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.

Photo-regulated PROTACs: A novel tool for temporal control of targeted protein degradation.

PROTACs (Proteolysis targeting chimeras) are chimeric molecules designed to induce targeted protein degradation via the ubiquitin-proteasome system. These molecules catalytically degrade target proteins and sustainably inhibit their function. Therefore, PROTAC's unique mechanism of action is not only beneficial in medicine but also serves as a valuable tool for molecular biological analysis in fields like chemical biology, biochemistry, and drug discovery. This study presents a novel turn-off (ON-OFF) type PROTAC development strategy utilizing a photocleavable linker. The inclusion of this linker enables temporal control of the degradation activity targeting BRD4 protein upon UV light exposure. PROTAC-2 demonstrated the most potent degradation activity against BRD4 among the other synthesized PROTACs with varying linker lengths. The UV light-induced cleavage of PROTAC-2 was confirmed, leading to a reduction in its BRD4 degradation activity. Notably, this study introduces a novel linker capable of nullifying degradation activity of PROTACs which is activated by light irradiation. These findings offer a promising strategy for the development of turn-off type PROTACs, providing enhanced temporal control over protein degradation. The approach holds significant potential for applications in molecular function studies and drug discovery.

Expansion of targeted degradation by Gilteritinib-Warheaded PROTACs to ALK fusion proteins.

Proteolysis targeting chimeras (PROTACs) induce the ubiquitination and subsequent proteasomal degradation of targeted proteins. Numerous PROTACs have emerged as promising drug candidates for various disease-related proteins. This study investigates PROTACs targeted to degrade anaplastic lymphoma kinase (ALK) fusion proteins, which are implicated in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. We recently reported the development of a gilteritinib-warheaded PROTAC to target and degrade the Fms-like tyrosine kinase 3 (FLT3) protein. Gilteritinib is a tyrosine kinase inhibitor that targets FLT3, and recent studies have revealed that it also functions as an ALK inhibitor. We conducted a structure-activity relationship (SAR) study and expanded the range of target proteins for gilteritinib-warheaded PROTACs to include echinoderm microtubule-associated protein-like 4 (EML4)-ALK and nucleophosmin (NPM)-ALK, in addition to FLT3. Our SAR study utilized three types of ligands for E3 ligase- inhibitor of apoptosis protein (IAP), cereblon (CRBN), and von Hippel-Lindau (VHL)- in the PROTAC designs and we observed varied efficacy in the degradation of target proteins. The CRBN-based PROTAC effectively reduced the protein expression of FLT3, EML4-ALK, and NPM-ALK. The IAP-based PROTAC reduced expression of both FLT3 and EML4-ALK proteins but not that of NPM-ALK, while the VHL-based PROTAC was ineffective against all target proteins. Several ALK-targeted PROTACs have already been developed using CRBN or VHL as E3 ligase, but this is the first report of an IAP-based ALK degrader. The length of the linker structure utilized in PROTAC also had a significant effect on their efficacy and activity. PROTACs formed with shorter linkers demonstrated an enhanced degradation activity to target proteins compared with those formed with longer linkers. These findings provide valuable insight for the development of effective PROTACs to target and degrade ALK fusion proteins.

Complement component C3 is associated with body composition parameters and sarcopenia in community-dwelling older adults: a cross-sectional study in Japan.

BACKGROUND: Chronic inflammation is a factor in the pathogenesis of sarcopenia, which is characterized by low muscle mass and reduced strength. Complement C3 is important in the management of the immune network system. This study seeks to determine the relationship between serum C3 levels and body composition and sarcopenia-related status in community-dwelling older adults. METHODS: Study participants were 269 older adults living in rural Japan. A bioelectrical impedance analysis device was used to measure body composition parameters including body mass index (BMI), body fat percentage, waist-hip-ratio, and appendicular skeletal muscle mass index (SMI). Muscle function was measured by handgrip strength and 6-m walking speed. The correlation coefficients for C3 level and measurements were calculated using Pearson correlation analysis. Participants were categorized into normal, pre-sarcopenia, dynapenia, or sarcopenia groups. Sarcopenia was defined according to 2019 Asian Working Group for Sarcopenia definition, dynapenia was defined as low muscle function without low muscle mass, and pre-sarcopenia was defined as the presence of low muscle mass only. The C3 threshold score for sarcopenia status was evaluated by receiver operating characteristic curve (ROC) analysis. RESULTS: Significant positive correlations were found between C3 and BMI, body fat percentage, and waist-hip ratio in both sexes, and further positive correlations with SMI were found in women. The relationship with body fat percentage was particularly strong. Body composition measurements (BMI, body fat percentage, and waist- hip ratio) and C3 levels were lowest in the sarcopenia group compared with the others. ROC analysis showed that the significant threshold of C3 for discriminating between the normal and sarcopenia groups was 105 mg/dL. Multiple logistic regression analysis showed that participants with C3 < 105 mg/dL had an odds ratio of 3.27 (95% confidence interval, 1.49-7.18) for sarcopenia adjusted by sex, age and body fat percentage. CONCLUSION: C3 levels are suggested to be related to body composition and pathophysiological functions of sarcopenia. C3 is expected to become a useful biomarker for sarcopenia, for predicting the onset of the disease and for predicting the effectiveness of interventions.

Rational Design of Amphipathic Antimicrobial Peptides with Alternating L-/D-Amino Acids That Form Helical Structures.

Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique ß6.3-helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a ß6.3-helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.

[Current Status and Regulatory Issues of Companion Diagnostics in Japan].

Companion diagnostics(CDx)are in vitro diagnostic products that are used to predict the efficacy and adverse effects of therapeutic drugs prior to administration, and are co-developed and co-approved with the therapeutic drugs in principle. In Japan, 40 CDx products have been approved by January 2024, and 39 products are used to determine if therapeutic drugs are applicable for cancer treatment. In the CDx products for cancer treatment, PCR, immunohistochemistry, or in situ hybridization is used to clarify the mutations(point mutations, insertions/deletions, fusions, etc.)in cancer-related genes or the expression levels of cancer-related molecules in the cancer tissues. The results of the analysis determine whether a particular therapeutic drug could be used or not for the treatment of the corresponding patient. Recently, several next-generation sequencing(NGS)-based CDx products have been approved and utilized for cancer treatment. The rise of NGS-based diagnostics has made it possible to comprehensively analyze mutations in many cancer-related genes in a single test and to determine whether each of several therapeutic drugs is applicable to the patient at once. On the other hand, with the increase in the number of CDx products, several regulatory issues have arisen, including an issue related to the co-development of CDx and a therapeutic drug and an issue related to the interchangeable use of CDx products that detect the same mutations of the cancer-related genes. The revision of CDx-related guidance is being considered in Japan and overseas in response to this situation.

Efficient intracellular drug delivery by co-administration of two antibodies against cell adhesion molecule 1.

Cell adhesion molecule 1 (CADM1), a single-pass transmembrane protein, is involved in oncogenesis. We previously demonstrated the therapeutic efficacy of anti-CADM1 ectodomain monoclonal antibodies against mesothelioma; however, the underlying mechanism is unclear. In the present study, we explored the molecular behavior of anti-CADM1 antibodies in CADM1-expressing tumor cells. Sequencing analyses revealed that the anti-CADM1 chicken monoclonal antibodies 3E1 and 9D2 are IgY and IgM isotype antibodies, respectively. Co-administration of 3E1 and 9D2 altered the subcellular distribution of CADM1 from the detergent-soluble fraction to the detergent-resistant fraction in tumor cells. Using recombinant chicken-mouse chimeric antibodies that had been isotype-switched from IgG to IgM, we demonstrated that the combination of the variable region of 3E1 and the constant region of IgM was required for CADM1 relocation. Cytochemical studies showed that 3E1 colocalized with late endosomes/lysosomes after co-administration with 9D2, suggesting that the CADM1-antibody complex is internalized from the cell surface to intracellular compartments by lipid-raft mediated endocytosis. Finally, 3E1 was conjugated with the antimitotic agent monomethyl auristatin E (MMAE) via a cathepsin-cleavable linker. Co-administration of 3E1-monomethyl auristatin E and 9D2 suppressed the growth of multiple types of tumor cells, and this anti-tumor activity was confirmed in a syngeneic mouse model of melanoma. 3E1 and 9D2 are promising drug delivery vehicles for CADM1-expressing tumor cells.

Relief of pain in mice by an antibody with high affinity for cell adhesion molecule 1 on nerves.

AIMS: Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin superfamily and is abundantly expressed on nerve fibers. Recently, the anti-CADM1 ectodomain antibody 3E1 has proven useful as a drug delivery vector for CADM1-expressing cells in vitro. When injected subcutaneously into mice, whether 3E1 accumulates on nerve fibers and serves as an analgesic was examined. MAIN METHODS: Injected 3E1 was detected by immunohistochemistry and double immunofluorescence. Analgesic effects were verified by a formalin-induced chemical-inflammatory pain test and video-recorded behavior analysis that were performed 6, 12, and 24 h after antibody injection. Primary cultures of mouse dorsal root ganglion (DRG) cells were incubated with 3E1 and expressions of CADM1 and its key downstream molecules were examined by Western blot analyses and live cell imaging. DRG cells were loaded with a Ca2+ fluorescent indicator Fluo-8 and a femtosecond laser pulse was irradiated near the cell body to mechanically stimulate the nerves. KEY FINDINGS: Subcutaneously injected 3E1 was widely localized almost exclusively on peripheral nerve fibers in the dermis. In formalin tests, 3E1-injected mice exhibited less pain-related behavior than control mice. When 3E1 was added to DRG cell cultures, it localized to neurites and resulted in decreased expression of CADM1, increased phosphorylation of Src and Akt, and CADM1-3E1 complex formation. Femtosecond laser-induced stimulation transmission along neurites was clearly visualized by Fluo-8 fluorescence in control cells, whereas it was markedly suppressed in 3E1-treated cells. SIGNIFICANCE: 3E1 was suggested to be a potential long-acting analgesic based on its high affinity for CADM1.

Psychophysiological and interpersonal effects of parallel group crafting: a multimodal study using EEG and ECG.

In occupational therapy, crafts and groups are used as therapeutic tools, but their electrophysiological effects have not been well described. This study aimed to investigate the effects of group crafting on the physiological synchrony (PS) of dyadic heartbeats and on the autonomic activity and electroencephalogram (EEG) of individuals. In this cross-sectional study, individuals' EEG and dyadic electrocardiogram (ECG) were measured during the task in a variety of conditions. The three conditions were alone, parallel, nonparallel. Autonomic activity from the subjects' ECG, PS from the dyadic ECG, and current source density from exact Low Resolution Brain Electromagnetic Tomography (eLORETA) from subjects' EEG were analyzed. Measurements from 30 healthy young adults showed that the parallel condition significantly increased subjects' parasympathetic activity and dyadic PS. Parallel condition and frontal midline theta influenced parasympathetic activity, whereas parasympathetic activity was not associated with PS. Dyadic lag value were correlated with frontal delta, beta, and gamma activity. The results suggest that crafting in parallel groups increases parasympathetic activity and PS through different mechanisms, despite the absence of direct interaction. They also explain the electrophysiological evidence for the use of crafts and groups in psychiatric occupational therapy, such as increased relaxation and PS.

Separation and Characterization of Therapeutic Oligonucleotide Isomer Impurities by Cyclic Ion Mobility Mass Spectrometry.

Therapeutic oligonucleotides such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA) are among the most remarkable modalities in modern medicine. ASOs and siRNA are composed of single- or double-stranded 15-25 mer synthesized oligonucleotides, which can be used to modulate gene expression. Liquid chromatography-mass spectrometry (LC/MS) is a necessary technique for the quality control of therapeutic oligonucleotides; it is used to evaluate the quantities of target oligonucleotides and their impurities. The widely applied oligonucleotide therapeutic quantitation method uses both ultraviolet (UV) absorbance and the MS signal intensity. Peaks separated from the main peak, which contains full-length product, are generally quantitated by UV. However, coeluting impurities, such as n - 1 shortmers, abasic oligonucleotides, and PS → PO (phosphorothiate to phosphodiester) oligonucleotides, are quantitated by MS. These coeluting impurities can also be comprised of various isomers with the same modification, thus increasing the difficulty in their separation and relative quantitation by LC/MS. It is possible that a specific isomer with a certain structural form induces toxicities. Therefore, characterization of each isomer separation is in high demand. In this study, we separated and characterized oligonucleotide isomers by employing a cyclic ion mobility mass spectrometry (cyclic IMS) system, which allows the separation of ions with the same m/z ratio based on their structural differences. Patisiran antisense and sense strands and their n - 1 and abasic isomers were used as sample sequences, and their ratio characterization was achieved by cyclic IMS. In addition, we evaluated the PS → PO conversion isomers of the antisense strand of givosiran, which originally contained four PS modification sites. The PS → PO isomers exhibited specific and distinguishable mobiligram patterns. We believe that cyclic IMS is a promising method for evaluating therapeutic oligonucleotide isomers.

Long-Term Efficacy and Safety of Lorlatinib in Japanese Patients With ALK-Positive Advanced NSCLC-A Brief Report From the CROWN Study.

Introduction: Lorlatinib was found to have improved efficacy versus crizotinib in the global phase 3 CROWN study (NCT03052608). Similar results were revealed for the Japanese population as for the overall population. We present results from the unplanned 3-year follow-up from the CROWN study in Japanese patients. Methods: Patients were randomized to either lorlatinib 100 mg once daily (n = 25) or crizotinib 250 mg twice daily (n = 23). The primary end point was progression-free survival assessed by blinded independent central review. Secondary end points included objective and intracranial responses assessed by blinded independent central review and safety. Results: At the data cutoff of September 20, 2021, median progression-free survival was not reached with lorlatinib and 11.1 months with crizotinib (hazard ratio = 0.36). Objective response rate was 72.0% with lorlatinib and 52.2% with crizotinib. For patients with baseline brain metastases, intracranial response rate was 100.0% versus 28.6% with lorlatinib versus crizotinib. Nine patients in the lorlatinib group received more than or equal to 1 subsequent anticancer systemic therapy, with ALK tyrosine kinase inhibitor as the most common first subsequent therapy. The safety profile was consistent with that reported previously, with no new safety signals. Conclusions: This updated analysis in the Japanese population revealed prolonged benefits of lorlatinib over crizotinib in patients with treatment-naive advanced ALK-positive NSCLC with and those without brain metastases.

Focal brain cooling suppresses spreading depolarization and reduces endothelial nitric oxide synthase expression in rats.

This study aimed to investigate the effects of focal brain cooling (FBC) on spreading depolarization (SD), which is associated with several neurological disorders. Although it has been studied from various aspects, no medication has been developed that can effectively control SD. As FBC can reduce neuronal damage and promote functional recovery in pathological conditions such as epilepsy, cerebral ischemia, and traumatic brain injury, it may also potentially suppress the onset and progression of SD. We created an experimental rat model of SD by administering 1 M potassium chloride (KCl) to the cortical surface. Changes in neuronal and vascular modalities were evaluated using multimodal recording, which simultaneously recorded brain temperature (BrT), wide range electrocorticogram, and two-dimensional cerebral blood flow. The rats were divided into two groups (cooling [CL] and non-cooling [NC]). Warm or cold saline was perfused on the surface of one hemisphere to maintain BrT at 37°C or 15°C in the NC and CL groups, respectively. Western blot analysis was performed to determine the effects of FBC on endothelial nitric oxide synthase (eNOS) expression. In the NC group, KCl administration triggered repetitive SDs (mean frequency = 11.57/h). In the CL group, FBC increased the duration of all KCl-induced events and gradually reduced their frequency. Additionally, eNOS expression decreased in the cooled brain regions compared to the non-cooled contralateral hemisphere. The results obtained by multimodal recording suggest that FBC suppresses SD and decreases eNOS expression. This study may contribute to developing new treatments for SD and related neurological disorders.

Machine Learning Approaches for Stroke Risk Prediction: Findings from the Suita Study.

Stroke constitutes a significant public health concern due to its impact on mortality and morbidity. This study investigates the utility of machine learning algorithms in predicting stroke and identifying key risk factors using data from the Suita study, comprising 7389 participants and 53 variables. Initially, unsupervised k-prototype clustering categorized participants into risk clusters, while five supervised models including Logistic Regression (LR), Random Forest (RF), Support Vector Machine (SVM), Extreme Gradient Boosting (XGBoost), and Light Gradient Boosted Machine (LightGBM) were employed to predict stroke outcomes. Stroke incidence disparities among identified risk clusters using the unsupervised k-prototype clustering method are substantial, according to the findings. Supervised learning, particularly RF, was a preferable option because of the higher levels of performance metrics. The Shapley Additive Explanations (SHAP) method identified age, systolic blood pressure, hypertension, estimated glomerular filtration rate, metabolic syndrome, and blood glucose level as key predictors of stroke, aligning with findings from the unsupervised clustering approach in high-risk groups. Additionally, previously unidentified risk factors such as elbow joint thickness, fructosamine, hemoglobin, and calcium level demonstrate potential for stroke prediction. In conclusion, machine learning facilitated accurate stroke risk predictions and highlighted potential biomarkers, offering a data-driven framework for risk assessment and biomarker discovery.

Effect of individualized occupational therapy on cognition among patients with schizophrenia: A randomized controlled trial.

This study aimed to evaluate the feasibility and efficacy of individualized occupational therapy (IOT) plus group occupational therapy (GOT) as standard care for cognition compared to GOT alone, and to determine which IOT component has the greatest effect on cognitive outcome in patients with schizophrenia. This study was conducted at 14 clinical sites across Japan and enrolled recently hospitalized patients with schizophrenia. The IOT consisted of motivational interview, self-monitoring, individualized visits, craft activities, individualized psychoeducation, and discharge planning. Among the 68 patients who were randomized to the GOT + IOT group (n = 34) and GOT alone group (n = 34), 67 completed the trial (GOT + IOT group, n = 34; GOT alone group, n = 33). There were significant improvements in change from baseline to post-treatment between the groups in verbal memory, working memory, verbal fluency, attention, executive function domains, and the composite score of the Brief Assessment of Cognition in Schizophrenia (BACS). The BACS composite score was significantly associated with the number of craft activity sessions. The addition of IOT to GOT has a favorable feasibility profile and efficacy for cognition in schizophrenia. Craft activity is the most effective IOT component in improving cognition.

Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.