Blockade of vasoactive intestinal peptide receptor 2 (VIPR2) signaling suppresses cyclin D1-dependent cell-cycle progression in MCF-7 cells.

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a G protein-coupled receptor that binds to Gαs, Gαi, and Gαq proteins to regulate various downstream signaling molecules, such as protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and phospholipase C. In this study, we examined the role of VIPR2 in cell cycle progression. KS-133, a newly developed VIPR2-selective antagonist peptide, attenuated VIP-induced cell proliferation in MCF-7 cells. The percentage of cells in the S-M phase was decreased in MCF-7 cells treated with KS-133. KS-133 in the presence of VIP decreased the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and glycogen synthase kinase-3ß (GSK3ß), resulting in a decrease in cyclin D1 levels. In MCF-7 cells stably-expressing VIPR2, KS-133 decreased PI3K activity and cAMP levels. Treatment with the ERK-specific kinase (MEK) inhibitor U0126 and the class I PI3K inhibitor ZSTK474 decreased the percentage of cells in the S phase. KS-133 reduced the percentage of cells in the S phase more than treatment with U0126 or ZSTK474 alone and did not affect the effect of the mixture of these inhibitors. Our findings suggest that VIPR2 signaling regulates cyclin D1 levels through the cAMP/PKA/ERK and PI3K/AKT/GSK3ß pathways, and mediates the G1/S transition to control cell proliferation.

Lipid bilayer membrane permeability mechanism of the K-Ras(G12D)-inhibitory bicyclic peptide KS-58 elucidated by molecular dynamics simulations.

Peptides are mid-size molecules (700-2000 g/mol) and have attracted particular interest as therapeutic modalities as they are superior in controlling protein-protein interactions, a process that is a typical drug target category, compared with small molecules (<500 g/mol). In 2020, we identified KS-58 (1333 g/mol) as a K-Ras(G12D)-inhibitory bicyclic peptide and suggested its cell membrane permeability. However, the membrane permeability mechanism had not been elucidated. In this study, we aim to clarify the mechanism by molecular dynamics (MD) simulations. Initially, we simulated the molecular conformations of KS-58 in water (a polar solvent) and in chloroform (a non-polar solvent). The identified stable conformations were significantly different in each solvent. KS-58 behaves as a chameleon-like molecule as it alters its polar surface area (PSA) depending on the solvent environment. It was also discovered that orientation of Asp's side chain is a critical energy barrier for KS-58 altering its conformation from hydrophilic to lipophilic. Taking these properties into consideration, we simulated its lipid bilayer membrane permeability. KS-58 shifted toward the inside of the lipid bilayer membrane with altering its conformations to lipophilic. When the simulation condition was set in deionized form of that carboxy group of Asp, KS-58 traveled deeper inside the cell membrane. PSA and the depth of the membrane penetration correlated. In vitro data suggested that cell membrane permeability of KS-58 is improved in weakly acidic conditions leading to partial deionization of the carboxy group. Our data provide an example of the molecular properties of mid-size peptides with membrane accessibility and propose an effective metadynamics approach to elucidate such molecular mechanisms by MD simulations.

ATNAS: Automatic Termination for Neural Architecture Search.

Neural architecture search (NAS) is a framework for automating the design process of a neural network structure. While the recent one-shot approaches have reduced the search cost, there still exists an inherent trade-off between cost and performance. It is important to appropriately stop the search and further reduce the high cost of NAS. Meanwhile, the differentiable architecture search (DARTS), a typical one-shot approach, is known to suffer from overfitting. Heuristic early-stopping strategies have been proposed to overcome such performance degradation. In this paper, we propose a more versatile and principled early-stopping criterion on the basis of the evaluation of a gap between expectation values of generalisation errors of the previous and current search steps with respect to the architecture parameters. The stopping threshold is automatically determined at each search epoch without cost. In numerical experiments, we demonstrate the effectiveness of the proposed method. We stop the one-shot NAS algorithms and evaluate the acquired architectures on the benchmark datasets: NAS-Bench-201 and NATS-Bench. Our algorithm is shown to reduce the cost of the search process while maintaining a high performance.

The VIPR2-selective antagonist KS-133 changes macrophage polarization and exerts potent anti-tumor effects as a single agent and in combination with an anti-PD-1 antibody.

We have previously demonstrated that KS-133 is a specific and potent antagonist of vasoactive intestinal peptide receptor 2 (VIPR2). We have also shown that vasoactive intestinal peptide-VIPR2 signaling affects the polarity and activation of tumor-associated macrophages, which is another strategy for cancer immunotherapy apart from the activation of effector T cells. In this study, we aimed to examine whether the selective blockade of VIPR2 by KS-133 changes the polarization of macrophages and induces anti-tumor effects. In the presence of KS-133, genetic markers indicative of tumor-aggressive M1-type macrophages were upregulated, and conversely, those of tumor-supportive M2-type macrophages were downregulated. Daily subcutaneous administration of KS-133 tended to suppress the growth of CT26 tumors (murine colorectal cancer-derived cells) implanted subcutaneously in Balb/c mice. To improve the pharmacological efficacy and reduce the number of doses, we examined a nanoformulation of KS-133 using the US Food and Drug Administration-approved pharmaceutical additive surfactant Cremophor® EL. KS-133 nanoparticles (NPs) were approximately 15 nm in size and stable at 4°C after preparation. Meanwhile, KS-133 was gradually released from the NPs as the temperature was increased. Subcutaneous administration of KS-133 NPs once every 3 days had stronger anti-tumor effects than daily subcutaneous administration of KS-133. Furthermore, KS-133 NPs significantly enhanced the pharmacological efficacy of an immune checkpoint-inhibiting anti-PD-1 antibody. A pharmacokinetic study suggested that the enhancement of anti-tumor activity was associated with improvement of the pharmacokinetic profile of KS-133 upon nanoformulation. Our data have revealed that specific blockade of VIPR2 by KS-133 has therapeutic potential for cancer both alone and in combination with immune checkpoint inhibitors.

Juniper berry extract containing Anthricin and Yatein suppresses lipofuscin accumulation in human epidermal keratinocytes through proteasome activation, increases brightness and decreases spots in human skin.

OBJECTIVE: Skin brightness and spot have a significant impact on youthful and beautiful appearance. One important factor influencing skin brightness is the amount of internal reflected light from the skin. Observers recognize the total surface-reflected light and internal reflected light as skin brightness. The more internal reflected light from the skin, the more attractive and brighter the skin appears. This study aims to identify a new natural cosmetic ingredient that increases the skin's internal reflected light, decreases spot and provides a youthful and beautiful skin appearance. METHODS: Lipofuscin in epidermal keratinocytes, the aggregating complex of denatured proteins and peroxidized lipids, is one factor that decreases skin brightness and causes of spot. Aggregates block light transmission, and peroxidized lipids lead to skin yellowness, dullness and age spot. Lipofuscin is known to accumulate intracellularly with ageing. Rapid removal of intracellular denatured proteins prevents lipofuscin formation and accumulation in cells. We focused a proteasome system that efficiently removes intracellular denatured proteins. To identify natural ingredients that increase proteasome activity, we screened 380 extracts derived from natural products. The extract with the desired activity was fractionated and purified to identify active compounds that lead to proteasome activation. Finally, the efficacy of the proteasome-activating extract was evaluated in a human clinical study. RESULTS: We discovered that Juniperus communis fruits (Juniper berry) extract (JBE) increases proteasome activity and suppresses lipofuscin accumulation in human epidermal keratinocytes. We found Anthricin and Yatein, which belong to the lignan family, to be major active compounds responsible for the proteasome-activating effect of JBE. In a human clinical study, an emulsion containing 1% JBE was applied to half of the face twice daily for 4 weeks, resulting in increased internal reflected light, brightness improvement (L-value) and reduction in yellowness (b-value) and spot in the cheek area. CONCLUSION: This is the first report demonstrating that JBE containing Anthricin and Yatein decreases lipofuscin accumulation in human epidermal keratinocytes through proteasome activation, increases brightness and decreases surface spots in human skin. JBE would be an ideal natural cosmetic ingredient for creating a more youthful and beautiful skin appearance with greater brightness and less spot.

OBJECTIF: La luminosité et les taches de peau ont un impact significatif sur la jeunesse et la beauté de l'apparence. L'un des facteurs importants influençant la luminosité de la peau est la quantité de lumière interne réfléchie par la peau. Pour les observateurs, la luminosité de la peau correspond à la somme de la lumière réfléchie par la surface et de la lumière réfléchie par l'intérieur de la peau. Plus la quantité de lumière interne réfléchie par la peau est importante, plus la peau semble attrayante et lumineuse. Cette étude vise à identifier un nouvel ingrédient cosmétique naturel qui augmente la lumière interne réfléchie par la peau, diminue les taches et donne à la peau une apparence jeune et belle. MÉTHODES: La lipofuscine dans les kératinocytes de l'épiderme, le complexe agrégé de protéines dénaturées et de lipides peroxydés, est un facteur qui diminue l'éclat de la peau et qui est à l'origine des taches. Les agrégats bloquent la transmission de la lumière et les lipides peroxydés entraînent une coloration jaune de la peau, un aspect terne et des taches de vieillesse. On sait que la lipofuscine s'accumule au niveau intracellulaire avec le vieillissement. L'élimination rapide des protéines dénaturées intracellulaires empêche la formation et l'accumulation de lipofuscine dans les cellules. Nous avons mis l'accent sur un système de protéasome qui élimine efficacement les protéines dénaturées intracellulaires. Pour identifier les ingrédients naturels qui augmentent l'activité du protéasome, nous avons passé au crible 380 extraits dérivés de produits naturels. L'extrait présentant l'activité souhaitée a été fractionné et purifié afin d'identifier les composés actifs qui conduisent à l'activation du protéasome. Enfin, l'efficacité de l'extrait activant le protéasome a été évaluée dans une étude clinique humaine. RÉSULTATS: Nous avons découvert que l'extrait de Juniperus communis fruits (baie de genièvre) augmente l'activité du protéasome et supprime l'accumulation de lipofuscine dans les kératinocytes épidermiques humains. Nous avons découvert que l'anthricine et la yateine, qui appartiennent à la famille des lignanes, sont les principaux composés actifs responsables de l'effet activateur du protéasome de l'extrait de baies de genévrier. Dans une étude clinique humaine, une émulsion contenant 1 % de JBE a été appliquée sur la moitié du visage deux fois par jour pendant 4 semaines, ce qui a entraîné une augmentation de la lumière interne réfléchie, une amélioration de la luminosité (valeur L) et une réduction de la jaunisse (valeur b) et des taches dans la zone des joues. CONCLUSION: Il s'agit du premier rapport démontrant que l'EBJ contenant de l'anthricine et de la yateine diminue l'accumulation de lipofuscine dans les kératinocytes épidermiques humains par l'activation du protéasome, augmente la luminosité et diminue les taches superficielles de la peau humaine. Le JBE serait un ingrédient cosmétique naturel idéal pour créer une peau plus jeune et plus belle, plus lumineuse et moins tachetée.

[Development of the vasoactive intestinal peptide receptor 2 (VIPR2) antagonist peptide for the treatment of schizophrenia].

Schizophrenia affects approximately 24 million people worldwide. Existing medications for the treatment of schizophrenia work primarily by improving positive symptoms such as agitation, hallucinations, delusions, and aggression. They possess common mechanism of action (MOA), blocking to neurotransmitter receptors such as dopamine, serotonin, and adrenaline receptors. Although multiple agents are available for the treatment of schizophrenia, the majority do not address negative symptoms or cognitive dysfunction. In other cases, patients have drug-related adverse effects. The vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2 receptor) might be an attractive drug target for the treatment of schizophrenia because both clinical and preclinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Despite these backgrounds, the proof-of-concept of VIPR2 inhibitors has not been examined clinically. A reason might be that VIPR2 belongs to class-B GPCRs, and the discovery of small-molecule drugs against class-B GPCRs is generally difficult. We have developed a bicyclic peptide KS-133, which shows VIPR2 antagonist activity and suppresses cognitive decline in a mouse model relevant to schizophrenia. KS-133 has a different MOA from current therapeutic drugs and exhibits high selectivity for VIPR2 and potent inhibitory activity against a single-target molecule. Therefore, it may contribute to both the development of a novel drug candidate for the treatment of psychiatric disorders such as schizophrenia and acceleration of basic studies on VIPR2.

Deep learning in random neural fields: Numerical experiments via neural tangent kernel.

A biological neural network in the cortex forms a neural field. Neurons in the field have their own receptive fields, and connection weights between two neurons are random but highly correlated when they are in close proximity in receptive fields. In this paper, we investigate such neural fields in a multilayer architecture to investigate the supervised learning of the fields. We empirically compare the performances of our field model with those of randomly connected deep networks. The behavior of a randomly connected network is investigated on the basis of the key idea of the neural tangent kernel regime, a recent development in the machine learning theory of over-parameterized networks; for most randomly connected neural networks, it is shown that global minima always exist in their small neighborhoods. We numerically show that this claim also holds for our neural fields. In more detail, our model has two structures: (i) each neuron in a field has a continuously distributed receptive field, and (ii) the initial connection weights are random but not independent, having correlations when the positions of neurons are close in each layer. We show that such a multilayer neural field is more robust than conventional models when input patterns are deformed by noise disturbances. Moreover, its generalization ability can be slightly superior to that of conventional models.

Nanoformulation of the K-Ras(G12D)-inhibitory peptide KS-58 suppresses colorectal and pancreatic cancer-derived tumors.

Single amino acid mutations of Ras occur in 30% of human cancers. In particular, K-Ras(G12D) has been detected in the majority of intractable colorectal and pancreatic cancers. Although efforts to target K-Ras(G12D) are currently underway, no effective drugs are available. We previously found that the K-Ras(G12D)-inhibitory bicyclic peptide KS-58 exhibits antitumor activity against syngeneic colon and orthotopic grafted pancreatic tumors; however, pristine KS-58 is difficult to handle because of low water solubility and it requires frequent administration to obtain sufficient antitumor activity. In this study, we used a nanoformulation of KS-58 prepared with the highly biocompatible surfactant Cremophor® EL (CrEL) to improve water solubility and reduce the dosing frequency. Nanoformulations of KS-58 with CrEL dramatically improved its water solubility and increased its stability. Weekly intravenous administration of KS-58 nanoparticles (NPs) suppressed the growth of CT26 and PANC-1 cell-derived tumors in vivo, and fluorescent bioimaging indicated that the NP-encapsulated near-infrared fluorescent probe indocyanine green selectively accumulated in the tumor and was safely excreted through the kidneys following intravenous injection. Histopathological analysis of CT26 tumors and Western blotting of PANC-1 tumors revealed that KS-58 NPs reduced ERK phosphorylation, a downstream signal of K-Ras(G12D). Our results suggest that KS-58 NPs represent a novel therapeutic agent for treating colorectal and pancreatic cancers.

Vasoactive intestinal peptide receptor 2 signaling promotes breast cancer cell proliferation by enhancing the ERK pathway.

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a class B G protein-coupled receptor with the neuropeptide VIP as a ligand. Increased VIPR2 mRNA expression and/or VIPR2 gene copy number has been documented in several cancers including breast carcinoma. However, the pathophysiological role of increased VIPR2 in the proliferation of breast cancer cells remains largely unknown. In this study, we found that VIPR2 overexpression in MCF-7 and MDA-MB-231 cells, human breast cancer cell lines, promoted cell proliferation. Increased VIPR2 also exacerbated intraperitoneal proliferation of breast cancer MDA-MB-231 cells in a tumor nude mouse model in vivo. Treatment with KS-133, a VIPR2-selective antagonist peptide, significantly inhibited VIP-induced cell proliferation in VIPR2-overexpressing MCF-7 and MDA-MB-231 cells. Overexpressed VIPR2 caused increases in the levels of cAMP and phosphorylated extracellular signal-regulated kinase (ERK), which involves a VIPR2 signaling pathway through Gs protein. Additionally, phosphorylation of vasodilator-stimulated phosphoprotein (Ser157) and cAMP response element binding protein (Ser133) in VIPR2-overexpressing MCF-7 cells was greater than that in control cells, suggesting the increased PKA activity. Moreover, an inhibitor of mitogen-activated protein kinase kinase, U0126, attenuated tumor proliferation in exogenous VIPR2-expressing MCF-7 and MDA-MB-231 cells at the same level as observed in EGFP-expressing cells treated with U0126. Together, these findings suggest that VIPR2 controls breast tumor growth by regulating the cAMP/PKA/ERK signaling pathway, and the excessive expression of VIPR2 may lead to an exacerbation of breast carcinoma.

Arnica montana L. extract containing 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin accelerates growth and differentiation of human subcutaneous preadipocytes and leads volumizing of skin.

OBJECTIVE: An important factor in the aging of the face is a reduction in the volume of adipose tissue. This reduction in adipose tissue contributes to decreased skin elasticity, which is also part of the aging process. Overall, these lead to wrinkle formation. Fat injection is a common means of addressing this issue and is used to reduce the effects of aging on the face and to increase the fullness of the lips and breasts. However, fat injection is an invasive surgical procedure. This study aimed to discover novel cosmetic ingredients that increase the volume of subcutaneous (pre)adipocytes to create the appearance of more youthful skin. METHODS: We focused on the number of subcutaneous preadipocytes and the accumulation of lipid droplets. To discover natural ingredients that increase both of these, extracts of 380 natural products were prepared and screened for their effects on both growth and differentiation (i.e., lipid droplet accumulation) of human subcutaneous preadipocytes. One extract was found to have the desired effects, and this was further studied to determine the active compounds. We then evaluated its efficacy in a human clinical study. RESULTS: We found that Arnica montana L. flower extract (AFE) accelerates both the growth and the differentiation of human subcutaneous preadipocytes. AFE was found to significantly increase the volume of adipocyte spheroids. The active compounds 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin were found to be responsible for the effects of AFE on preadipocytes. In a human clinical study, gels containing 1% AFE successfully enhanced the volume of the lips and face with reduction of wrinkles with no adverse reactions. CONCLUSION: This is the first report to demonstrate that AFE and the included compounds, 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin, act on preadipocytes. AFE would be ideal for use in products that plump the face to reduce wrinkles and create a more youthful appearance.

OBJECTIF: Un facteur important du vieillissement du visage réside dans la réduction du volume du tissu adipeux. Cette réduction du tissu adipeux contribue à une diminution de l'élasticité de la peau qui fait également partie du processus de vieillissement. Globalement, ces facteurs induisent la formation des rides. L'injection de graisse est un moyen courant pour remédier à ce problème et sert à réduire les effets du vieillissement sur le visage et à augmenter la plénitude des lèvres et des seins. Cependant, l'injection de graisse est une intervention chirurgicale invasive. Cette étude visait à découvrir des ingrédients cosmétiques innovants qui augmentent le volume des (pré)adipocytes sous-cutanés pour créer l'apparence d'une peau plus jeune. MÉTHODES: Nous avons mis l'accent sur le nombre de préadipocytes sous-cutanés et sur l'accumulation de gouttelettes lipidiques. Pour découvrir des ingrédients naturels qui augmentent ces deux facteurs, des extraits de 380 produits naturels ont été préparés et analysés en vue de la détermination de leurs effets sur la croissance et la différenciation (c'est-à-dire l'accumulation de gouttelettes lipidiques) des préadipocytes humains sous-cutanés. Un extrait s'est avéré avoir les effets escomptés et il a fait l'objet d'études approfondies visant à déterminer les composés actifs. Nous avons ensuite évalué son efficacité dans une étude clinique chez l'homme. RÉSULTATS: Nous avons découvert que l'extrait de fleur de l'Arnica montana L. (AFE) accélère à la fois la croissance et la différenciation des préadipocytes humains sous-cutanés. L'AFE s'est avéré augmenter considérablement le volume des sphéroïdes des adipocytes. Les composés actifs 6-Ométhacryloyl-hélénaline et 6-O-isobutyryl-hélénaline se sont avérés responsables des effets de l'AFE sur les préadipocytes. Dans une étude clinique chez l'homme, des gels contenant 1 % d'AFE ont permis d'améliorer le volume des lèvres et du visage avec une réduction des rides sans effets indésirables. CONCLUSION: Il s'agit du premier rapport démontrant que l'AFE et les composés inclus, 6-O-méthacryloyl-hélénaline et 6-O-isobutyryl-hélénaline, agissent sur les préadipocytes. L'AFE serait idéal pour les produits qui repulpent le visage afin de réduire les rides et de donner un aspect rajeuni.

AlphaFold version 2.0 elucidates the binding mechanism between VIPR2 and KS-133, and reveals an S-S bond (Cys25-Cys192) formation of functional significance for VIPR2.

The vasoactive intestinal peptide receptor 2 (VIPR2) has attracted attention as a drug target for the treatment of mental disorders, cancer, and immune diseases. In 2021, we identified the peptide KS-133 as a VIPR2-selective antagonist. In this study, we aimed to elucidate the binding mechanism between VIPR2 and KS-133. To this end, VIPR2/KS-133 and VIPR2/vasoactive intestinal peptide (VIP) complex models were constructed through AlphaFold version 2.0 and molecular dynamic simulations. Our models revealed that: (i) both KS-133 and VIP have helical structures, (ii) the interaction residues on VIPR2 for both peptides are similar, and (iii) the orientation of their helices upon their binding to VIPR2 are different by ∼45°. Interestingly, in the process of constructing the aforementioned models, an S-S bond formation between Cys25 and Cys192 of the human VIPR2 was identified. Although these two Cys residues are highly conserved among species (i.e., corresponding to Cys24 and Cys191 in the mouse), no previous reports regarding this S-S bond formation exist. In order to clarify the potential role of this S-S bond in the VIPR2 has functional consequences, a cell line expressing the mouse VIPR2(Cys24Ala, Cys191Ala) was generated. During the VIP stimulation of this cell line, the phosphorylation of AKT (a downstream signal marker of VIPR2) was found to be significantly attenuated, thereby suggesting that the S-S bond has a functional significance for VIPR2. Our study not only elucidates the VIPR2-binding mechanism of KS-133 for the first time, but also provides new insights into the structural biology of VIPR2.

Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.

Phosphoinositide metabolism is critically involved in human cancer cell migration and metastatic growth. The formation of lamellipodia at the leading edge of migrating cells is regulated by metabolism of the inositol phospholipid PI(4,5)P2 into PI(3,4,5)P3. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane and regulates guanine nucleotide exchange factor Rac-mediated actin filament remodeling. Here, we investigated if VIPR2, a receptor for vasoactive intestinal peptide (VIP), has a potential role in regulating cell migration via this pathway. We found that silencing of VIPR2 in MDA-MB-231 and MCF-7 human breast cancer cells inhibited VIP-induced cell migration. In contrast, stable expression of exogenous VIPR2 promoted VIP-induced tumor cell migration, an effect that was inhibited by the addition of a PI3-kinase (PI3K)γ inhibitor or a VIPR2-selective antagonist. VIPR2 stably-expressing cells exhibited increased PI3K activity. Membrane localization of PI(3,4,5)P3 was significantly attenuated by VIPR2-silencing. VIPR2-silencing in MDA-MB-231 cells suppressed lamellipodium extension; in VIPR2-overexpressing cells, VIPR2 accumulated in the cell membrane on lamellipodia and co-localized with WAVE2. Conversely, VIPR2-silencing reduced WAVE2 level on the cell membrane and inhibited the interaction between WAVE2, actin-related protein 3, and actin. These findings suggest that VIP-VIPR2 signaling controls cancer migration by regulating WAVE2-mediated actin nucleation and elongation for lamellipodium formation through the synthesis of PI(3,4,5)P3.

Generation of KS-487 as a novel LRP1-binding cyclic peptide with higher affinity, higher stability and BBB permeability.

The blood-brain barrier (BBB) is a major hurdle in drug discovery for central nervous system (CNS) disorders. Particularly, mid-size molecules and macromolecules (e.g., peptides and antibodies) that modulate intractable drug targets such as protein-protein interaction are prevented from entering the CNS via BBB. The receptor-mediated transcytosis (RMT) pathway has been examined to deliver these molecules to CNS. Among the receptors, low-density lipoprotein receptor-related protein 1 (LRP1) has been emerged as one of the promising receptors for RMT. Although several LRP1-binding peptides have been reported, no drugs are available on the market based on the combination of reported LRP1-binding peptides and therapeutic molecules. One reason may be stability in vivo and BBB-permeability of the peptides. The present study aims to identify a novel LRP1-binding peptide for RMT, where we successfully generated a 15-mer cyclic peptide named KS-487. It explicitly bound to Cluster 4 domain of LRP1 with the binding EC50 value of 10.5 nM and was relatively stable in mouse plasma within 24 h. Moreover, its high BBB permeability was demonstrated using in vitro rat and monkey BBB models. By 24 h incubation, 13% and 17% of the added amount of KS-487 (10 µM) penetrated rat BBB and monkey BBB, respectively. KS-487 would be a potential candidate for the LRP1-mediated transcytosis-based drug delivery to CNS, as these values were significantly higher than those of the known LRP1-binding peptides-Angiopep-2 and L57.

The ambiguous feeling between "mine" and "not-mine" measured by integrated information theory during rubber hand illusion.

Human body awareness is adaptive to context changes. The illusory sense of body ownership has been studied since the publication of the rubber hand illusion, where ambiguous body ownership feeling was first defined. Phenomenologically, the ambiguous body ownership is attributed to a conflict between feeling and judgement: it characterises a discrepancy between first- and third-person processes. Although Bayesian inference can explain this malleability of body image, it still fails to relate the subjective feeling to physiological data. This study attempts to explain subjective experience during rubber hand illusions by using integrated information theory (IIT). The integrated information [Formula: see text] in IIT measures the difference between the whole system and its subsystems. By analysing seven different time-series of physiological data representing a small body-brain system, we demonstrate that the integrity of the whole system during the illusion decreases, while the integrity of its subsystems increases. These general tendencies agree with many brain-image analyses and subjective reports; furthermore, we found that subjective ratings as ambiguous body ownership were associated with [Formula: see text]. Our result suggests that IIT can explain the general tendency of the sense of ownership illusions and individual differences in subjective experience during the illusions.

The K-Ras(G12D)-inhibitory peptide KS-58 suppresses growth of murine CT26 colorectal cancer cell-derived tumors.

Mutations in the cell proliferation regulator K-Ras are found with a variety of cancer types, so drugs targeting these mutant proteins could hold great clinical potential. Very recently, a drug targeting the K-Ras(G12C) mutant observed in lung cancer gained regulatory approval and several clinical trials are currently underway to examine the efficacy of this agent when combined with other drugs such as a monoclonal antibody inhibitor of programmed cell death 1 receptor (anti-PD-1). Alternatively, there are currently no approved drugs targeting K-Ras(G12D), the most common cancer-associated K-Ras mutant. In 2020, we described the development of the K-Ras(G12D) inhibitory bicyclic peptide KS-58 and presented evidence for anticancer activity against mouse xenografts derived from the human pancreatic cancer cell line PANC-1 stably expressing K-Ras(G12D). Here, we show that KS-58 also possess anticancer activity against mouse tumors derived from the colorectal cancer cell line CT26 stably expressing K-Ras(G12D). Further, KS-58 treatment reduced phosphorylation of ERK, a major downstream signaling factor in the Ras pathway, confirming that KS-58 inhibits K-Ras(G12D) function. Unexpectedly; however, KS-58 did not show additive or synergistic anticancer activity with mouse anti-PD-1. Morphological analysis and immunostaining demonstrated no obvious differences in CD8+ cells infiltration or PD-L1 expression levels in CT26-derived tumors exposed to monotherapy or combination treatment. Nonetheless, KS-58 demonstrated reasonable stability in blood (t1/2 ≈ 30 min) and no obvious systemic adverse effects, suggesting clinical potential as a lead molecule against colorectal cancer.

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Generation of KS-133 as a Novel Bicyclic Peptide with a Potent and Selective VIPR2 Antagonist Activity that Counteracts Cognitive Decline in a Mouse Model of Psychiatric Disorders.

Worldwide, more than 20 million people suffer from schizophrenia, but effective and definitive new therapeutic drugs/treatments have not been established. Vasoactive intestinal peptide receptor 2 (VIPR2) might be an attractive drug target for the treatment of schizophrenia because both preclinical and clinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Nevertheless, VIPR2-targeting drugs are not yet available. VIPR2 is a class-B G protein-coupled receptor that possesses high structural homology to its subtypes, vasoactive intestinal peptide receptor 1 (VIPR1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). These biological and structural properties have made it difficult to discover small molecule drugs against VIPR2. In 2018, cyclic peptide VIpep-3, a VIPR2-selective antagonist, was reported. The aim of this study was to generate a VIpep-3 derivative for in vivo experiments. After amino acid substitution and structure optimization, we successfully generated KS-133 with 1) a VIPR2-selective and potent antagonistic activity, 2) at least 24 h of stability in plasma, and 3) in vivo pharmacological efficacies in a mouse model of psychiatric disorders through early postnatal activation of VIPR2. To the best of our knowledge, this is the first report of a VIPR2-selective antagonistic peptide that counteracts cognitive decline, a central feature of schizophrenia. KS-133 may contribute to studies and development of novel schizophrenia therapeutic drugs that target VIPR2.

A selective delta opioid receptor agonist SNC80, but not KNT-127, induced tremor-like behaviors via hippocampal glutamatergic system in mice.

Although delta opioid receptors (DOP) are now known to play a major role in modulating chronic pain and controlling emotional processes, unfortunately, some DOP agonists, such as SNC80, reportedly produced convulsive-like behaviors manifesting as tremor-like behaviors in a preclinical study. Therefore, these induced convulsions limit the progress of the clinical development of DOP agonists. However, mechanisms underlying DOP-induced convulsant activity remain unclarified. Thus, the study aimed to elucidate mechanisms that could cause tremor-like behaviors of SNC80. These drugs were microinjected into the ventral hippocampus CA3 (vCA3), amygdala (AMY), and insular cortex (IC) of mice. In addition, we examined the extracellular glutamate levels after DOP agonist local treatment. Microinjection of SNC80 into the vCA3 increased the number of tremor-like behaviors and extracellular glutamate levels but did not cause tremor-like behaviors in mice when microinjected into IC and AMY. Pretreatment with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainite receptor antagonist CNQX into vCA3 totally inhibited the SNC80-induced increases in tremor-like behaviors. In contrast, another DOP agonist, KNT-127, did not cause tremor-like behaviors in any of the tested brain areas. Further, the extracellular glutamate levels in the hippocampus were significantly lower in the KNT-127-treated mice than in the SNC80-treated mice. Our results showed that the administration of SNC80, but not KNT-127, into vCA3 induced tremor-like behaviors by activating glutamatergic neurons in mice. We propose that KNT-127 should be further studied clinically as a DOP agonist that is expected to have a low risk for convulsions than those resulting in antinociceptive and antidepressant effects.

Generation of KS-58 as the first K-Ras(G12D)-inhibitory peptide presenting anti-cancer activity in vivo.

Ras mutations (e.g., occur in K-Ras, N-Ras, and H-Ras) are one of the most desirable and promising drug targets in chemotherapy treatments for cancer. However, there are still no approved drugs directly targeting mutated Ras. In 2017, an artificial cyclic peptide, KRpep-2d, was discovered as the first selective inhibitor of K-Ras(G12D), the most frequent K-Ras mutation. Here, we report the generation of KS-58, a KRpep-2d derivative that is identified as a bicyclic peptide and possess unnatural amino acid structures. Our in vitro data and molecular dynamics simulations suggest that KS-58 enters cells and blocks intracellular Ras-effector protein interactions. KS-58 selectively binds to K-Ras(G12D) and suppresses the in vitro proliferation of the human lung cancer cell line A427 and the human pancreatic cancer cell line PANC-1, both of which express K-Ras(G12D). Moreover, KS-58 exhibits anti-cancer activity when given as an intravenous injection to mice with subcutaneous or orthotropic PANC-1 cell xenografts. The anti-cancer activity is further improved by combination with gemcitabine. To the best of our knowledge, this is the first report of K-Ras(G12D)-selective inhibitory peptide presenting in vivo anti-cancer activity. KS-58 is an attractive lead molecule for the development of novel cancer drugs that target K-Ras(G12D).

Four-Types of IIT-Induced Group Integrity of Plecoglossus altivelis.

Integrated information theory (IIT) was initially proposed to describe human consciousness in terms of intrinsic-causal brain network structures. Particularly, IIT 3.0 targets the system's cause-effect structure from spatio-temporal grain and reveals the system's irreducibility. In a previous study, we tried to apply IIT 3.0 to an actual collective behaviour in Plecoglossus altivelis. We found that IIT 3.0 exhibits qualitative discontinuity between three and four schools of fish in terms of Φ value distributions. Other measures did not show similar characteristics. In this study, we followed up on our previous findings and introduced two new factors. First, we defined the global parameter settings to determine a different kind of group integrity. Second, we set several timescales (from Δ t = 5 / 120 to Δ t = 120 / 120 s). The results showed that we succeeded in classifying fish schools according to their group sizes and the degree of group integrity around the reaction time scale of the fish, despite the small group sizes. Compared with the short time scale, the interaction heterogeneity observed in the long time scale seems to diminish. Finally, we discuss one of the longstanding paradoxes in collective behaviour, known as the heap paradox, for which two tentative answers could be provided through our IIT 3.0 analysis.