The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice.

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

Multi-dimensional analysis identified rheumatoid arthritis-driving pathway in human T cell.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease accompanied by lymphocyte infiltration into joint synovium. While T cells are considered to be important for its pathogenesis, the features that are the most relevant to disease and how they change after treatment remain unclear. The aim of this study was to clarify the characteristics of T cells in RA, comprehensively. METHODS: We enrolled a total of 311 patients with RA and 73 healthy participants, and carefully classified them by disease state, constructed multiple cohorts and analysed clinical samples from them in a stepwise manner. We performed immunophenotyping with multiple evaluation axes, and two independent transcriptome analyses complementary to each other. RESULTS: We identified that 'effector memory-Tfh' subset was specifically expanded in the peripheral blood (PB) of patients with RA in correlation with disease activity, and reverted after treatment. Besides, we revealed distinct features of T cells in synovial fluid (SF) that the expression of Tfh/Tph-related genes and pro-inflammatory cytokines and chemokines, including CXCL13, were significantly enriched, whereas these phenotype were Th1-like. Finally, we identified specific pathways, such as mTORC1, IL-2-stat5, E2F, cell cycle and interferon-related genes, that were significantly enriched in SF, in particular, as well as PB of untreated patients with RA, and notably, these features reverted after treatment. CONCLUSION: Our multi-dimensional investigation identified disease relevant T-cell subsets and gene signatures deeply involved in pathogenesis of RA. These findings could aid in our understanding of essential roles of T cells in RA and will facilitate to development better diagnostic and therapeutic interventions.

Gene Signature-Based Approach Identified MEK1/2 as a Potential Target Associated With Relapse After Anti-TNFα Treatment for Crohn's Disease.

Background: Anti-tumor necrosis factor alpha (anti-TNFα) therapy has become the mainstay of therapy for Crohn's disease (CD). However, post-therapy, the recurrence rate is still high. The aim of this study was to dissect the molecular mechanism for recurrence of CD treated with anti-TNFα therapy and investigate novel therapeutic options that could induce complete remission. Methods: We re-analyzed publicly available mucosal gene expression data from CD patients pre- and post-infliximab therapy to extract the transcriptional differences between responders and healthy controls. We used a systematic computational approach based on identified differences to discover novel therapies and validated this prediction through in vitro and in vivo experimentation. Results: We identified a set of 3545 anti-TNFα therapy-untreatable genes (TUGs) that are significantly regulated in intestinal epithelial cells, which remain altered during remission. Pathway enrichment analysis of these genes clearly showed excessive growth state and suppressed terminal differentiation, whereas immune components were clearly resolved. Through in silico screening strategy, we observed that MEK inhibitors were predicted to revert expression of genes dysregulated in infliximab responders. In vitro transcriptome analysis demonstrated that selective MEK1/2 inhibitor significantly normalized reference genes from TUGs. In addition, in vitro functional study proved that MEK1/2 inhibitor facilitated intestinal epithelial differentiation. Finally, using murine colitis model, administration of MEK1/2 inhibitor significantly improved diarrhea and histological score. Conclusions: Our data revealed the abnormalities in anti-TNFα responders' CD colons that would be cause of recurrence of CD. Also, we provided evidence regarding MEK1/2 inhibitor as a potential treatment against CD to achieve sustainable remission.

A Novel LSD1 Inhibitor T-3775440 Disrupts GFI1B-Containing Complex Leading to Transdifferentiation and Impaired Growth of AML Cells.

Dysregulation of lysine (K)-specific demethylase 1A (LSD1), also known as KDM1A, has been implicated in the development of various cancers, including leukemia. Here, we describe the antileukemic activity and mechanism of action of T-3775440, a novel irreversible LSD1 inhibitor. Cell growth analysis of leukemia cell lines revealed that acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia cells (AMKL) were highly sensitive to this compound. T-3775440 treatment enforced transdifferentiation of erythroid/megakaryocytic lineages into granulomonocytic-like lineage cells. Mechanistically, T-3775440 disrupted the interaction between LSD1 and growth factor-independent 1B (GFI1B), a transcription factor critical for the differentiation processes of erythroid and megakaryocytic lineage cells. Knockdown of LSD1 and GFI1B recapitulated T-3775440-induced transdifferentiation and cell growth suppression, highlighting the significance of LSD1-GFI1B axis inhibition with regard to the anti-AML effects of T-3775440. Moreover, T-3775440 exhibited significant antitumor efficacy in AEL and AMKL xenograft models. Our findings provide a rationale for evaluating LSD1 inhibitors as potential treatments and indicate a novel mechanism of action against AML, particularly AEL and AMKL. Mol Cancer Ther; 16(2); 273-84. ©2016 AACR.

Peroxisome proliferator-activated receptor gamma (PPARγ) has multiple binding points that accommodate ligands in various conformations: Structurally similar PPARγ partial agonists bind to PPARγ LBD in different conformations.

In the course of studies directed toward the creation of human peroxisome proliferator-activated receptor gamma (hPPARγ) partial agonists, we designed and synthesized benzylsulfonylaminocarbonyl derivative (3) by structural modification of our reported hPPARγ partial agonist 2. Co-crystallization of 3 with the hPPARγ ligand-binding domain (LBD) afforded a homodimeric complex in which one of the LBDs adopts a fully active structure without bound 3, while the other LBD exhibits a non-fully active structure containing one molecule of bound 3. Interestingly, 2 and 3 are structurally similar, but bind to hPPARγ LBD in distinct conformations, that is, the sulfonylaminocarbonyl moiety of bound 3 is directed at 180° away from that of bound 2. These results support our previous proposal that the hPPARγ LBD has multiple binding points that can be utilized to accommodate structurally flexible hPPAR ligands.

Structural design and synthesis of arylalkynyl amide-type peroxisome proliferator-activated receptor γ (PPARγ)-selective antagonists based on the helix12-folding inhibition hypothesis.

Peroxisome proliferator-activated receptor γ (PPARγ) antagonists are candidates for treatment of type 2 diabetes, obesity and osteoporosis. However, few rational design strategies are currently available. Here, we utilized the helix12 (H12)-folding inhibition hypothesis, in combination with our previously determined X-ray crystal structure of PPARγ agonist MEKT-21 (6) complexed with the PPARγ ligand-binding domain, to design and develop a potent phenylalkynyl amide-type PPARγ antagonist 9i, focusing initially on pinpoint structural modification of the propanoic acid moiety of 6. Since 9i retained very weak, but distinct, PPARγ agonist activity, we next modified the distal benzene ring of 9i, aiming to delete the residual PPARγ agonist activity while retaining the antagonist activity. Introduction of a chlorine atom at the 2-position of the distal benzene ring afforded 9p, which exhibited potent, PPARγ-selective full antagonist activity without detectable agonist activity. We found that 9p stabilized the corepressor-PPARγ complex and suppressed basal PPARγ activity. This compound showed anti-adipogenesis activity at the cellular level. This agonist-antagonist switching concept based on the H12-folding inhibition hypothesis should also be applicable for designing other classes of PPARγ full antagonists.

Hesperetin glucuronides induce adipocyte differentiation via activation and expression of peroxisome proliferator-activated receptor-γ.

In previous reports, hesperidin, a flavonoid glucoside from citrus fruit, is hydrolyzed to hesperetin, an aglycone of hesperidin, and converted to the hesperetin glucuronides (H7-OG and H3'-OG) in vivo and depresses blood glucose levels. But there are no reports on the activity of hesperetin glucuronides. To determine the activity of hesperetin glucuronides, H7-OG and H3'-OG were synthesized and peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity was observed at 250 µM. These glucuronides accelerated the differentiation of 3T3-L1 cells into adipocytes at 10 µM. Furthermore, H7-OG showed additive effects in reporter gene assays and caused noncompetitive reactions in time-resolved fluorescence resonance energy transfer assays with a thiazolidinedione derivative. Our results indicated that hesperetin glucuronides activated PPARγ, accelerated adipocyte differentiation.

Molecular dynamics study-guided identification of cyclic amine structures as novel hydrophobic tail components of hPPARγ agonists.

We previously reported that a α-benzylphenylpropanoic acid-type hPPARγ-selective agonist with a piperidine ring as the hydrophobic tail part (3) exhibited sub-micromolar-order hPPARγ agonistic activity. In order to enhance the activity, we planned to carry out structural development based on information obtained from the X-ray crystal structure of hPPARγ ligand binding domain (LBD) complexed with 3. However, the shape and/or nature of the binding pocket surrounding the piperidine ring of 3 could not be precisely delineated because the structure of the omega loop of the LBD was poorly defined. Therefore, we constructed and inserted a plausible omega loop by means of molecular dynamics simulation. We then used the reconstructed LBD structure to design new mono-, bi- and tricyclic amine-bearing compounds that might be expected to show greater binding affinity for the LBD. Here, we describe synthesis and evaluation of α-benzylphenylpropanoic acid derivatives 8. As expected, most of the newly synthesized compounds exhibited more potent hPPARγ agonistic activity and greater hPPARγ binding affinity than 3. Some of these compounds also showed comparable aqueous solubility to 3.

Potent protein glycation inhibition of plantagoside in Plantago major seeds.

Plantagoside (5,7,4',5'-tetrahydroxyflavanone-3'-O-glucoside) and its aglycone (5,7,3',4',5'-pentahydroxyflavanone), isolated from a 50% ethanol extract of Plantago major seeds (Plantaginaceae), were established to be potent inhibitors of the Maillard reaction. These compounds also inhibited the formation of advanced glycation end products in proteins in physiological conditions and inhibited protein cross-linking glycation. These results indicate that P. major seeds have potential therapeutic applications in the prevention of diabetic complications.

Mechanism of peroxisome proliferator-activated receptor gamma (PPARγ) transactivation by hesperetin glucuronides is distinct from that by a thiazolidine-2,4-dione agent.

Hesperidin, a flavanone glycoside present abundantly in citrus fruits, is predominantly metabolized to hesperetin-7-O-ß-D-glucuronide (H7-OG) and hesperetin-3'-O-ß-D-glucuronide (H3'-OG), which exhibit partial agonistic activity towards peroxisome proliferator-activated receptor gamma (PPARγ). Here, in order to understand the mechanism(s) of action of PPARγ transactivation elicited by hesperetin glucuronides, we compared the transactivation activities of PPARγ (ligand-binding domain (LBD)) mutants by hesperetin glucuronides and troglitazone, a thiazolidine-2,4-dione class PPARγ full agonist. The assay results indicated that the mechanisms of activation of PPARγ by hesperetin glucuronides and by troglitazone are distinct, probably due to a difference in the binding sites of these compounds on the PPARγ LBD. Flavanone-class PPARγ partial agonists, luteolin and hesperetin glucuronides, showed similar activation profiles of the PPARγ LBD mutants, even though they have different side chain functionalities.

γ-Mangostin from Garcinia mangostana pericarps as a dual agonist that activates Both PPARα and PPARδ.

We tested the peroxisome proliferator-activated receptor (PPAR)δ agonistic activity of a Garcinia mangostana pericarp extract to develop a treatment for the metabolic syndrome, and demonstrated γ-mangostin to be an active compound on the basis of a luciferase reporter gene assay. γ-Mangostin induced the expression of the uncoupling protein-3 (UCP-3) gene which is related to energy expenditure and fat metabolism in L6 cells. We showed that γ-mangostin is a dual agonist that activates both PPARδ and PPARα. γ-Mangostin also induced the expression of acyl-CoA synthase and carnitine palmitoyl-transferase 1A genes in HepG2 cells. These results suggest the potential of γ-mangostin as a preventive agent of the metabolic syndrome.