Upregulation of a Disintegrin and Metalloproteinase With Thrombospondin Motifs-7 by miR-29 Repression Mediates Vascular Smooth Muscle Calcification

Objective-Vascular calcification significantly increases cardiovascular morbidity and mortality. We recently reported that the deficiency of cartilage oligomeric matrix protein (COMP) leads to vascular mineralization. We characterized the COMP-degrading metalloproteinase, a disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7). Here, we tested whether ADAMTS-7 facilitates vascular calcification. Methods and Results-ADAMTS-7 expression was markedly upregulated in calcifying rat vascular smooth muscle cells (VSMCs) in vitro, calcified arteries of rats with chronic renal failure in vivo, and radial arteries of uraemic patients. Silencing of ADAMTS-7 markedly reduced COMP degradation and ameliorated VSMC calcification, whereas ectopic expression of ADAMTS-7 greatly enhanced COMP degradation and exacerbated mineralization. The transcriptional activity of ADAMTS-7 promoter was not altered by high phosphate. We used bioinformatics and quantitative polymerase chain reaction analysis to demonstrate that high-phosphate upregulated ADAMTS-7 mRNA and protein via miR-29a/b repression, which directly targeted the 3' untranslated region of ADAMTS-7 in VSMCs. MicroRNA (MiR)-29a/b mimic markedly inhibited but miR-29a/b inhibitor greatly enhanced high-phosphate-induced ADAMTS-7 expression, COMP degradation, and subsequent VSMC calcification. ADAMTS-7 silencing significantly diminished miR-29a/b repression-exaggerated VSMC calcification. Conclusion-Our data reveal a novel mechanism by which ADAMTS-7 upregulation by miR-29a/b repression mediates vascular calcification, which may shed light on preventing cardiovascular morbidity and mortality. (Arterioscler Thromb Vasc Biol. 2012;32:2580-2588.)HematologyPeripheral Vascular DiseaseSCI(E)PubMed20ARTICLE112580-+3

Class I HDAC Inhibitors Display Different Antitumor Mechanism in Leukemia and Prostatic Cancer Cells Depending on Their p53 Status

Previously, we designed and synthesized a series of <i>o</i>-aminobenzamide-based histone deacetylase (HDAC) inhibitors, among which the representative compound <b>11a</b> exhibited potent inhibitory activity against class I HDACs. In this study, we report the development of more potent hydrazide-based class I selective HDAC inhibitors using <b>11a</b> as a lead. Representative compound <b>13b</b> showed a mixed, slow, and tight binding inhibition mechanism for HDAC1, 2, and 3. The most potent compound <b>13e</b> exhibited low nanomolar IC₅₀s toward HDAC1, 2, and 3 and could down-regulate HDAC6 in acute myeloid leukemia MV4-11 cells. The EC₅₀ of <b>13e</b> against MV4-11 cells was 34.7 nM, which is 26 times lower than its parent compound <b>11a</b>. <i>In vitro</i> responses to <b>13e</b> vary significantly and interestingly based on cell type: in p53 wild-type MV4-11 cells, <b>13e</b> induced cell death via apoptosis and G1/S cell cycle arrest, which is likely mediated by a p53-dependent pathway, while in p53-null PC-3 cells, <b>13e</b> caused G2/M arrest and inhibited cell proliferation without inducing caspase-3-dependent apoptosis

Class I HDAC Inhibitors Display Different Antitumor Mechanism in Leukemia and Prostatic Cancer Cells Depending on Their p53 Status

Previously, we designed and synthesized a series of <i>o</i>-aminobenzamide-based histone deacetylase (HDAC) inhibitors, among which the representative compound <b>11a</b> exhibited potent inhibitory activity against class I HDACs. In this study, we report the development of more potent hydrazide-based class I selective HDAC inhibitors using <b>11a</b> as a lead. Representative compound <b>13b</b> showed a mixed, slow, and tight binding inhibition mechanism for HDAC1, 2, and 3. The most potent compound <b>13e</b> exhibited low nanomolar IC₅₀s toward HDAC1, 2, and 3 and could down-regulate HDAC6 in acute myeloid leukemia MV4-11 cells. The EC₅₀ of <b>13e</b> against MV4-11 cells was 34.7 nM, which is 26 times lower than its parent compound <b>11a</b>. <i>In vitro</i> responses to <b>13e</b> vary significantly and interestingly based on cell type: in p53 wild-type MV4-11 cells, <b>13e</b> induced cell death via apoptosis and G1/S cell cycle arrest, which is likely mediated by a p53-dependent pathway, while in p53-null PC-3 cells, <b>13e</b> caused G2/M arrest and inhibited cell proliferation without inducing caspase-3-dependent apoptosis

Mixed Lineage Leukemia 5 (MLL5) Protein Stability Is Cooperatively Regulated by O-GlcNac Transferase (OGT) and Ubiquitin Specific Protease 7 (USP7).

Mixed lineage leukemia 5 (MLL5) protein is a trithorax family histone 3 lysine 4 (H3K4) methyltransferase that regulates diverse biological processes, including cell cycle progression, hematopoiesis and cancer. The mechanisms by which MLL5 protein stability is regulated have remained unclear to date. Here, we showed that MLL5 protein stability is cooperatively regulated by O-GlcNAc transferase (OGT) and ubiquitin-specific protease 7 (USP7). Depletion of OGT in cells led to a decrease in the MLL5 protein level through ubiquitin/proteasome-dependent proteolytic degradation, whereas ectopic expression of OGT protein suppressed MLL5 ubiquitylation. We further identified deubiquitinase USP7 as a novel MLL5-associated protein using mass spectrometry. USP7 stabilized the MLL5 protein through direct binding and deubiquitylation. Loss of USP7 induced degradation of MLL5 protein. Conversely, overexpression of USP7, but not a catalytically inactive USP7 mutant, led to decreased ubiquitylation and increased MLL5 stability. Co-immunoprecipitation and co-immunostaining assays revealed that MLL5, OGT and USP7 interact with each other to form a stable ternary complex that is predominantly located in the nucleus. In addition, upregulation of MLL5 expression was correlated with increased expression of OGT and USP7 in human primary cervical adenocarcinomas. Our results collectively reveal a novel molecular mechanism underlying regulation of MLL5 protein stability and provide new insights into the functional interplay among O-GlcNAc transferase, deubiquitinase and histone methyltransferase

Iterative Optimization and Structure-Activity Relationship Studies of Oseltamivir Amino Derivatives as Potent and Selective Neuraminidase Inhibitors via Targeting 150-Cavity

With our continuous endeavors in seeking neuraminidase (NA) inhibitors, we reported herein three series of novel oseltamivir amino derivatives with the goal of exploring the druggable chemical space inside the 150-cavity of influenza virus NAs. Among them, around half of the compounds in series C were demonstrated to be better inhibitors against both wild-type and oseltamivir-resistant group-1 NAs than oseltamivir carboxylate (OSC). Notably, compounds 12d, 12e, 15e, and 15i showed more potent or equipotent antiviral activity against H1N1, H5N1, and H5N8 viruses compared to OSC in cellular assays. Furthermore, compounds 12e and 15e exhibited high metabolic stability in human liver microsomes (HLMs) and low inhibitory effect on main cytochrome P450 (CYP) enzymes, as well as low acute/subacute toxicity and certain antiviral efficacy in vivo. Also, pharmacokinetic (PK) and molecular docking studies were performed. Overall, 12e and 15e possess great potential to serve as anti-influenza candidates and are worthy of further investigation

Structure-Based Optimization of N-Substituted Oseltamivir Derivatives as Potent Anti-Influenza A Virus Agents with Significantly Improved Potency against Oseltamivir-Resistant N1-H274Y Variant

Due to the emergence of highly pathogenic and oseltamivir-resistant influenza viruses, there is an urgent need to develop new anti-influenza agents. Herein, five subseries of oseltamivir derivatives were designed and synthesized to improve their activity toward drug-resistant viral strains by further exploiting the 150-cavity in the neuraminidases (NAs). The bioassay results showed that compound 21h exhibited antiviral activities similar to or better than those of oseltamivir carboxylate (OSC) against H5N1, H5N2, H5N6, and H5N8. Besides, 21h was 5- to 86-fold more potent than OSC toward N1, N8, and N1-H274Y mutant NAs in the inhibitory assays. Computational studies provided a plausible rationale for the high potency of 21h against group-1 and N1-H274Y NAs. In addition, 21h demonstrated acceptable oral bioavailability, low acute toxicity, potent antiviral activity in vivo, and high metabolic stability. Overall, the above excellent profiles make 21h a promising drug candidate for the treatment of influenza virus infection

Modulation of nitrate-induced phosphate response by the MYB transcription factor RLI1/HINGE1 in the nucleus

The coordinated utilization of nitrogen (N) and phosphorus (P) is vital for plants to maintain nutrient balance and achieve optimal growth. Previously, we revealed a mechanism by which nitrate induces genes for phosphate utilization; this mechanism depends on NRT1.1B-facilitated degradation of cytoplasmic SPX4, which in turn promotes cytoplasmic-nuclear shuttling of PHR2, the central transcription factor of phosphate signaling, and triggers the nitrate-induced phosphate response (NIPR) and N-P coordinated utilization in rice. In this study, we unveiled a fine-tuning mechanism of NIPR in the nucleus regulated by Highly Induced by Nitrate Gene 1 (HINGE1, also known as RLI1), a MYB-transcription factor closely related to PHR2. RLI1/HINGE1, which is transcriptionally activated by PHR2 under nitrate induction, can directly activate the expression of phosphate starvation-induced genes. More importantly, RLI1/HINGE1 competes with PHR2 for binding to its repressor proteins in the nucleus (SPX proteins), and consequently releases PHR2 to further enhance phosphate response. Therefore, RLI1/HINGE1 amplifies the phosphate response in the nucleus downstream of the cytoplasmic SPX4-PHR2 cascade, thereby enabling fine-tuning of N-P balance when nitrate supply is sufficient

Optimization of N-Substituted Oseltamivir Derivatives as Potent Inhibitors of Group-1 and -2 Influenza A Neuraminidases, Including a Drug-Resistant Variant

none25simixedZhang, Jian; Poongavanam, Vasanthanathan; Kang, Dongwei; Bertagnin, Chiara; Lu, Huamei; Kong, Xiujie; Ju, Han; Lu, Xueyi; Gao, Ping; Tian, Ye; Jia, Haiyong; Desta, Samuel; Ding, Xiao; Sun, Lin; Fang, Zengjun; Huang, Boshi*; Liang, Xuewu; Jia, Ruifang; Ma, Xiuli; Xu, Wenfang; Murugan, Natarajan Arul; Loregian, Arianna; Huang, Bing; Zhan, Peng; Liu, XinyongZhang, Jian; Poongavanam, Vasanthanathan; Kang, Dongwei; Bertagnin, Chiara; Lu, Huamei; Kong, Xiujie; Ju, Han; Lu, Xueyi; Gao, Ping; Tian, Ye; Jia, Haiyong; Desta, Samuel; Ding, Xiao; Sun, Lin; Fang, Zengjun; Huang, Boshi; Liang, Xuewu; Jia, Ruifang; Ma, Xiuli; Xu, Wenfang; Murugan, Natarajan Arul; Loregian, Arianna; Huang, Bing; Zhan, Peng; Liu, Xinyon

OGT interacts with and stabilizes MLL5 protein.

<p><b>(A)</b> HEK293T cells were transiently co-transfected with expression vectors for HA-tagged MLL5 and FLAG-tagged OGT, and extracts prepared and immunoprecipitated with anti-HA or anti-FLAG antibodies. The presence of FLAG-tagged OGT or HA-tagged MLL5 protein were examined by western blotting using anti-FLAG or anti-HA antibodies, respectively. <b>(B)</b> Mapping the MLL5 interacting domains in the OGT protein. <i>Upper panel</i>, a schematic representation of the domain structure of the full length OGT and its truncated <i>Δ</i>TPR mutant. <i>Lower panel</i>, HEK293T cells were co-transfected with expression vectors for FLAG-tagged OGT or its truncated ΔTPR mutant and HA-tagged MLL5 and truncated mutants. 48h after transfection, cell lysates were harvested and FLAG-tagged OGT or its truncated <i>Δ</i>TPR mutant proteins were immunoprecipitated (IP) with anti-FLAG antibody, and the presence of MLL5 protein were examined by western blotting using anti-HA antibody. The asterisk indicate a nonspecific band. <b>(C)</b> Mapping the OGT interacting domains in the MLL5 protein. <i>Upper panel</i>, a schematic representation of the domain structure of the full length MLL5 and its truncated mutants. <i>Lower panel</i>, HEK293T cells were co-transfected with expression vector encoding MLL5-HA and OGT-FLAG or truncated mutants. Cells were lysed for co-immunoprecipitation as indicated. <b>(D)</b> Knockdown of OGT in HeLa cells leads to down-regulation of MLL5 protein. Two different lentivirus-based shRNAs target to an mRNA sequence located 1275 and 1412 bp downstream of the translation start site were used to knockdown OGT expression in HeLa cells. Whole cell lysates were harvested at day 6 post-infection. The expression of endogenous MLL5 and the global levels of O-GlcNAcylation were detected by anti-MLL5 and anti-GlcNAc antibodies. Actin was used as a loading control. <b>(E)</b> The mRNA levels of MLL5 remain unchanged by OGT knockdown. The mRNA levels of OGT and MLL5 in OGT knockdown cells were analyzed by quantitative RT-PCR. Two-tailed unpaired Student’s <i>t</i> tests were performed, p < 0.001. <b>(F-H)</b> Control or OGT knockdown cells were treated with CHX (100μg/ml), MG132 (30μM) or NH₄Cl (5mM) for the indicated times before harvesting. The expression of endogenous MLL5 proteins were detected by anti-MLL5 antibody. β-actin was used as a loading control. Quantification of relative MLL5 levels is shown in the bottom panel. Numbers below lanes indicate densitometry of the protein presented relative to β-actin.</p