNF-α levels in the LPS-induced murine neuroinflammation model. In vitro and in vivo pharmacokinetic studies of selonsertib were conducted in support of central nervous system (CNS) drug discovery. In both Caco-2 and MDR-MDCK cells, selonsertib exhibited a high efflux ratio, showing that it is a P-gp substrate. Selonsertib was rapidly and effectively absorbed into the systemic circulation after oral treatment, with a Tmax of 0.5 h and oral bioavailability of 74%. In comparison with high systemic exposure with Cmax of 16.2 µg/ml and AUC of 64 µg·h/mL following oral dosing of 10 mg/kg, the brain disposition of selonsertib was limited, with Cmax of 0.08 µg/g and Kp value of 0.004. This study demonstrates that selonsertib can be a therapeutic agent for neuroinflammatory diseases.

Early postnatal serotonin modulation prevents adult-stage deficits in Arid1b-deficient mice through synaptic transcriptional reprogramming.

Autism spectrum disorder is characterized by early postnatal symptoms, although little is known about the mechanistic deviations that produce them and whether correcting them has long-lasting preventive effects on adult-stage deficits. ARID1B, a chromatin remodeler implicated in neurodevelopmental disorders, including autism spectrum disorder, exhibits strong embryonic- and early postnatal-stage expression. We report here that Arid1b-happloinsufficient (Arid1b+/-) mice display autistic-like behaviors at juvenile and adult stages accompanied by persistent decreases in excitatory synaptic density and transmission. Chronic treatment of Arid1b+/- mice with fluoxetine, a selective serotonin-reuptake inhibitor, during the first three postnatal weeks prevents synaptic and behavioral deficits in adults. Mechanistically, these rescues accompany transcriptomic changes, including upregulation of FMRP targets and normalization of HDAC4/MEF2A-related transcriptional regulation of the synaptic proteins, SynGAP1 and Arc. These results suggest that chronic modulation of serotonergic receptors during critical early postnatal periods prevents synaptic and behavioral deficits in adult Arid1b+/- mice through transcriptional reprogramming.

Antibacterial Activity against Clinical Isolates and In Vivo Efficacy of Coralmycins.

Coralmycins, such as coralmycin A and DH-coralmycin A, have novel molecular skeletons and have been reported to exhibit potent antibacterial activity against standard Gram-positive bacterial strains. Here, the in vitro antibacterial activity against an extensive clinical isolate collection, time-kill kinetics, pharmacokinetics (PK), and in vivo efficacy of coralmycins were studied. Coralmycin A showed potent antibacterial activity with an MIC90 of 1 mg/L against 73 clinical methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci isolates, which was 2-8 times higher than the corresponding activities of DH-coralmycin A, vancomycin, daptomycin, and linezolid, and against 73 vancomycin-resistant Enterococcus and Streptococcus pneumoniae isolates, which was 4-16 times higher than the corresponding activities of DH-coralmycin A, daptomycin, and linezolid. Pharmacokinetic analysis after i.v. injection showed that coralmycins have a moderate volume of distribution and moderate-to-high clearance in mice. The coralmycin A and DH-coralmycin A bioavailability values were 61.3% and 11.7%, respectively, after s.c. administration. In a mouse respiratory tract infection model, coralmycin A showed bacteriostatic and bactericidal in vivo efficacies at an s.c. administration of 4 and 100 mg/kg bid, respectively; these efficacies were similar to those of vancomycin at 4 and 20 mg/kg bid, respectively. The present findings indicate that coralmycin A has great potential as a new class of antibiotic for treating infections caused by multidrug-resistant Gram-positive bacteria.

Discovery and synthesis of 1,2,4-oxadiazole derivatives as novel inhibitors of Zika, dengue, Japanese encephalitis, and classical swine fever virus infections.

Zika virus (ZIKV), an arbovirus of the Flaviviridae family, has emerged as a significant public health concern owing to its association with congenital abnormalities and severe neurological sequelae. Thus, there is an urgent need to develop effective therapeutic approaches to efficiently treat ZIKV infections. This study used phenotypic screening to identify a series of 1,2,4-oxadiazole derivatives that possess antiviral activity against ZIKV infection. Subsequently, 28 new derivatives were designed, synthesized, and evaluated for this purpose. Among these compounds, 4-(5-phenyl-1,2,4-oxadiazol-3-yl)-N-(pyridin-3-ylmethyl)aniline (5d) had potent antiviral activity against ZIKV infections. Furthermore, a structure-activity relationship analysis indicated that a benzyl substitution on the aniline nitrogen of this compound improved potency while augmenting its drug-like properties. In addition, 5d exhibited antiviral activity against various viruses of Flaviviridae family of worldwide public health importance, such as dengue, Japanese encephalitis and classical swine fever viruses, indicating its potential as a lead compound for generating 1,2,4-oxadiazole derivatives with broad-spectrum anti-flaviviral properties.

PredMS: a random forest model for predicting metabolic stability of drug candidates in human liver microsomes.

MOTIVATION: Poor metabolic stability leads to drug development failure. Therefore, it is essential to evaluate the metabolic stability of small compounds for successful drug discovery and development. However, evaluating metabolic stability in vitro and in vivo is expensive, time-consuming and laborious. In addition, only a few free software programs are available for metabolic stability data and prediction. Therefore, in this study, we aimed to develop a prediction model that predicts the metabolic stability of small compounds. RESULTS: We developed a computational model, PredMS, which predicts the metabolic stability of small compounds as stable or unstable in human liver microsomes. PredMS is based on a random forest model using an in-house database of metabolic stability data of 1917 compounds. To validate the prediction performance of PredMS, we generated external test data of 61 compounds. PredMS achieved an accuracy of 0.74, Matthew's correlation coefficient of 0.48, sensitivity of 0.70, specificity of 0.86, positive predictive value of 0.94 and negative predictive value of 0.46 on the external test dataset. PredMS will be a useful tool to predict the metabolic stability of small compounds in the early stages of drug discovery and development. AVAILABILITY AND IMPLEMENTATION: The source code for PredMS is available at https://bitbucket.org/krictai/predms, and the PredMS web server is available at https://predms.netlify.app. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pharmacokinetic Characterization of Supinoxin and Its Physiologically Based Pharmacokinetic Modeling in Rats.

Supinoxin is a novel anticancer drug candidate targeting the Y593 phospho-p68 RNA helicase, by exhibiting antiproliferative activity and/or suppression of tumor growth. This study aimed to characterize the in vitro and in vivo pharmacokinetics of supinoxin and attempt physiologically based pharmacokinetic (PBPK) modeling in rats. Supinoxin has good permeability, comparable to that of metoprolol (high permeability compound) in Caco-2 cells, with negligible net absorptive or secretory transport observed. After an intravenous injection at a dose range of 0.5-5 mg/kg, the terminal half-life (i.e., 2.54-2.80 h), systemic clearance (i.e., 691-865 mL/h/kg), and steady state volume of distribution (i.e., 2040-3500 mL/kg) of supinoxin remained unchanged, suggesting dose-independent (i.e., dose-proportional) pharmacokinetics for the dose ranges studied. After oral administration, supinoxin showed modest absorption with an absolute oral bioavailability of 56.9-57.4%. The fecal recovery following intravenous and oral administration was 16.5% and 46.8%, respectively, whereas the urinary recoveries in both administration routes were negligible. Supinoxin was mainly eliminated via NADPH-dependent phase I metabolism (i.e., 58.5% of total clearance), while UDPGA-dependent phase II metabolism appeared negligible in the rat liver microsome. Supinoxin was most abundantly distributed in the adipose tissue, gut, and liver among the nine major tissues studied (i.e., the brain, liver, kidneys, heart, lungs, spleen, gut, muscles, and adipose tissue), and the tissue exposure profiles of supinoxin were well predicted with physiologically based pharmacokinetics.

SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function.

Glycine transporters (GlyT1 and GlyT2) that regulate levels of brain glycine, an inhibitory neurotransmitter with co-agonist activity for NMDA receptors (NMDARs), have been considered to be important targets for the treatment of brain disorders with suppressed NMDAR function such as schizophrenia. However, it remains unclear whether other amino acid transporters expressed in the brain can also regulate brain glycine levels and NMDAR function. Here, we report that SLC6A20A, an amino acid transporter known to transport proline based on in vitro data but is understudied in the brain, regulates proline and glycine levels and NMDAR function in the mouse brain. SLC6A20A transcript and protein levels were abnormally increased in mice carrying a mutant PTEN protein lacking the C terminus through enhanced ß-catenin binding to the Slc6a20a gene. These mice displayed reduced extracellular levels of brain proline and glycine and decreased NMDAR currents. Elevating glycine levels back to normal ranges by antisense oligonucleotide-induced SLC6A20 knockdown, or the competitive GlyT1 antagonist sarcosine, normalized NMDAR currents and repetitive climbing behavior observed in these mice. Conversely, mice lacking SLC6A20A displayed increased extracellular glycine levels and NMDAR currents. Lastly, both mouse and human SLC6A20 proteins mediated proline and glycine transports, and SLC6A20 proteins could be detected in human neurons. These results suggest that SLC6A20 regulates proline and glycine homeostasis in the brain and that SLC6A20 inhibition has therapeutic potential for brain disorders involving NMDAR hypofunction.

LightBBB: computational prediction model of blood-brain-barrier penetration based on LightGBM.

MOTIVATION: Identification of blood-brain barrier (BBB) permeability of a compound is a major challenge in neurotherapeutic drug discovery. Conventional approaches for BBB permeability measurement are expensive, time-consuming and labor-intensive. BBB permeability is associated with diverse chemical properties of compounds. However, BBB permeability prediction models have been developed using small datasets and limited features, which are usually not practical due to their low coverage of chemical diversity of compounds. Aim of this study is to develop a BBB permeability prediction model using a large dataset for practical applications. This model can be used for facilitated compound screening in the early stage of brain drug discovery. RESULTS: A dataset of 7162 compounds with BBB permeability (5453 BBB+ and 1709 BBB-) was compiled from the literature, where BBB+ and BBB- denote BBB-permeable and non-permeable compounds, respectively. We trained a machine learning model based on Light Gradient Boosting Machine (LightGBM) algorithm and achieved an overall accuracy of 89%, an area under the curve (AUC) of 0.93, specificity of 0.77 and sensitivity of 0.93, when 10-fold cross-validation was performed. The model was further evaluated using 74 central nerve system compounds (39 BBB+ and 35 BBB-) obtained from the literature and showed an accuracy of 90%, sensitivity of 0.85 and specificity of 0.94. Our model outperforms over existing BBB permeability prediction models. AVAILABILITYAND IMPLEMENTATION: The prediction server is available at http://ssbio.cau.ac.kr/software/bbb.

Discovery of a Novel Chemical Scaffold Against Mutant Isocitrate Dehydrogenase 1 (IDH1).

BACKGROUND: Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are frequently found in various cancer types. IDH1 mutants produce 2-hydroxyglutarate (2-HG), an oncometabolite, from alpha-ketoglutarate (α-KG). This 2-HG plays a key role in tumorigenesis via inhibition of α-KG dependent enzymes. For this reason, IDH1 mutant could be an ideal target for the treatment of cancer. MATERIALS AND METHODS: To find a new IDH1 inhibitor, 8,364 compounds were obtained from Korea Chemical Bank. Using high-throughput screening (HTS) of a chemical library, we unveiled a compound that could inhibit the IDH1 mutant. RESULTS: According to the enzyme assay, our compound (KRC-09) effectively inhibited the activity of IDH1 R132H mutant. In addition, KRC-09 decreased the concentration of intracellular 2-HG in the U-87 MG cell line harboring IDH1 R132H. CONCLUSION: In this article, we present a novel chemical scaffold that suppresses the activity of an IDH1 mutant.

Design and characterization of cereblon-mediated androgen receptor proteolysis-targeting chimeras.

Proteolysis-targeting chimera (PROTAC)-mediated protein degradation is a rapidly emerging therapeutic intervention that induces the degradation of targeted proteins. Herein, we report the design and biological evaluation of a series of androgen receptor (AR) PROTAC degraders for the treatment of metastatic castration-resistant prostate cancer. Predominantly, instead of thalidomide, we utilized the TD-106 scaffold, a novel cereblon (CRBN) binder that was identified in our previous study. Our results suggest that the linker position in the TD-106 CRBN binder is critical for the efficiency of AR degradation. The compounds attached to the 6-position of TD-106 promoted better degradation of AR than those at the 5- and 7-positions. Among the synthesized AR PROTACs, the representative degrader 33c (TD-802) effectively induced AR protein degradation, with a degradation concentration 50% of 12.5 nM and a maximum degradation of 93% in LNCaP prostate cancer cells. Additionally, most AR PROTAC degraders, including TD-802, displayed good liver microsomal stability and in vivo pharmacokinetic properties. Finally, we showed that TD-802 effectively inhibited tumor growth in an in vivo xenograft study.

Neurochemical and behavioral analysis by acute exposure to bisphenol A in zebrafish larvae model.

Bisphenol A (BPA) is a chemical monomer widely used in the production of hard plastics for food containers and personal items. Through improper industrial control and disposal, BPA has become a pervasive environmental contaminant, and toxicological studies have shown potent xenobiotic endocrine disruptor activity. Prenatal exposure in particular can lead to infertility and nervous system disorders characterized by behavioral aggression, depression, and cognitive impairment, thus necessitating careful hazard assessment. In this study, we evaluated BPA accumulation rate, blood-brain barrier (BBB) permeability, lethality, cardiotoxicity, behavioral effects, and impacts on multiple neurochemical pathways in zebrafish larvae. The bioconcentration factor (BCF) ranged from 1.95 to 10.0, resulting in a high rate of accumulation in the larval body. Also, high BBB permeability allowed BPA to accumulate at similar rates in both zebrafish and adult mouse (blood to brain concentration ratios of 3.2-6.7 and 1.8 to 5.5, respectively). In addition, BPA-exposed zebrafish larvae exhibited developmental deformities, reduced heart rate, and impaired behavioral patterns, including decreased total distance traveled, slower movement velocity, and altered color-preference. These impairments were associated with inhibition of the phenylalanine to dopamine synthesis pathway and an imbalance between excitatory and inhibitory neurotransmitter systems. Our results suggest that behavioral alteration in BPA-exposed zebrafish result from high accumulation and ensuing dysregulation of serotonergic, kynurenergic, dopaminergic, cholinergic, and GABAergic neurotransmitter systems. In conclusion, similarities in toxic responses to mammalian models highlight the utility of the zebrafish larva as a convenient model for screening environmental toxins.

Disordered region of cereblon is required for efficient degradation by proteolysis-targeting chimera.

Proteolysis targeting chimeras (PROTACs) are an emerging strategy for promoting targeted protein degradation by inducing the proximity between targeted proteins and E3 ubiquitin ligases. Although successful degradation of numerous proteins by PROTACs has been demonstrated, the elements that determine the degradability of PROTAC-targeted proteins have not yet been explored. In this study, we developed von Hippel-Lindau-Cereblon (VHL-CRBN) heterodimerizing PROTACs that induce the degradation of CRBN, but not VHL. A quantitative proteomic analysis further revealed that VHL-CRBN heterodimerizing PROTACs induced the degradation of CRBN, but not the well-known immunomodulatory drug (IMiD) neo-substrates, IKAROS family zinc finger 1 (IKZF1) and -3 (IZKF3). Moreover, truncation of disordered regions of CRBN and the androgen receptor (AR) attenuated their PROTAC-induced degradation, and attachment of the disordered region to stable CRBN or AR facilitated PROTAC-induced degradation. Thus, these results suggest that the intrinsically disordered region of targeted proteins is essential for efficient proteolysis, providing a novel criterion for choosing degradable protein targets.

αVß3-Targeted Delivery of Camptothecin-Encapsulated Carbon Nanotube-Cyclic RGD in 2D and 3D Cancer Cell Culture.

Integrin αvß3 is widely expressed in various types of human cancer lines and plays a key role in angiogenesis for tumor growth and metastasis. Delivery of therapeutics to αvß3-expressing tumors can thus be a promising approach for treating cancer. For targeted delivery of anticancer therapeutics to αvß3-expressing tumor cells, cyclic arginylglycylaspartic acid (RGD) peptide was covalently conjugated to the surface of carboxylic acid-functionalized carbon nanotubes (fCNTs), and the topoisomerase I inhibitor camptothecin (CPT) was encapsulated in the fCNTs (CPT@fCNT-RGD). CPT@fCNT-RGD was successfully delivered to αvß3-expressing A375 cells, and compared with nontargeted CPT@fCNT, it provided 3.78- and 3.02-fold increases in the anticancer effect in 2D and 3D culture. Analysis of apoptosis-related gene expression shows that the expression levels of Bax, cleaved caspase-3, and nuclear factor kappa-light-chain-enhancer of activated B cells were significantly increased in A375 cells incubated with CPT@fCNT-RGD compared with those incubated with CPT@fCNT. These results suggest that cyclic RGD-conjugated CNTs encapsulating an anticancer therapeutic can be a promising platform for treating cancer.