The polygenic background of selectively bred diabetes models mimics the etiology of type 2 diabetes. So far, three different rodent models (Goto-Kakizaki rats, Nagoya-Shibata-Yasuda mice, and ...Oikawa-Nagao mice) have been established in the diabetes research field by continuous selective breeding for glucose tolerance from outbred rodent stocks. The origin of hyperglycemia in these rodents is mainly insulin secretion deficiency from the pancreatic β-cells and mild insulin resistance in insulin target organs. In this chapter, we summarize backgrounds and phenotypes of these rodent models to highlight their importance in diabetes research. Then, we introduce experimental methodologies to evaluate β-cell exocytosis as a putative common defect observed in these rodent models.
Signal transducer and activator of transcription 3 (STAT3) is a validated anticancer target due to the relationship between its constitutive activation and malignant tumors. Through a virtual ...screening approach on the STAT3-SH2 domain, 5,6-dimethyl-1
,3
-2,1,3-benzothiadiazole-2,2-dioxide (
) was identified as a potential STAT3 inhibitor. Some benzothiadiazole derivatives were synthesized by employing a versatile methodology, and they were tested by an AlphaScreen-based assay. Among them, benzosulfamide
showed a significant activity with an IC
= 15.8 ± 0.6 µM as a direct STAT3 inhibitor. Notably, we discovered that compound
was also able to interact with cysteine residues located around the SH2 domain. By applying mass spectrometry, liquid chromatography, NMR, and UV spectroscopy, an in-depth investigation was carried out, shedding light on its intriguing and unexpected mechanism of interaction.
Kinesin spindle protein Eg5 is a target for anticancer therapies, and small molecule inhibitors of its ATPase activity have been developed. We herein report for the first time the crystal structure ...of and biochemical studies on the Eg5 motor domain in complex with a new type of allosteric inhibitor. The biphenyl-type inhibitor PVZB1194 binds to the α4/α6 allosteric pocket 15 Å from the ATP-binding pocket, which differs from conventional allosteric inhibitors that bind to the allosteric L5/α2/α3 pocket of Eg5. Binding of the inhibitor is involved in the neck-linker conformation and also causes conformational changes around the ATP-binding pocket through Tyr104 to affect the interaction of ATP with the pocket. This structure provides useful information for the development of novel types of allosteric drugs as well as a novel insight into the molecular mechanism responsible for regulating the motor activity of kinesins.
Dimerization in signal transduction is a dynamically regulated process and a key regulatory mechanism. Signal transducer and activator of transcription 3 (STAT3) dimerizes after tyrosine ...phosphorylation upon cytokine stimulation. Because only the STAT3 dimer possesses the trans-activation activity, dimerization is an indispensable process for cytokine signaling. Here we report the detection of dynamic STAT3 dimerization in living cells using the homoFluoppi system. This method allowed us to validate the presence of an intact Src homology 2 domain and STAT3 Tyr705 phosphorylation, which facilitate puncta formation and homodimerization. Puncta formation was reversible, as determined by a decreased punctate signal after washout of oncostatin M. We analyzed STAT3 mutants, which have been reported in patients with hyper IgE syndrome and inflammatory hepatocellular adenoma (IHCA). Analysis of the IHCA mutants using homoFluoppi revealed constitutive activity independent of cytokine stimulation and novel insight into kinetics of dimer dissociation process. Next, we used homoFluoppi to screen for inhibitors of STAT3 dimerization, and identified 3,4-methylenedioxy-β-nitrostyrene as a novel inhibitor. The results of this study show that homoFluoppi is a useful research tool for the analysis of proteins like STAT3 that dynamically dimerize, and is applicable for the screening of dimerization modulators.
Increased susceptibility of mice 10–30 days postburn to MRSA infection is controlled through the intervention of CCL1‐producing M2bMΦ, appearing in association with burn injuries.
Patients with 10–30 ...days postburn injury are greatly susceptible to infections. M1MΦ (IL‐10−IL‐12+ MΦ) are essential cells in host antibacterial innate immunity against MRSA infections. However, these effector cells are not easily generated in hosts who are carriers of M2bMΦ (IL‐12−IL‐10+CCL1+LIGHT+ MΦ). M2bMΦ are inhibitory on M1MΦ generation. In this study, the antibacterial resistance of mice, 10–30 days postburn injury against MRSA infection, was improved by the modulation of M2bMΦ activities. Unburned mice inoculated with MΦ preparations from mice, 10–30 days after burn injury, were susceptible to MRSA infection, whereas unburned mice, inoculated with MΦ preparations from the same mice that were previously treated with CCL1 antisense ODN, were resistant to the infection. M2bMΦ, isolated from Day 15 burn mice, lost their M2bMΦ properties 3 days after cultivation under frequent medium changes, whereas their M2bMΦ properties remained in the same cultures supplemented with rCCL1. In cultures, MΦ preparations from Day 15 burn mice treated with CCL1 antisense ODN did not produce CCL1 and did convert to M1MΦ after heat‐killed MRSA stimulation. Also, Day 15 burn mice treated with the ODN became resistant against MRSA infection. These results indicate that CCL1 released from M2bMΦ is essentially required for the maintenance of their properties. The increased susceptibility of mice, 10–30 days after burn injury to MRSA infection, may be controlled through the intervention of CCL1 production by M2bMΦ appearing in association with severe burn injuries.
Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in blood plasma has been observed in various pathological conditions, including ...atherosclerosis. In this study, we investigated the use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) to develop a method for accurate quantification of PCOOH (1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC), focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH (M+Na+, m/z 812) provided not only a known product ion (m/z 147) but also characteristic product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Thus, three multiple reaction monitorings (MRMs) could be performed. MRM (812/147) enabled determination of 16:0/HpODE PC, and MRM (812/541) and MRM (812/388) allowed specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this method, we could determine plasma PCOOH concentrations in healthy subjects and patients with angiographically significant stenosis. In healthy subject and patient plasma, the concentration of 16:0/HpODE PC was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC. This finding shows that radical and/or enzymatic oxidation, rather than singlet oxygen oxidation, is recognized to cause peroxidation of PC. The newly developed LC–MS/MS method appears to be a powerful tool for developing a better understanding of in vivo lipid peroxidation and its involvement in human diseases.
There is an urgent need for structurally novel anti-norovirus agents. In this study, we describe the synthesis, anti-norovirus activity, and structure–activity relationship (SAR) of a series of ...heterocyclic carboxamide derivatives. Heterocyclic carboxamide 1 (50% effective concentration (EC50)=37 µM) was identified by our screening campaign using the cytopathic effect reduction assay. Initial SAR studies suggested the importance of halogen substituents on the heterocyclic scaffold and identified 3,5-di-boromo-thiophene derivative 2j (EC50=24 µM) and 4,6-di-fluoro-benzothiazole derivative 3j (EC50=5.6 µM) as more potent inhibitors than 1. Moreover, their hybrid compound, 3,5-di-bromo-thiophen-4,6-di-fluoro-benzothiazole 4b, showed the most potent anti-norovirus activity with a EC50 value of 0.53 µM (70-fold more potent than 1). Further investigation suggested that 4b might inhibit intracellular viral replication or the late stage of viral infection.
Kynurenine (Kyn), a metabolite of tryptophan (Trp), is a key regulator of mammal immune responses such as cancer immune tolerance. Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase ...(TDO) are main enzymes regulating the first and rate-limiting step of the Kyn pathway. To identify new small molecule inhibitors of TDO, we selected A172 glioblastoma cell line constitutively expressed TDO. Characterization of this cell line using kinase inhibitor library resulted in identification of MEK/ERK pathway-dependent TDO expression. After knowing the properties for TDO expression, we further proceeded to screen chemical library for TDO inhibitors. We previously determined that S-benzylisothiourea derivatives are enzymatic inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and suggested that the isothiourea moiety could be an important pharmacophore for binding to heme. Based on this premise, we screened an in-house library composed of various isothiourea derivatives and identified a bisisothiourea derivative, PVZB3001, as an inhibitor of TDO. Interestingly, PVZB3001 also inhibited the enzymatic activity of IDO1 in both cell-based and cell-free assays but did not inhibit other heme enzymes. Molecular docking studies suggested the importance of isothiourea moieties at the ortho position of the phenyl ring for the inhibition of catalytic activity. PVZB3001 showed competitive inhibition against TDO, and this was supported by the docking simulation. PVZB3001 recovered natural killer (NK) cell viability and functions by inhibiting Kyn accumulation in conditioned medium of both IDO1- and TDO-expressing cells. Furthermore, oral administration of IDO1-overexpressing tumor-bearing mice with PVZB3001 significantly inhibited tumor growth. Thus, we identified a novel selective dual inhibitor of IDO1 and TDO using the Kyn production assay with a glioblastoma cell line. This inhibitor could be a useful pharmacological tool for modulating the Kyn pathway in a variety of experimental systems.
Patients with advanced anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer who are prescribed ALK-tyrosine kinase inhibitors (ALK-TKIs) rarely have complete responses, with ...residual tumors relapsing as heterogeneous resistant phenotypes. Herein, we investigated new therapeutic strategies to reduce and eliminate residual tumors in the early treatment phase. Functional genomic screening using small guide RNA libraries showed that treatment-induced adaptive survival of ALK-rearranged lung cancer cells was predominantly dependent on STAT3 activity upon ALK inhibition. STAT3 inhibition effectively suppressed the adaptive survival of ALK-rearranged lung cancer cells by enhancing ALK inhibition-induced apoptosis. The combined effects were characterized by treatment-induced STAT3 dependence and transcriptional regulation of anti-apoptotic factor BCL-X
. In xenograft study, the combination of YHO-1701 (STAT3 inhibitor) and alectinib significantly suppressed tumor regrowth after treatment cessation with near tumor remission compared with alectinib alone. Hence, this study provides new insights into combined therapeutic strategies for patients with ALK-rearranged lung cancer.
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