3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2 as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis.

Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.

TRPA1 channel in the airway underlies protection against airborne threats by modulating respiration and behaviour.

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of cation channels, is broadly expressed in sensory neural pathways, including the trigeminal neurons innervating the nasal cavity and vagal neurons innervating the trachea and the lung. TRPA1 acts as a detector of various irritant chemicals as well as hypoxia and hyperoxia. For the past 15 years, we have characterised its role in respiratory and behavioural modulation in vivo using Trpa1 knockout (KO) mice and wild-type (WT) littermates. Trpa1 KO mice failed to detect, wake up from sleeping, and escape from formalin vapour and a mild hypoxic (15% O2 ) environment. Respiratory augmentation induced by mild hypoxia was absent in either Trpa1 KO mice or WT mice treated with a TRPA1 antagonist. Irritant gas introduced into the nasal cavity inhibited respiratory responses in WT mice but not in the KO mice. The effect of TRPA1 on the olfactory system seemed minimal because olfactory bulbectomized WT mice reacted similarly to the intact mice. Immunohistological analyses using a cellar activation marker, the phosphorylated form of extracellular signal-regulated kinase, confirmed activation of trigeminal neurons in WT mice but not in Trpa1 KO mice in response to irritant chemicals and mild hypoxia. These data collectively show that TRPA1 is necessary for multiple chemical-induced protective responses in respiration and behaviour. We propose that TRPA1 channels in the airway may play a sentinel role for environmental threats and prevent incoming damage.

Novel cancer-specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long-term survival.

Although esophageal cancer has a poor prognosis after recurrence, some patients have shown long-term survival despite recurrence. We hypothesized that induction of either antitumor Abs or antitumor-specific CTLs could play a role in long-term survival (5 years or longer) in patients with recurrence and/or distant metastases. Therefore, we aimed to obtain Abs that specifically bind to cancer cells by using serum samples from patients with a good prognosis. A phage library was prepared using PBMC mRNA of the patients, and cell panning was carried out using an esophageal cancer cell line. Results showed the presence of an epidermal growth factor receptor (EGFR) Ab, KT112, that specifically bound to the cancer cell line. Notably, KT112 bound to only EGFR-positive cancer cells but failed to bind to normal esophageal cells. Furthermore, KT112 was characterized by responses to EGFR expressed on cancer cells but not to the recombinant extracellular domain of EGFR. Immunohistochemical analysis showed that KT112 reacted with 17.4% of esophageal squamous cell carcinoma tissue but not with any other cancer or normal tissue, suggesting that the Ab recognizes cancer-specific forms of EGFR and might have contributed to tumor suppression in patients with esophageal cancer. Furthermore, because of its high cancer specificity, KT112 could be a promising therapeutic option (e.g., in Ab-drug conjugates) for esophageal cancer.

Retro-aza-Michael reaction of an o-aminophenol adduct in protic solvents inspired by natural products.

α,ß-Unsaturated carbonyls are reactive group often found in bioactive small molecules. Their non-specific reaction with biomolecules can be the cause of the low efficacy and unexpected side-effects of the molecule. Accordingly, unprotected α,ß-unsaturated carbonyls are not often found in drugs. Here, we report that o-aminophenol is a new masking group of α,ß-unsaturated ketone, which is inspired by natural products saccharothriolides. o-Aminophenol adduct of α,ß-unsaturated ketone, but not those of α,ß-unsaturated amide or ester, undergoes a retro-Michael reaction to yield o-aminophenol and the Michael acceptor. This reaction was observed only in protic solvents, such as MeOH and aqueous MeOH. In contrast, o-anisidine was not eliminated from its Michael adduct. o-Aminophenol may be a promising masking tool of highly-reactive bioactive α,ß-unsaturated carbonyl compounds.

Design, synthesis, and target identification of new hypoxia-inducible factor 1 (HIF-1) inhibitors containing 1-alkyl-1H-pyrazole-3-carboxamide moiety.

Hypoxia-inducible factor 1 (HIF-1) is a promising drug target for cancer chemotherapy. In our screening program aimed at identifying new HIF-1 inhibitors by using a hypoxia-responsive luciferase reporter gene assay, KUSC-5001 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety was found as a potential hit molecule. During an extensive structure-activity relationship (SAR) study, we developed a more effective HIF-1 inhibitor KUSC-5037 (IC50 = 1.2 µM). Under hypoxic conditions, KUSC-5037 suppressed the HIF-1α (a regulatory subunit of HIF-1) mRNA, causing decreases in the gene expression of HIF-1 target genes such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) genes. Furthermore, by applying our fluorescent and bifunctional probes, ATP5B, a catalytic ß subunit of mitochondrial FoF1-ATP synthase, was identified as a target protein of KUSC-5037. These results indicate that the derivatives of KUSC-5037 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety are promising lead molecules for the inhibition of HIF-1 signaling via FoF1-ATP synthase suppression.

The Ventral Striatum is a Key Node for Functional Recovery of Finger Dexterity After Spinal Cord Injury in Monkeys.

In a recent study, we demonstrated that the ventral striatum (VSt) controls finger movements directly during the early recovery stage after spinal cord injury (SCI), implying that the VSt may be a part of neural substrates responsible for the recovery of dexterous finger movements. The VSt is accepted widely as a key node for motivation, but is not thought to be involved in the direct control of limb movements. Therefore, whether a causal relationship exists between the VSt and motor recovery after SCI is unknown, and the role of the VSt in the recovery of dexterous finger movements orfinger movements in general after SCI remains unclear. In the present study, functional brain imaging in a macaque model of SCI revealed a strengthened functional connectivity between motor-related areas and the VSt during the recovery process for precision grip, but not whole finger grip after SCI. Furthermore, permanent lesion of the VSt impeded the recoveryof precision grip, but not coarse grip. Thus, the VSt was needed specifically for functional recovery of dexterous finger movements. These results suggest that the VSt is the key node of the cortical reorganization required for functional recovery of finger dexterity.

Total Synthesis and Antimicrobial Activity of Tumescenamide C and Its Derivatives.

Tumescenamide C (1) is an antimicrobial compound produced by Streptomyces sp. KUSC_F05 and consists of a cyclic depsipeptide core and a polyketide side chain with branched methyl groups. Here, we report the total synthesis of tumescenamide C and two derivatives, mainly using Fmoc solid-phase peptide synthesis (SPPS). In addition, a biological evaluation of these compounds revealed the critical partial structure in 1 for antimicrobial activity.

Development of an anti-hepatitis B virus (HBV) agent through the structure-activity relationship of the interferon-like small compound CDM-3008.

Hepatitis B, a viral infectious disease caused by hepatitis B virus (HBV), is a life-threatening disease that leads liver cirrhosis and liver cancer. Because the current treatments for HBV, such as an interferon (IFN) formulation or nucleoside/nucleotide analogues, are not sufficient, the development of a more effective agent for HBV is urgent required. CDM-3008 (1, 2-(2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridin-8-yl)-1,3,4-oxadiazole) (RO8191)) is a small molecule with an imidazo[1,2-a][1,8]naphthyridine scaffold that shows anti-HCV activity with an IFN-like effect. Here, we report that 1 was also effective for HBV, although the solubility and metabolic stability were insufficient for clinical use. Through the structure-activity relationship (SAR), we discovered that CDM-3032 (11, N-(piperidine-4-yl)-2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridine-8-carboxamide hydrochloride) was more soluble than 1 (>30 mg/mL for 11 versus 0.92 mg/mL for 1). In addition, the half-life period of 11 was dramatically improved in both mouse and human hepatic microsomes (T1/2, >120 min versus 58.2 min in mouse, and >120 min versus 34.1 min in human, for 11 and 1, respectively).

Functional and Structural Divergence in Human TRPV1 Channel Subunits by Oxidative Cysteine Modification.

Transient receptor potential vanilloid 1 (TRPV1) channel is a tetrameric protein that acts as a sensor for noxious stimuli such as heat and for diverse inflammatory mediators such as oxidative stress to mediate nociception in a subset of sensory neurons. In TRPV1 oxidation sensing, cysteine (Cys) oxidation has been considered as the principle mechanism; however, its biochemical basis remains elusive. Here, we characterize the oxidative status of Cys residues in differential redox environments and propose a model of TRPV1 activation by oxidation. Through employing a combination of non-reducing SDS-PAGE, electrophysiology, and mass spectrometry we have identified the formation of subunit dimers carrying a stable intersubunit disulfide bond between Cys-258 and Cys-742 of human TRPV1 (hTRPV1). C258S and C742S hTRPV1 mutants have a decreased protein half-life, reflecting the role of the intersubunit disulfide bond in supporting channel stability. Interestingly, the C258S hTRPV1 mutant shows an abolished response to oxidants. Mass spectrometric analysis of Cys residues of hTRPV1 treated with hydrogen peroxide shows that Cys-258 is highly sensitive to oxidation. Our results suggest that Cys-258 residues are heterogeneously modified in the hTRPV1 tetrameric complex and comprise Cys-258 with free thiol for oxidation sensing and Cys-258, which is involved in the disulfide bond for assisting subunit dimerization. Thus, the hTRPV1 channel has a heterogeneous subunit composition in terms of both redox status and function.

Accumulation of immunoglobulin G against Dermatophagoides farinae tropomyosin in dorsal root ganglia of NC/Nga mice with atopic dermatitis-like symptoms.

Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as intractable itch, but its underlying mechanisms are poorly understood. This study assessed the relationship between immunoglobulin G (IgG) and dorsal root ganglia (DRG) in NC/Nga mice, a model of AD that manifests AD-like symptoms including itch. Immunohistochemical analysis showed large amounts of IgG in DRG extracts of NC/Nga mice with AD-like dermatitis, with a large fraction of the IgG distributed in satellite glial cells of the DRG. Proteomic analysis showed that this IgG was reactive against tropomyosin of Dermatophagoides farinae. These findings indicate that the accumulation of anti-tropomyosin IgG in DRG of atopic NC/Nga mice may be associated with the pathogenesis of AD-like symptoms, including itch.

Involvement of µ-opioid Receptors and κ-opioid Receptors in Itch-related Scratching Behaviour of Imiquimod-induced Psoriasis-like Dermatitis in Mice.

The pathogenesis of psoriatic itch is poorly understood. The aim of this study was to investigate the involvement of opioid receptors in scratching behaviour of imiquimod-induced psoriasis-like dermatitis model mice. Topical application of 5% imiquimod cream to the rostral back skin of mice induced antihistamine-resistant scratching behaviour. The expression of µ-opioid receptor (MOR) protein increased in the epidermis, dorsal root ganglia (DRG) and spinal cord of imiquimod-treated mice. In contrast, the expression of κ-opioid receptor (KOR) protein decreased in the DRG and spinal cord of imiquimod-treated mice, and was undetectable in the epidermis of both groups. Topical or intraperitoneal administration of the MOR antagonist naloxone and oral administration of the centrally acting KOR agonist ICI-199,441 inhibited scratching behaviour, whereas oral administration of the peri-pherally-selective KOR agonist asimadoline did not. These results suggest that peripheral and central MOR and central KOR may be involved in the modulation of scratching behaviour in imiquimod-treated mice.

Curcumin ß-D-Glucuronide Plays an Important Role to Keep High Levels of Free-Form Curcumin in the Blood.

Curcumin, a polyphenol derived from the rhizome of the naturally occurring plant Curcuma longa, has various pharmacological actions such as antioxidant and anti-inflammatory effects. In this paper, we evaluated the role of its internal metabolite, curcumin ß-D-glucuronide (curcumin monoglucuronide, CMG), by investigating curcumin kinetics and metabolism in the blood. Firstly, we orally administered highly bioavailable curcumin to rats to elucidate its kinetics, and observed not only the free-form of curcumin, but also, curcumin in a conjugated form, within the portal vein. We confirmed that curcumin is conjugated when it passes through the intestinal wall. CMG, one of the metabolites, was then orally administered to rats. Despite its high aqueous solubility compared to free-form curcumin, it was not well absorbed. In addition, CMG was injected intravenously into rats in order to assess its metabolic behavior in the blood. Interestingly, high levels of free-form curcumin, thought to be sufficiently high to be pharmacologically active, were observed. The in vivo antitumor effects of CMG following intravenous injection were then evaluated in tumor-bearing mice with the HCT116 human colon cancer cell line. The tumor volume within the CMG group was significantly less than that of the control group. Moreover, there was no significant loss of body weight in the CMG group compared to the control group. These results suggest that CMG could be used as an anticancer agent without the serious side effects that most anticancer agents have.

TRP channels in oxygen physiology: distinctive functional properties and roles of TRPA1 in O2 sensing.

Transient Receptor Potential (TRP) proteins form cation channels characterized by a wide variety of activation triggers. Here, we overview a group of TRP channels that respond to reactive redox species to transduce physiological signals, with a focus on TRPA1 and its role in oxygen physiology. Our systematic evaluation of oxidation sensitivity using cysteine-selective reactive disulphides with different redox potentials reveals that TRPA1 has the highest sensitivity to oxidants/electrophiles among the TRP channels, which enables it to sense O2. Proline hydroxylation by O2-dependent hydroxylases also regulates the O2-sensing function by inhibiting TRPA1 in normoxia; TRPA1 is activated by hypoxia through relief from the inhibition and by hyperoxia through cysteine oxidation that overrides the inhibition. TRPA1 enhances neuronal discharges induced by hyperoxia and hypoxia in the vagus to underlie respiratory adaptation to changes in O2 availability. This importance of TRPA1 in non-carotid body O2 sensors can be extended to the universal significance of redox-sensitive TRP channels in O2 adaptation.

Redox-sensitive transient receptor potential channels in oxygen sensing and adaptation.

Regulation of ion channels is central to the mechanisms that underlie immediate acute physiological responses to changes in the availability of molecular oxygen (O2). A group of cation-permeable channels that are formed by transient receptor potential (TRP) proteins have been characterized as exquisite sensors of redox reactive species and as efficient actuators of electric/ionic signals in vivo. In this review, we first discuss how redox-sensitive TRP channels such as TRPA1 have recently emerged as sensors of the relatively inert oxidant O2. With regard to the physiological significance of O2 sensor TRP channels, vagal TRPA1 channels are mainly discussed with respect to their role in respiratory regulation in comparison with canonical pathways in glomus cells of the carotid body, which is a well-established O2-sensing organ. TRPM7 channels are discussed regarding hypoxia-sensing function in ischemic cell death. Also, ubiquitous expression of TRPA1 and TRPM7 together with their physiological relevance in the body is examined. Finally, based upon these studies on TRP channels, we propose a hypothesis of "O2 remodeling." The hypothesis is that cells detect deviation of O2 availability from appropriate levels via sensors and adjust local O2 environments in vivo by controlling supply and consumption of O2 via pathways comprising cellular signals and transcription factors downstream of sensors, which consequently optimize physiological functions. This new insight into O2 adaptation through ion channels, particularly TRPs, may foster a paradigm shift in our understanding in the biological significance of O2.

Possible Antipruritic Mechanism of Cyclosporine A in Atopic Dermatitis.

Cyclosporine A is an immunosuppressive agent that suppresses pruritus and is currently used in the treatment of patients with severe atopic dermatitis. The aim of this study was to elucidate the antipruritic mechanism of cyclosporine A using a mouse model of atopic dermatitis. Intraperitoneal injection of cyclosporine A (5 mg/kg) significantly reduced epidermal nerve density, number of scratching bouts, dermatitis scores, and transepidermal water loss, as well as decreasing the numbers of inflammatory cells in the dermis and decreasing epidermal thickness. Intraperitoneal injection of cyclosporine A dose-dependently inhibited increased itch-related receptor gene expression, such as interleukin-31 receptor A and neurokinin-1 receptor, in the dorsal root ganglion of atopic dermatitis model mice. Thus, the antipruritic efficacy of cyclosporine A may involve reduced epidermal nerve density and expression levels of itch-related receptor genes in the dorsal root ganglion, as well as improvement in acanthosis and reduction in cutaneous inflammatory cell number.

Anti-influenza virus effects of cocoa.

BACKGROUND: Cocoa contains biologically active ingredients that have broad-spectrum antimicrobial activity, which includes an inhibitory effect on influenza virus infection. RESULTS: A cocoa extract (CE) was prepared by treating defatted cocoa powder with boiling water. The extract demonstrated dose-dependent inhibition of infection in Madin-Darby canine kidney (MDCK) cells infected with human influenza virus A (H1N1, H3N2), human influenza virus B and avian influenza viruses (H5N1, H5N9). CE inhibited viral adsorption to MDCK cells. Animal experiments showed that CE significantly improved survival in mice after intra-nasal administration of a lethal dose of influenza virus. In human intervention trials, participants were allocated to two groups, one in which the participants ingested cocoa for 3 weeks before and after vaccination against A(H1N1)pdm2009 influenza virus and another in which the participants did not ingest cocoa. Neutralizing antibody titers against A(H1N1)pdm2009 influenza virus increased significantly in both groups; however, the extent of the increase was not significantly different between the two groups. Although natural killer cell activity was also elevated in both groups, the increase was more substantial in the cocoa intake group. CONCLUSION: Drinking cocoa activates natural immunity and enhances vaccination-induced immune response, providing stronger protection against influenza virus infection and disease onset.

2-(Isopropylamino)thieno[3,2-d]pyrimidin-4(3H)-one derivatives as selective phosphodiesterase 7 inhibitors with potent in vivo efficacy.

A new series of thienopyrimidinones is synthesized and evaluated as selective phosphodiesterase 7 (PDE7) inhibitors for the treatment of inflammatory diseases. The modification of the substituents on thienopyrimidinone revealed that an isopropylamino group at the 2-position was favorable for aqueous solubility. The introduction of 3-pyrrolidines at the 7-position resulted in good solubility, highly potent activity, and good PDE7 selectivity. Among the synthesized compounds, compound 46 exhibited the greatest inhibition of ear edema in a phorbol ester-induced mouse model.

Discovery and SAR study of 2-(4-pyridylamino)thieno[3,2-d]pyrimidin-4(3H)-ones as soluble and highly potent PDE7 inhibitors.

The discovery and SAR study of a new series of soluble and highly potent phosphodiesterase (PDE) 7 inhibitors are described herein. We explored a new lead compound with improved solubility, which led to the discovery of a 2-(4-pyridylamino)thieno[3,2-d]pyrimidin-4(3H)-one series. The introduction of 3-piperidines at the 7-position resulted in the significant enhancement of PDE7 activity. In particular, compound 32 also showed strong PDE7 inhibitory activity; good selectivity against PDE3, 4, and 5; and good aqueous solubility.

Transnitrosylation directs TRPA1 selectivity in N-nitrosamine activators.

S-Nitrosylation, the addition of a nitrosyl group to cysteine thiols, regulates various protein functions to mediate nitric oxide (NO) bioactivity. Recent studies have demonstrated that selectivity in protein S-nitrosylation signaling pathways is conferred through transnitrosylation, a transfer of the NO group, between proteins via interaction. We previously demonstrated that sensitivity to activation by synthetic NO-releasing agents via S-nitrosylation is a common feature of members of the transient receptor potential (TRP) family of Ca(2+)-permeable cation channels. However, strategies to confer subtype selectivity to nitrosylating agents targeted to TRP channels are yet to be developed. Here, we show selective activation of TRPA1 channels by novel NO donors derived from the ABBH (7-azabenzobicyclo[2.2.1]heptane) N-nitrosamines, which exhibit transnitrosylation reactivity to thiols without releasing NO. The NNO-ABBH1 (N-nitroso-2-exo,3-exo-ditrifluoromethyl-7-azabenzobicyclo[2.2.1]heptane) elicits S-nitrosylation of TRPA1 proteins, and dose-dependently induces robust Ca(2+) influx via both recombinant and native TRPA1 channels, but not via other NO-activated TRP channels. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, suggesting that cysteine transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by the correlation of N-NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Interestingly, nonelectrophilic derivatives of ABBH also activate TRPA1 selectively, but less potently, compared with NNO-ABBH1. Thus, ABBH N-nitrosamines confer subtype selectivity on S-nitrosylation in TRP channels through synergetic effects of two chemical processes: cysteine transnitrosylation and molecular recognition of the nonelectrophilic moiety.