[Comparison of DNA Extraction Methods for Processed Foods Containing Soybean or Maize].

Processed foods containing soybean or maize are subject to labeling regulations pertinent to genetically modified (GM) foods in Japan. To confirm the reliability of the labeling procedure of GM foods, the Japanese standard analytical methods (standard methods) using real-time PCR technique have been established. Although certain DNA extraction protocols are stipulated as standard in these methods, the use of other protocols confirmed to be equivalent to the existing ones was permitted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or corn was conducted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or maize was conducted. The silica membrane-based DNA extraction kits, GM quicker 4 and DNeasy Plant Maxi Kit (Maxi Kit), as an existing method were compared. GM quicker 4 was considered to be equivalent to or better than Maxi Kit.

Development of STING degrader with double covalent ligands.

Stimulator of interferon genes (STING), a homodimeric membrane receptor localized in the endoplasmic reticulum, plays a pivotal role in signaling innate immune responses. Inhibitors and proteolysis-targeting chimeras (PROTACs) targeting STING are promising compounds for addressing autoinflammatory and autoimmune disorders. In this study, we used a minimal covalent handle recently developed as the ligand portion of an E3 ligase. The engineered STING degrader with a low molecular weight compound covalently binds to STING and E3 ligase. Degrader 2 showed sustained STING degradation activity at lower concentrations (3 µM, 48 h, about 75 % degradation) compared to a reported STING PROTAC, SP23. This discovery holds significance for its potential in treating autoinflammatory and autoimmune diseases, offering promising avenues for developing more efficacious STING-targeted therapies.

Providing appropriate information to consumers boosts the acceptability of genome-edited foods in Japan.

The Japanese Health Ministry recently granted permission for the market distribution of genome-edited (GE) foods, yet there remains a lack of full understanding among consumers regarding this technology. In this study, we conducted a survey to assess the acceptability of GE foods among Japanese consumers and examined the impact of providing information about GE foods on their acceptability. We conducted a web-based survey among 3,408 consumers aged 20-69 years, focusing on three aspects: (1) the commercial availability of GE foods, (2) the consumption of GE foods by others, and (3) your own consumption of GE foods. The survey findings revealed that participants were most accepting of the consumption of GE foods by others, followed by their acceptance of GE foods being commercially available. Notably, participants' acceptance of GE foods increased in all three aspects after they viewed an informative video. The video had a particularly strong impact on participants who fully or partially understood its content, compared to those who did not. Furthermore, regression analyses showed that participants' understanding of two key areas, namely "Why are GE foods important" and "What procedures are in place to ensure the safety of GE foods," played a crucial role in increasing acceptability. Overall, these results indicate that providing information about GE foods to Japanese consumers can effectively enhance their acceptance of such foods. The findings highlight the importance of understanding the benefits and safety measures associated with GE foods in influencing consumer attitudes.

Rapid Screening Detection of Genetically Modified Papaya by Loop-Mediated Isothermal Amplification.

A loop-mediated isothermal amplification (LAMP)-mediated screening detection method for genetically modified (GM) papaya was developed targeting the 35S promoter (P35S) of the cauliflower mosaic virus. LAMP products were detected using a Genie II real-time fluorometer. The limit of detection (LOD) was evaluated and found to be ≤0.05% for papaya seeds. We also designed a primer set for the detection of the papaya endogenous reference sequence, chymopapain, and the species-specificity was confirmed. To improve cost-effectiveness, single-stranded tag hybridization (STH) on a chromatography printed-array strip (C-PAS) system, which is a lateral flow DNA chromatography technology, was applied. LAMP amplification was clearly detected by the system at the LOD level, and a duplex detection of P35S and chymopapain was successfully applied. This simple and quick method for the screening of GM papaya will be useful for the prevention of environmental contamination of unauthorized GM crops.

CRBN ligand expansion for hematopoietic prostaglandin D2 synthase (H-PGDS) targeting PROTAC design and their in vitro ADME profiles.

An increasing number of research reports are describing modifications of the E3 ligand, in particular, cereblon (CRBN) ligands, to improve the chemical and metabolic stabilities as well as the physical properties of PROTACs. In this study, phenyl-glutarimide (PG) and 6-fluoropomalidomide (6-F-POM), recently used as CRBN ligands for PROTAC design, were applied to hematopoietic prostaglandin D2 synthase (H-PGDS)-targeted PROTACs. Both PROTAC-5 containing PG and PROTAC-6 containing 6-F-POM were found to have potent activities to induce H-PGDS degradation. Furthermore, we obtained in vitro ADME data on the newly designed PROTACS as well as our previously reported PROTACs(H-PGDS) series. Although all PROTACs(H-PGDS) are relatively stable toward metabolism, they had poor PAMPA values. Nevertheless, PROTAC-5 showed Papp values similar to TAS-205, which is in Phase 3 clinical trials, and is expected to be the key to improving the pharmacokinetics of PROTACs.

[Investigation of Genetically Modified Maize Imported into Japan in 2021/2022 and the Applicability of Japanese Official Methods].

Given that the number of genetically modified (GM) maize events that have been announced as having undergone safety assessment procedures in Japan is increasing yearly, more information is needed about their actual recent domestic distribution in Japan. In this study, we investigated whether current Japanese official qualitative and quantitative methods (the current official methods) for GM maize can comprehensively target events in domestically distributed maize. For samples with the identity-preserved (IP) handling system and non-IP samples from the United States (US) and non-IP samples from Brazil, we performed event-specific real-time PCR targeting 25 authorized single GM maize events in addition to the current official methods. According to our results, 15 events targeted by the current official methods were detected, but insect-resistance (IR) Event5307 and herbicide-tolerant (HT) DAS40278, not targeted by the current official methods, were detected in the US (one out of 5 lots) and Brazilian (four out of 5 lots) non-IP samples, respectively. Nevertheless, a survey of recent GM maize acreage in recent years has revealed that more than 95% of the acreage in US maize is occupied by HT or IR/HT stacked events, and that more than 95% of the acreage in Brazilian maize is occupied by IR or IR/HT stacked events. Because the current official methods can target all stacked events related to Event5307 and DAS40278, the only undetectable events are the single Event5307 and DAS40278, whose production is estimated to be less than 5% of the total production in the producing country. Therefore, we conclude that the current official methods for the labelling of GM maize should be maintained in view of practicability.

Structure-activity relationship study of PROTACs against hematopoietic prostaglandin D2 synthase.

Degradation of hematopoietic prostaglandin D2 synthase (H-PGDS) by proteolysis-targeting chimeras (PROTACs) is expected to be important in the treatment of allergic diseases and Duchenne's muscular dystrophy. We recently reported that PROTAC(H-PGDS)-7 (PROTAC1), which is composed of H-PGDS inhibitor (TFC-007) and cereblon (CRBN) E3 ligase ligand (pomalidomide), showed potent H-PGDS degradation activity. Here, we investigated the structure-activity relationships of PROTAC1, focusing on the C4- or C5-conjugation of pomalidomide, in addition, the H-PGDS ligand exchanging from TFC-007 with the biaryl ether to TAS-205 with the pyrrole. Three new PROTACs were evaluated for H-PGDS affinity, H-PGDS degrading activity, and inhibition of prostaglandin D2 production. All compounds showed high H-PGDS degrading activities, but PROTAC(H-PGDS)-4-TAS-205 (PROTAC3) was slightly less active than the other compounds. Molecular dynamics simulations suggested that the decrease in activity of PROTAC3 may be due to the lower stability of the CRBN-PROTAC-H-PGDS ternary complex.

Development and Validation of a New Robust Detection Method for Low-Content DNA Using ΔΔCq-Based Real-Time PCR with Optimized Standard Plasmids as a Control Sample.

Real-time polymerase chain reaction (PCR) is the gold standard for DNA detection in many fields, including food analysis. However, robust detection using a real-time PCR for low-content DNA samples remains challenging. In this study, we developed a robust real-time PCR method for low-content DNA using genetically modified (GM) maize at concentrations near the limit of detection (LOD) as a model. We evaluated the LOD of real-time PCR targeting two common GM maize sequences (P35S and TNOS) using GM maize event MON863 containing a copy of P35S and TNOS. The interlaboratory study revealed that the LOD differed among laboratories partly because DNA input amounts were variable depending on measurements of DNA concentrations. To minimize this variability for low-content DNA samples, we developed ΔΔCq-based real-time PCR. In this study, ΔCq and ΔΔCq are as follows: ΔCq = Cq (P35S or TNOS) - Cq (SSIIb; maize endogenous gene), ΔΔCq = ΔCq (analytical sample) - ΔCq (control sample at concentrations near the LOD). The presence of GM maize was determined based on ΔΔCq values. In addition, we used optimized standard plasmids containing SSIIb, P35S, and TNOS with ΔCq equal to the MON863 genomic DNA (gDNA) at concentrations near the LOD as a control sample. A validation study indicated that at least 0.2% MON863 gDNA could be robustly detected. Using several GM maize certified reference materials, we have demonstrated that this method was practical for detecting low-content GM crops and thus for validating GM food labeling. With appropriate standards, this method would be applicable in many fields, not just food.

Unbiased prediction of off-target sites in genome-edited rice using SITE-Seq analysis on a web-based platform.

Genome-editing using the CRISPR-Cas9 system has the potential to substantially accelerate crop breeding. Since off-target editing is one of problems, a reliable method for comprehensively detecting off-target sites is needed. A number of in silico methods based on homology to on-target sequence have been developed, however the prediction without false negative is still under discussion. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq analysis is a comprehensive method that can detect double-strand breaks in vitro. Furthermore, we developed a systematic method using SITE-Seq in combination with web-based Galaxy system (Galaxy for Cut Site Detection), which can perform reproducible analyses without command line operations. We conducted a SITE-Seq analysis of a rice genome targeted by OsFH15 gRNA-Cas9 as a model, and found 41 candidate off-target sites in the annotated regions. Detailed amplicon-sequencing revealed mutations at one off-target site in actual genome-edited rice. Since this off-target site has an uncommon protospacer adjacent motif, it is difficult to predict using in silico methods alone. Therefore, we propose a novel off-target assessment scheme for genome-edited crops that combines the prediction of off-target candidates by SITE-Seq and in silico programs and the validation of off-target sites by amplicon-sequencing.

Development and Interlaboratory Validation of a Novel Reproducible Qualitative Method for GM Soybeans Using Comparative Cq-Based Analysis for the Revised Non-GMO Labeling System in Japan.

Many countries have implemented the labeling system of genetically modified organisms (GMO). In Japan, the regulatory threshold for non-GMO labeling will be revised and restricted to undetectable by April 2023. The practical criterion for the revised system is based on the limit of detection (LOD). However, determining whether the commingling of GMO levels exceeds the LOD is challenging because GM contents close to the LOD are usually below the limit of quantification. In this study, we developed a qualitative method based on comparative Cq-based analysis targeting cauliflower mosaic virus 35S promoter and GM soybean MON89788 event-specific sequences that could be applicable to the revised non-GMO labeling. ΔCq values between the target and endogenous sequences were calculated, and the ΔΔCq value obtained was used as a criterion to determine analytical samples with GM contents exceeding the threshold. To improve the reproducibility of the method, we used a standard plasmid that yields equivalent and stable ΔCq values comparable with those obtained from LOD samples. The developed method was validated with an interlaboratory study. The new qualitative detection concept would be useful for ensuring robust and reproducible results among laboratories, particularly for detecting low-copy-number DNA samples.

Development of Rapid and Facile Solid-Phase Synthesis of PROTACs via a Variety of Binding Styles.

Optimizing linker design is important for ensuring efficient degradation activity of proteolysis-targeting chimeras (PROTACs). Therefore, developing a straightforward synthetic approach that combines the protein-of-interest ligand (POI ligand) and the ligand for E3 ubiquitin ligase (E3 ligand) in various binding styles through a linker is essential for rapid PROTAC syntheses. Herein, a solid-phase approach for convenient PROTAC synthesis is presented. We designed azide intermediates with different linker lengths to which the E3 ligand, pomalidomide, is attached and performed facile PROTACs synthesis by forming triazole, amide, and urea bonds from the intermediates.

Control of STING Agonistic/Antagonistic Activity Using Amine-Skeleton-Based c-di-GMP Analogues.

Stimulator of Interferon Genes (STING) is a type of endoplasmic reticulum (ER)-membrane receptor. STING is activated by a ligand binding, which leads to an enhancement of the immune-system response. Therefore, a STING ligand can be used to regulate the immune system in therapeutic strategies. However, the natural (or native) STING ligand, cyclic-di-nucleotide (CDN), is unsuitable for pharmaceutical use because of its susceptibility to degradation by enzymes and its low cell-membrane permeability. In this study, we designed and synthesized CDN derivatives by replacing the sugar-phosphodiester moiety, which is responsible for various problems of natural CDNs, with an amine skeleton. As a result, we identified novel STING ligands that activate or inhibit STING. The cyclic ligand 7, with a cyclic amine structure containing two guanines, was found to have agonistic activity, whereas the linear ligand 12 showed antagonistic activity. In addition, these synthetic ligands were more chemically stable than the natural ligands.

Development of a degrader against oncogenic fusion protein FGFR3-TACC3.

Fibroblast growth factor receptor 3-transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3), which has been identified in many cancers such as glioblastoma and bladder cancer, is a potent oncogenic fusion protein that induces constitutive activation of FGFR signaling, resulting in uncontrolled cell proliferation. Although several tyrosine kinase inhibitors against FGFR are currently under development, resistance to such types of inhibitors in patients has become a concern. In this study, a chimeric molecule SNIPER(TACC3)-11 (5a) was developed and found to reduce FGFR3-TACC3 levels effectively. Compound 5a conjugated KHS108 (a TACC3 ligand) to an LCL161 derivative (11) (an inhibitor of apoptosis protein [IAP] ligand) with a PEG linker (n = 2). Mechanistical analysis showed that cellular IAP1 was required for the reduction of FGFR3-TACC3 levels. Consistent with the decrease in FGFR3-TACC3 levels, compound 5a suppressed the growth of FGFR3-TACC3 positive cells. Thus, compound 5a is a candidate therapeutic with a novel drug modality against cancers that exhibit FGFR3-TACC3-dependent proliferation and exerts pharmacological effects distinct from FGFR3 kinase inhibitors because it lacks substructures crucial for kinase inhibition.

Discovery of a Highly Potent and Selective Degrader Targeting Hematopoietic Prostaglandin D Synthase via In Silico Design.

Targeted protein degradation by proteolysis-targeting chimera (PROTAC) is one of the exciting modalities for drug discovery and biological discovery. It is important to select an appropriate linker, an E3 ligase ligand, and a target protein ligand in the development; however, it is necessary to synthesize a large number of PROTACs through trial and error. Herein, using a docking simulation of the ternary complex of a hematopoietic prostaglandin D synthase (H-PGDS) degrader, H-PGDS, and cereblon, we have succeeded in developing PROTAC(H-PGDS)-7 (6), which showed potent and selective degradation activity (DC50 = 17.3 pM) and potent suppression of prostaglandin D2 production in KU812 cells. Additionally, in a Duchenne muscular dystrophy model using mdx mice with cardiac hypertrophy, compound 6 showed better inhibition of inflammatory cytokines than a potent H-PGDS inhibitor TFC-007. Thus, our results demonstrated that in silico simulation would be useful for the rational development of PROTACs.

Protocols for Synthesis of SNIPERs and the Methods to Evaluate the Anticancer Effects.

Inducing degradation of undruggable target proteins by the use of chimeric small molecules, represented by proteolysis-targeting chimeras, is a promising strategy for drug development. We developed a series of chimeric molecules, termed "specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers" (SNIPERs) that recruit IAP ubiquitin ligases to induce degradation of target proteins. SNIPERs also induce degradation of some IAPs, including cIAP1 and XIAP, which are antiapoptotic proteins that are overexpressed in many cancers. Such protein degraders have unique properties that could be especially useful in cancer therapy. This chapter describes (1) the design and synthesis of SNIPER compounds, (2) the methods used for the detection of target protein degradation and ubiquitylation, and (3) the protocol to evaluate the antitumor activity of SNIPER.

Development of a Hematopoietic Prostaglandin D Synthase-Degradation Inducer.

Although hematopoietic prostaglandin D synthase (H-PGDS) is an attractive target for treatment of a variety of diseases, including allergic diseases and Duchenne muscular dystrophy, no H-PGDS inhibitors have yet been approved for treatment of these diseases. Therefore, the development of novel agents having other modes of action to modulate the activity of H-PGDS is required. In this study, a chimeric small molecule that degrades H-PGDS via the ubiquitin-proteasome system, PROTAC(H-PGDS)-1, was developed. PROTAC(H-PGDS)-1 is composed of two ligands, TFC-007 (that binds to H-PGDS) and pomalidomide (that binds to cereblon). PROTAC(H-PGDS)-1 showed potent activity in the degradation of H-PGDS protein via the ubiquitin-proteasome system and in the suppression of prostaglandin D2 (PGD2) production. Notably, PROTAC(H-PGDS)-1 showed sustained suppression of PGD2 production after the drug removal, whereas PGD2 production recovered following removal of TFC-007. Thus, the H-PGDS degrader-PROTAC(H-PGDS)-1-is expected to be useful in biological research and clinical therapies.

Deubiquitylase USP25 prevents degradation of BCR-ABL protein and ensures proliferation of Ph-positive leukemia cells.

Fusion genes resulting from chromosomal rearrangements are frequently found in a variety of cancer cells. Some of these are known to be driver oncogenes, such as BCR-ABL in chronic myelogenous leukemia (CML). The products of such fusion genes are abnormal proteins that are ordinarily degraded in cells by a mechanism known as protein quality control. This suggests that the degradation of BCR-ABL protein is suppressed in CML cells to ensure their proliferative activity. Here, we show that ubiquitin-specific protease 25 (USP25) suppresses the degradation of BCR-ABL protein in cells harboring Philadelphia chromosome (Ph). USP25 was found proximal to BCR-ABL protein in cells. Depletion of USP25 using shRNA-mediated gene silencing increased the ubiquitylated BCR-ABL, and reduced the level of BCR-ABL protein. Accordingly, BCR-ABL-mediated signaling and cell proliferation were suppressed in BCR-ABL-positive leukemia cells by the depletion of USP25. We further found that pharmacological inhibition of USP25 induced rapid degradation of BCR-ABL protein in Ph-positive leukemia cells, regardless of their sensitivity to tyrosine kinase inhibitors. These results indicate that USP25 is a novel target for inducing the degradation of oncogenic BCR-ABL protein in Ph-positive leukemia cells. This could be an effective approach to overcome resistance to kinase inhibitors.

Rational design of novel amphipathic antimicrobial peptides focused on the distribution of cationic amino acid residues.

Antimicrobial peptides (AMPs) have garnered much attention as novel therapeutic agents against infectious diseases. They exhibit antimicrobial activity through microbial membrane disruption based on their amphipathic properties. In this study, we rationally designed and synthesized a series of novel AMPs Block, Stripe, and Random, and revealed that Stripe exhibits potent antimicrobial activity against Gram-positive and Gram-negative microbes. Moreover, we also demonstrated that Stripe disrupts both Gram-positive and Gram-negative mimetic bacterial membranes. Finally, we investigated the hemolytic activity and cytotoxicity in human blood cells and human cell lines, and found that Stripe exhibited neither. These data indicated that Stripe is a promising antimicrobial reagent that does not display significant cytotoxicity.

SNIPERs-Hijacking IAP activity to induce protein degradation.

The induction of protein degradation by chimeric small molecules represented by proteolysis-targeting chimeras (PROTACs) is an emerging approach for novel drug development. We have developed a series of chimeric molecules termed specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to effect targeted degradation. Unlike the chimeric molecules that recruit von Hippel-Lindau and cereblon ubiquitin ligases, SNIPERs induce simultaneous degradation of IAPs such as cIAP1 and XIAP along with the target proteins. Because cancer cells often overexpress IAPs-a mechanism involved in the resistance to cancer therapy-SNIPERs could be used to kill cancer cells efficiently.

Development of a Potent Protein Degrader against Oncogenic BCR-ABL Protein.

Chromosomal translocation occurs in some cancer cells, resulting in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate expression of the BCR-ABL protein. Recently, we have devised a protein knockdown system by hybrid molecules named Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER). This system is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins. In this review, we describe the development of SNIPER against BCR-ABL, and discuss the features and prospect for treatment of CML.