Search

Canine histiocytic sarcoma (CHS) is a malignant tumor derived from macrophages and dendritic cells. Since effective chemotherapy is needed for CHS cases, we conducted this prospective study to evaluate the efficacy and adverse events of vincristine treatment as a rescue therapy for this disease. We administered vincristine to nine CHS cases that acquired resistance to lomustine or nimustine. Complete remission was achieved in one dog, partial remission in two dogs, stable disease in five dogs, and progressive disease in one dog. The median progression-free survival was 21 days (range: 7-71 days). Severe adverse effect was observed in one dog (Grade 3 thrombocytopenia). It is essential to establish novel effective treatments for CHS.

Waldiomycin is an inhibitor of histidine kinases (HKs). Although most HK inhibitors target the ATP-binding region, waldiomycin binds to the intracellular dimerization domain (DHp domain) with its naphthoquinone moiety presumed to interact with the conserved H-box region. To further develop inhibitors targeting the H-box, various 2-aminonaphthoquinones with cyclic, aliphatic, or aromatic amino groups and naphtho [2,3-d] isoxazole-4,9-diones were synthesized. These compounds were tested for their inhibitory activity (IC 50 ) against WalK, an essential HK for Bacillus subtilis growth, and their minimum inhibitory concentrations (MIC) against B. subtilis. As a result, 11 novel HK inhibitors were obtained as naphthoquinone derivatives (IC 50 : 12.6-305 µM, MIC: 0.5-128 µg ml -1 ). The effect of representative compounds on the expression of WalK/WalR regulated genes in B. subtilis was investigated. Four naphthoquinone derivatives induced the expression of iseA (formerly yoeB), whose expression is negatively regulated by the WalK/WalR system. This suggests that these compounds inhibit WalK in B. subtilis cells, resulting in antibacterial activity. Affinity selection/mass spectrometry analysis was performed to identify whether these naphthoquinone derivatives interact with WalK in a manner similar to waldiomycin. Three compounds were found to competitively inhibit the binding of waldiomycin to WalK, suggesting that they bind to the H-box region conserved in HKs and inhibit HK activity., (© 2024. The Author(s).)

Non-neoplastic bone marrow disorders are main causes of non-regenerative anemia in dogs. Despite the high incidence of the diseases, their molecular pathophysiology has not been elucidated. We previously reported that Miniature Dachshund (MD) was a predisposed breed to be diagnosed with non-neoplastic bone marrow disorders in Japan, and immunosuppressive treatment-resistant MDs showed higher number of platelets and morphological abnormalities in peripheral blood cells. These data implied that treatment-resistant MDs might possess distinct pathophysiological features from treatment-responsive MDs. Therefore, we conducted transcriptomic analysis of bone marrow specimens to investigate the pathophysiology of treatment-resistant MDs. Transcriptomic analysis comparing treatment-resistant MDs and healthy control dogs identified 179 differentially expressed genes (DEGs). Pathway analysis using these DEGs showed that "Wnt signaling pathway" was a significantly enriched pathway. We further examined the expression levels of DEGs associated with Wnt signaling pathway and confirmed the upregulation of AXIN2 and CCND2 and the downregulation of SFRP2 in treatment-resistant MDs compared with treatment-responsive MDs and healthy control dogs. This alteration implied the activation of Wnt signaling pathway in treatment-resistant MDs. The activation of Wnt signaling pathway has been reported in human patients with myelodysplastic syndrome (MDS), which is characterized by dysplastic features of blood cells. Therefore, the results of this study implied that treatment-resistant MDs have distinct molecular pathological features from treatment-responsive MDs and the pathophysiology of treatment-resistant MDs might be similar to that of human MDS patients.

Background: Cancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we demonstrated that tetracyclines enhanced T-cell responses. Therefore, we herein investigated the efficacy of tetracyclines on antitumor T-cell responses by human peripheral T cells, murine models, and the lung tumor tissues of patients with non-small cell lung cancer (NSCLC), with a focus on signaling pathways in T cells., Methods: The cytotoxicity of peripheral and lung tumor-infiltrated human T cells against tumor cells was assessed by using bispecific T-cell engager (BiTE) technology (BiTE-assay system). The effects of tetracyclines on T cells in the peripheral blood of healthy donors and the tumor tissues of patients with NSCLC were examined using the BiTE-assay system in comparison with anti-programmed cell death-1 (PD-1) antibody, nivolumab. T-cell signaling molecules were analyzed by flow cytometry, ELISA, and qRT-PCR. To investigate the in vivo antitumor effects of tetracyclines, tetracyclines were administered orally to BALB/c mice engrafted with murine tumor cell lines, either in the presence or absence of anti-mouse CD8 inhibitors., Results: The results obtained revealed that tetracyclines enhanced antitumor T-cell cytotoxicity with the upregulation of granzyme B and increased secretion of interferon-γ in human peripheral T cells and the lung tumor tissues of patients with NSCLC. The analysis of T-cell signaling showed that CD69 in both CD4 + and CD8 + T cells was upregulated by minocycline. Downstream of T-cell receptor signaling, Zap70 phosphorylation and Nur77 were also upregulated by minocycline in the early phase after T-cell activation. These changes were not observed in T cells treated with anti-PD-1 antibodies under the same conditions. The administration of tetracyclines exhibited antitumor efficacy with the upregulation of CD69 and increases in tumor antigen-specific T cells in murine tumor models. These changes were canceled by the administration of anti-mouse CD8 inhibitors., Conclusions: In conclusion, tetracyclines enhanced antitumor T-cell immunity via Zap70 signaling. These results will contribute to the development of novel cancer immunotherapy., Competing Interests: Competing interests: There are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

Background: Tumor-derived extracellular vesicles (EVs) have been proposed as the essential mediator between host immunity and cancer development. These EVs conduct cellular communication to facilitate tumor growth, enable invasion and metastasis, and shape the favorable tumor microenvironment. Lymphoma is one of the most common hematological malignancies in humans and dogs. Effective T-cell responses are required for the control of these malignancies. However, the immune crosstalk between CD8 + T-cells, which dominates anti-tumor responses, and canine lymphoma has rarely been described., Methods: This study investigates the immune manipulating effects of EVs, produced from the clinical cases and cell line of canine B cell lymphoma, on CD8 + T-cells isolated from canine donors., Results: Lymphoma-derived EVs lead to the apoptosis of CD8 + T-cells. Furthermore, EVs trigger the overexpression of CTLA-4 on CD8 + T-cells, which indicates that EV blockade could serve as a potential therapeutic strategy for lymphoma patients. Notably, EVs transform the CD8 + T-cells into regulatory phenotypes by upregulating their PD-1, PD-L1, and FoxP3 mRNA expression. The regulatory CD8 + T-cells secret the panel of inhibitory cytokines and angiogenic factors and thus create a pro-tumorigenic microenvironment., Conclusion: In summary, the current study demonstrated that the EVs derived from canine B cell lymphoma impaired the anti-tumor activity of CD8 + T-cells and manipulated the possible induction of regulatory CD8 + T-cells to fail the activation of host cellular immunity., (© 2023. The Author(s).)

Background: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3 , regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure., Methods: We generated the knock-in mice ( Mylk3 +/fs and Mylk 3 fs/fs ) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation ( MYLK3 +/fs ) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154)., Results: Both mice ( Mylk3 +/fs and Mylk 3 fs/fs ) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_ MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3 +/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_ MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the V max for ventricular myosin regulatory light chain phosphorylation without affecting the K m . LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3 +/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3 / PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes., Conclusions: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure., Competing Interests: Disclosures None.

Histiocytic sarcoma (HS) is an incurable aggressive tumor, and no consensus has been made on the treatment due to its rare occurrence. Since dogs spontaneously develop the disease and several cell lines are available, they have been advocated as translational animal models. In the present study, therefore, we explored gene mutations and aberrant molecular pathways in canine HS by next generation sequencing to identify molecular targets for treatment. Whole exome sequencing and RNA-sequencing revealed gene mutations related to receptor tyrosine kinase pathways and activation of ERK1/2, PI3K-AKT, and STAT3 pathways. Analysis by quantitative PCR and immunohistochemistry revealed that fibroblast growth factor receptor 1 (FGFR1) is over-expressed. Moreover, activation of ERK and Akt signaling were confirmed in all HS cell lines, and FGFR1 inhibitors showed dose-dependent growth inhibitory effects in two of the twelve canine HS cell lines. The findings obtained in the present study indicated that ERK and Akt signaling were activated in canine HS and drugs targeting FGFR1 might be effective in part of the cases. The present study provides translational evidence that leads to establishment of novel therapeutic strategies targeting ERK and Akt signaling in HS patients., (© 2023. The Author(s).)

Celastrol, a bioactive molecule extracted from the Tripterygium wilfordii plant, has been shown to exhibit anti-inflammatory properties. However, its mechanism of action has not been fully elucidated. Here, we show that celastrol suppresses humoral immune responses and autoimmunity by disabling a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and COMMD8 (COMMD3/8 complex), a signaling adaptor for chemoattractant receptors. Having demonstrated the involvement of the COMMD3/8 complex in a mouse model of rheumatoid arthritis, we identified celastrol as a compound that covalently bound to and dissociated the COMMD3/8 complex. Celastrol inhibited B cell migration, reduced antibody responses, and blocked arthritis progression, recapitulating deficiency of the COMMD3/8 complex. These effects of celastrol were abolished in mice expressing a celastrol-resistant mutant of the COMMD3/8 complex. These findings establish that celastrol exerts immunosuppressive activity by targeting the COMMD3/8 complex. Our study suggests that the COMMD3/8 complex is a potentially druggable target in autoimmune diseases and points to celastrol as a lead pharmacologic candidate in this capacity.

Cats urgently visit emergency hospitals due to respiratory distress, and the chief cause is cardiogenic pulmonary edema (CPE). Although cats with CPE were frequently encountered in clinics, the prognostic factors were poorly reported. The objective of this retrospective study was to investigate the association of physical examination and venous blood gas parameters with the survival of cats with CPE in an emergency hospital. Thirty-six cats with CPE were ultimately included in the present study, and eight of them died within 12 h after their presentation to our hospital. Statistical analyses of clinical parameters between cats that died within 12 h and those that survived for 12 h were conducted using Mann-Whitney U test with Bonferroni correction. Cats that died within 12 h had significantly lower rectal temperatures and higher PvCO 2 than those that did not die within 12 h. Moreover, hypotension and vasoconstrictor use were related to death within 12 h of presentation and higher PvCO 2 . These findings indicated the prognostic utility of body temperature and PvCO 2 , and the association between hypercapnia and the severity of CPE or hypotension. A large number of prospective studies should be performed to validate these results.

Canine histiocytic proliferative disorders include aggressive and fatal diseases, such as histiocytic sarcoma (HS) and histiocytosis (SyH). The molecular mechanisms underlying cell proliferation need to be elucidated for the development of effective treatments. In the present study, mRNA expression levels were comprehensively analysed in cell lines derived from localized HS, disseminated HS, SyH and Langerhans cell histiocytosis (LCH) in dogs. Based on the results obtained, the growth inhibitory effects of palbociclib, a CDK4/6 inhibitor, were verified with the cell lines in vitro and in xenograft mouse model. Hierarchical clustering and principal component analysis plots of mRNA expression profiles divided the cell lines into three groups: a localized HS group, disseminated HS/SyH group, and LCH. The results of an ingenuity pathway analysis suggested that the MAPK signalling pathway was activated in the localized HS and LCH cell lines, and the PI3K signalling pathway in the disseminated and localized HS cell lines. In all cell lines, the expression of the tumour suppressor genes TP53, CDKN2A and CDKN1A was down-regulated, whereas that of Rb was preserved. In vitro assessments revealed the growth inhibitory effects of palbociclib in all cell lines examined. In a xenograft mouse model using a cell line from disseminated HS, palbociclib exerted significant growth inhibitory effects. These results suggest the potential of palbociclib as a therapeutic drug candidate for the treatment of malignant histiocytic proliferative disorders of the dog., (© 2022 John Wiley & Sons Ltd.)

Interactions between tumor and immune cells within the tumor microenvironment play an important role in tumor progression, and small extracellular vesicles (EVs) derived from these tumor cells have been shown to exert immunomodulatory effects on various immune cells, including macrophages and lymphocytes. Although the immunomodulatory effects of small EVs derived from human cancer cells have been intensively investigated, few studies have investigated the effects of lymphoma-derived small EVs on macrophages in both human and veterinary medicine. Here, we evaluated the effects of canine lymphoma-derived small EVs on canine primary monocytes, which are the major source of macrophages in neoplastic tissues. Comprehensive gene expression analysis of these treated monocytes revealed their distinct activation via the Toll-like receptor (TLR) and NF-κβ signaling pathways. In addition, treatment with lymphoma small EVs increased the secretion of MCP-1, which induces the infiltration and migration of monocytes and lymphocytes in neoplastic and cancer tissues. The results of this study indicate that canine lymphoma small EVs activate monocytes, possibly through the activation of TLR and NF-κβ signaling pathways, and induce monocytes to secrete of MCP-1, which might contribute to immune cell infiltration within the tumor microenvironment.

Ectopic gene expression is an indispensable tool in biology and medicine, but is often limited by the low efficiency of DNA transfection. We previously reported that depletion of the autophagy receptor p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target for drugs to increase transfection efficiency. To identify such drugs, a nonbiased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62 dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the p62-dependent mechanisms, the time required for p62-dependent ubiquitination, which is required for autophagosome formation, was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. Microtubule inhibitors caused a delay in ubiquitination. Furthermore, the level of phosphorylated p62 at S405 was markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings demonstrate for the first time that microtubule inhibitors suppress p62 activation as a mechanism for increasing DNA transfection efficiency and provide solutions to increase efficiency., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Non-neoplastic bone marrow disorders such as non-regenerative immune-mediated anemia, pure red cell aplasia, and myelodysplastic syndrome are major causes of non-regenerative anemia in dogs. However, there has been no study on the clinical and clinicopathological features of canine non-neoplastic bone marrow disorders in Japan. Hence, we first investigated the breed disposition of non-neoplastic bone marrow disorders that induce anemia as a retrospective study and found that Miniature Dachshund (MD) was a predisposed breed. Based on this finding, we investigated the clinical and clinicopathological features of non-neoplastic bone marrow disorders in MDs as a preliminary retrospective study, and we compared them between immunosuppressive treatment-responsive and -resistant MDs. We found that treatment-resistant MDs showed thrombocytosis and increased frequencies of dysplastic features in the peripheral blood. These results indicate that bone marrow disorders in treatment-resistant MDs might manifest distinct features compared with those in treatment-sensitive MDs, and sensitivity to immunosuppressive treatments could be predicted based on thrombocytosis and dysplastic features in the peripheral blood. Further studies that examine aberrations in the genome are needed to elucidate the pathophysiology of bone marrow disorders in MDs.

Inflammatory colorectal polyps (ICRPs) are frequently observed in miniature dachshunds in Japan and treated by prednisolone and immunosuppressive drugs such as cyclosporine and leflunomide. The purpose of this retrospective study was to compare the treatment efficacy, such as response rate, response interval, recurrence rate, and adverse events between cyclosporine and leflunomide. While the response rates were significantly higher in dogs treated with leflunomide, no significant differences were observed in the response interval or recurrence rate. Two of the 11 dogs treated with leflunomide showed hematological or gastrointestinal adverse events, while no dog treated with cyclosporine showed any adverse events. A case-controlled prospective study to compare the treatment efficacy of leflunomide with that of cyclosporine should be conducted.

Approximately 60-70% of EWSR1-negative small blue round cell sarcomas harbour a rearrangement of CIC, most commonly CIC-DUX 4 . CIC-DUX 4 sarcoma (CDS) is an aggressive and often fatal high-grade sarcoma appearing predominantly in children and young adults. Although cell lines and their xenograft models are essential tools for basic research and development of antitumour drugs, few cell lines currently exist for CDS. We successfully established a novel human CDS cell line designated Kitra-SRS and developed orthotopic tumour xenografts in nude mice. The CIC-DUX 4 fusion gene in Kitra-SRS cells was generated by t(12;19) complex chromosomal rearrangements with an insertion of a chromosome segment including a DUX 4 pseudogene component. Kitra-SRS xenografts were histologically similar to the original tumour and exhibited metastatic potential to the lungs. Kitra-SRS cells displayed autocrine activation of the insulin-like growth factor 1 (IGF-1)/IGF-1 receptor (IGF-1R) pathway. Accordingly, treatment with the IGF-1R inhibitor, linsitinib, attenuated Kitra-SRS cell growth and IGF-1-induced activation of IGF-1R/AKT signalling both in vitro and in vivo. Furthermore, upon screening 1134 FDA-approved drugs, the responses of Kitra-SRS cells to anticancer drugs appeared to reflect those of the primary tumour. Our model will be a useful modality for investigating the molecular pathology and therapy of CDS.

Vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2) is a class B G-protein coupled receptor (GPCR) and plays important roles in the physiology of central nervous system (CNS) by interaction with natural ligands; vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Because it has been reported that high-expression and/or overactivation of VIPR2 link to schizophrenic symptoms, VIPR2 antagonists could be good drug candidates for schizophrenia therapeutics. In this study, we discovered several artificial peptides that antagonize both human and rodent VIPR2 with selectivities against receptor subtypes VIPR1 (also known as VPAC1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). Of them, the representative 16-mer cyclic peptide VIpep-3 (Ac-CPPYLPRRLCTLLLRS-OH) exhibited strong binding affinity with K D value of 41 nM to extracellular domain of human VIPR2 in SPR analysis and showed potent antagonist activity with IC 50 values of 47 nM (human), 180 nM (mouse), and 44 nM (rat) against VIP-VIPR2 signal in cell-based Ca influx assay. This is not only the first report on artificial VIPR2-selective antagonist peptides but also good example of the effective approach to discover novel antagonist against class B GPCR. Our peptides will contribute to study and development of the novel CNS drugs targeting to VIPR2., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cytidine triphosphate synthase 1 (CTPS1) is an enzyme expressed in activated lymphocytes that catalyzes the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP) with ATP-dependent amination, using either L-glutamine or ammonia as the nitrogen source. Since CTP plays an important role in DNA/RNA synthesis, phospholipid synthesis, and protein sialyation, CTPS1-inhibition is expected to control lymphocyte proliferation and size expansion in inflammatory diseases. In contrast, CTPS2, an isozyme of CTPS1 possessing 74% amino acid sequence homology, is expressed in normal lymphocytes. Thus, CTPS1-selective inhibition is important to avoid undesirable side effects. Here, we report the discovery of CTpep-3: Ac-FRLGLLKAFRRLF-OH from random peptide libraries displayed on T7 phage, which exhibited CTPS1-selective binding with a K D value of 210nM in SPR analysis and CTPS1-selective inhibition with an IC 50 value of 110nM in the enzyme assay. Furthermore, two fundamentally different approaches, enzyme inhibition assay and HDX-MS, provided the same conclusion that CTpep-3 acts by binding to the amidoligase (ALase) domain on CTPS1. To our knowledge, CTpep-3 is the first CTPS1-selective inhibitor., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We report a design strategy to obtain potent DPP-4 inhibitors by incorporating salt bridge formation with Lys554 in the S1' pocket. By applying the strategy to the previously identified templates, quinoline 4 and pyridines 16a, 16b, and 17 have been identified as subnanomolar or nanomolar inhibitors of human DPP-4. Docking studies suggested that a hydrophobic interaction with Tyr547 as well as the salt bridge interaction is important for the extremely high potency. The design strategy would be useful to explore a novel design for DPP-4 inhibitors having a distinct structure with a unique binding mode., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Protein-protein interaction between dedicator of cytokinesis 2 (DOCK2) and Ras-related C3 botulinum toxin substrate 1 (Rac1) is an attractive intracellular target for transplant rejection and inflammatory diseases. Recently, DOCK2-selective inhibitory peptides have been discovered, and conjugation with oligoarginine cell-penetrating peptide (CPP) improved inhibitory activity in a cell migration assay. Although a number of CPPs have been reported, oligoarginine was only one example introduced to the inhibitory peptides. In this study, we aimed to confirm the feasibility of CPP-conjugation approach for DOCK2-inhibitory peptides, and select preferable sequences as CPP moiety. First, we evaluated cell permeability of thirteen known CPPs and partial sequences of influenza A viral protein PB1-F2 using an internalization assay system based on luciferin-luciferase reaction, and then selected four CPPs with efficient cellular uptake. Among four conjugates of these CPPs and a DOCK2-inhibitory peptide, the inhibitory activity of a novel CPP, PB1-F2 fragment 5 (PF5), conjugate was comparable to oligoarginine conjugate and higher than that of the non-conjugated peptide. Finally, internalization assay revealed that oligoarginine and PF5 increased the cellular uptake of inhibitory peptides to the same extent. Hence, we demonstrated that CPP-conjugation approach is applicable to the development of novel anti-inflammatory drugs based on DOCK2 inhibition by investigating both cellular uptake and bioactivity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Amino-acid mutations of Gly 12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH 2 ) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K D and IC 50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH 2 ) that inhibited enzyme activity of K-Ras(G12D) with IC 50  = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The phospholipid hydroperoxidase glutathione peroxidase (GPX4) is an enzyme that reduces lipid hydroperoxides in lipid membranes. Recently, GPX4 has been investigated as a target molecule that induces iron-dependent cell death (ferroptosis) selectively in cancer cells that express mutant Ras. GPX4 inhibitors have the potential to become novel anti-cancer drugs. However, there are no druggable pockets for conventional small molecules on the molecular surface of GPX4. To generate GPX4 inhibitors, we examined the use of peptides as an alternative to small molecules. By screening peptide libraries displayed on T7 phages, and analyzing the X-ray crystal structures of the peptides, we successfully identified one peptide that binds to near Sec73 of catalytic site and two peptides that bind to another site on GPX4. To our knowledge, this is the first study reporting GPX4 inhibitory peptides and their structural information., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Trelagliptin (SYR-472), a novel dipeptidyl peptidase-4 inhibitor, shows sustained efficacy by once-weekly dosing in type 2 diabetes patients. In this study, we characterized in vitro properties of trelagliptin, which exhibited approximately 4- and 12-fold more potent inhibition against human dipeptidyl peptidase-4 than alogliptin and sitagliptin, respectively, and >10,000-fold selectivity over related proteases including dipeptidyl peptidase-8 and dipeptidyl peptidase-9. Kinetic analysis revealed reversible, competitive and slow-binding inhibition of dipeptidyl peptidase-4 by trelagliptin (t1/2 for dissociation ≈ 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. Taken together, potent dipeptidyl peptidase inhibition may partially contribute to sustained efficacy of trelagliptin.

Spiro-pyrazolidinedione derivatives without quaternary chiral center were discovered by structure-based drug design and characterized as potent acetyl-CoA carboxylase (ACC) inhibitors. The high metabolic stability of the spiro-pyrazolo[1,2-a]pyridazine scaffold and enhancement of the activity by incorporation of a 7-methoxy group on the benzothiophene core successfully led to the identification of compound 4c as an orally bioavailable and highly potent ACC inhibitor. Oral administration of 4c significantly decreased the values of the respiratory quotient in rats, indicating the stimulation of fatty acid oxidation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Spiro-lactone (S)-1 is a potent acetyl-CoA carboxylase (ACC) inhibitor and was found to be metabolically liable in human hepatic microsomes. To remove one of the risk factors in human study by improving the metabolic stability, we focused on modifying the spiro-lactone ring and the benzothiophene portion of the molecule. Spiro-imide derivative 8c containing a 6-methylthieno[2,3-b]pyridine core exhibited potent ACC inhibitory activity and favorable pharmacokinetic profiles in rats., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The co-crystal structure of the human acetyl-coenzyme A 2 (ACC2) carboxyl transferase domain and the reported compound CP-640186 (1b) suggested that two carbonyl groups are essential for potent ACC2 inhibition. By focusing on enhancing the interactions between the two carbonyl groups and the amino acid residues Gly(2162) and Glu(2230), we used ligand- and structure-based drug design to discover spirolactones bearing a 2-ureidobenzothiophene moiety., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The design, synthesis, and structure-activity relationships of a new class of potent and orally active non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones, are described. We hypothesized that the 4-phenyl group of the isoquinolone occupies the S1 pocket of the enzyme, the 3-aminomethyl group forms an electrostatic interaction with the S2 pocket, and the introduction of a hydrogen bond donor onto the 6- or 7-substituent provides interaction with the hydrophilic region of the enzyme. Based on this hypothesis, intensive research focused on developing new non-peptide DPP-4 inhibitors has been carried out. Among the compounds designed in this study, we identified 2-[(3-aminomethyl-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-6-isoquinolinyl)oxy]acetamide (35a) as a potent, selective, and orally bioavailable DPP-4 inhibitor, which exhibited in vivo efficacy in diabetic model rats. Finally, X-ray crystallography of 35a in a complex with the enzyme validated our hypothesized binding mode and identified Lys554 as a new target-binding site available for DPP-4 inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Dipeptidyl peptidase IV (DPP-4) inhibition is a validated therapeutic option for type 2 diabetes, exhibiting multiple antidiabetic effects with little or no risk of hypoglycemia. In our studies involving non-covalent DPP-4 inhibitors, a novel series of quinoline-based inhibitors were designed based on the co-crystal structure of isoquinolone 2 in complex with DPP-4 to target the side chain of Lys554. Synthesis and evaluation of designed compounds revealed 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]piperazine-2,5-dione (1) as a potent, selective, and orally active DPP-4 inhibitor (IC₅₀=1.3 nM) with long-lasting ex vivo activity in dogs and excellent antihyperglycemic effects in rats. A docking study of compound 1 revealed a hydrogen-bonding interaction with the side chain of Lys554, suggesting this residue as a potential target site useful for enhancing DPP-4 inhibition., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Inhibition of dipeptidyl peptidase IV (DPP-4) is an exciting new approach for the treatment of diabetes. To date there has been no DPP-4 chemotype possessing a carboxy group that has progressed into clinical trials. Originating from the discovery of the structurally novel quinoline derivative 1, we designed novel pyridine derivatives containing a carboxy group. In our design, the carboxy group interacted with the targeted amino acid residues around the catalytic region and thereby increased the inhibitory activity. After further optimization, we identified a hydrate of [5-(aminomethyl)-6-(2,2-dimethylpropyl)-2-ethyl-4-(4-methylphenyl)pyridin-3-yl]acetic acid (30c) as a potent and selective DPP-4 inhibitor. The desired interactions with the critical active-site residues, such as a salt-bridge interaction with Arg125, were confirmed by X-ray cocrystal structure analysis. In addition, compound 30c showed a desired preclinical safety profile, and it was encoded as TAK-100.

We have previously discovered nicotinic acid derivative 1 as a structurally novel dipeptidyl peptidase IV (DPP-4) inhibitor. In this study, we obtained the X-ray co-crystal structure between nicotinic acid derivative 1 and DPP-4. From these X-ray co-crystallography results, to achieve more potent inhibitory activity, we targeted Arg125 as a potential amino acid residue because it was located near the pyridine core, and some known DPP-4 inhibitors were reported to interact with this residue. We hypothesized that the guanidino group of Arg125 could interact with two hydrogen-bond acceptors in a bidentate manner. Therefore, we designed a series of 3-pyridylacetamide derivatives possessing an additional hydrogen-bond acceptor that could have the desired bidentate interaction with Arg125. We discovered the dihydrochloride of 1-{[5-(aminomethyl)-2-methyl-4-(4-methylphenyl)-6-(2-methylpropyl)pyridin-3-yl]acetyl}-l-prolinamide (13j) to be a potent and selective DPP-4 inhibitor that could interact with the guanidino group of Arg125 in a unique bidentate manner., (Copyright © 2010 Elsevier Ltd. All rights reserved.)