Strategy to Target the Substrate Binding site of SET Domain Protein Methyltransferases

Protein methyltransferases (PMTs) are a novel gene family of therapeutic relevance involved in chromatin-mediated signaling and other biological mechanisms. Most PMTs are organized around the structurally conserved SET domain that catalyzes the methylation of a substrate lysine. A few potent chemical inhibitors compete with the protein substrate, and all are anchored in the channel recruiting the methyl-accepting lysine. We propose a novel strategy to design focused chemical libraries targeting the substrate binding site, where a limited number of warheads each occupying the lysine-channel of multiple enzymes would be decorated by different substituents. A variety of sequence and structure-based approaches used to analyze the diversity of the lysine channel of SET domain PMTs support the relevance of this strategy. We show that chemical fragments derived from published inhibitors are valid warheads that can be used in the design of novel focused libraries targeting other PMTs

Tacrolimus inhibits IL-12 p40 protein and mRNA expression.

<p>(<b>A</b>) Murine bone marrow-derived macrophages (BMDMs) were pretreated with the indicated concentrations of tacrolimus for 1 h. Cells were then either left untreated (NS) or stimulated with LPS (100 ng/ml) or LPS and IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. (<b>B</b>) BMDMs were incubated with the indicated concentrations of tacrolimus for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml, where indicated) prior to LPS (100 ng/ml) stimulation for 4 h or left untreated (NS). Cells were harvested and total RNA was assayed for <i>Il12b</i> mRNA levels by real-time RT-PCR. Each result represents the mean ± standard error (SD) for duplicate assays from three independent experiments. * p<0.05.</p

11R-VIVIT inhibits pro-inflammatory cytokine expression in murine macrophages.

<p>BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. IL-12 p70 (<b>A</b>), IL-23 (<b>B</b>) and TNF (<b>C</b>) protein secretion were assayed from supernatants by ELISA. Each result represents mean ± SE of duplicate assays from three independent experiments. * p<0.05.</p

NFAT binds to <i>Nos2</i> promoter and its selective inhibition abrogates nitric oxide secretion in macrophages.

<p>(<b>A</b>) Nuclear extracts were prepared from BMDMs stimulated with LPS (lane 1) or LPS and IFN-γ (lanes 2–7). Extracts were either incubated with a labeled probe NFAT/ISRE (element from region II of the promoter scheme), a competitor ISRE oligonucleotide, or with the indicated antibodies. DNA-protein complexes (indicated by arrow) were separated by electrophoresis. EMSA revealed binding of both IRF8 and NFATc1 to the same <i>Nos2</i> promoter element. (<b>B</b>) BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. Nitric oxide secretion was assayed from supernatants by Greiss reaction. Experiments were performed in duplicate and repeated three times (mean ± SEM). (<b>C</b>) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with LPS alone or IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for <i>Nos2</i> mRNA levels by real-time RT PCR. Data is representative of three independent experiments. * p<0.05.</p

11R-VIVIT inhibits IL-12 p40 protein and mRNA expression.

<p>(<b>A</b>) Murine bone marrow-derived macrophages (BMDMs) were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide 11R-VEET for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. (<b>B</b>) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for <i>Il12b</i> mRNA levels by real-time RT-PCR. Results were normalized with the respect to the levels of <i>β-actin</i> mRNA, and represent the mean ± SE for duplicate assays from three independent experiments. * p<0.05, ** p<0.01.</p

Inhibition of IL-12 p40 by 11R-VIVIT is independent of IL-10.

<p>(<b>A</b>) Murine BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT (white bars) or 11R-VEET (black bars) for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-10 protein secretion was assayed from supernatants by ELISA. (<b>B</b>) BMDMs from <i>Il10</i>^−/− mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and LPS plus IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. Results represent the mean ± SD for duplicate assays from three independent experiments. * p<0.05, ** p<0.01.</p

11R-VIVIT reduces DNA binding to the NFAT/IRF8 site in the murine IL-12 p40 promoter.

<p>BMDMs were either untreated (lane 1) or pretreated with the indicated concentrations of 11R-VEET (lanes 2–4) or 11R-VIVIT (lanes 5–7) for 1 h. Cells were then treated for 1 h with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) stimulation for 4 h. Cells were harvested and nuclear extracts were prepared for EMSA. ³²P labeled oligonucleotide probe spanning the NFAT/IRF8 site of the IL-12 p40 promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034172#pone.0034172-Zhu1" target="_blank">[10]</a> was incubated with 10 mg of nuclear extracts on ice for 30 min prior to electrophoresis. For supershift assays (lanes 3,4 and 6,7), 10 mg nuclear extract were incubated with 3 mg of anti-NFAT c1 (lanes 3 and 6) or anti-IRF8 (lanes 4 and 7) antibodies for 30 min on ice prior to addition of the ³²P labeled probe and 30 min incubation on ice followed by electrophoresis. The arrows represent DNA-protein complexes formed before and after incubation with the indicated antibodies. The above result is representative of five independent experiments.</p

Discovery of Bisubstrate Inhibitors of Nicotinamide <i>N</i>‑Methyltransferase (NNMT)

Nicotinamide <i>N</i>-methyltransferase (NNMT) catalyzes the N-methylation of pyridine-containing compounds using the cofactor <i>S</i>-5′-adenosyl-l-methionine (SAM) as the methyl group donor. Through the regulation of the levels of its substrates, cofactor, and products, NNMT plays an important role in physiology and pathophysiology. Overexpression of NNMT has been implicated in various human diseases. Potent and selective small-molecule NNMT inhibitors are valuable chemical tools for testing biological and therapeutic hypotheses. However, very few NNMT inhibitors have been reported. Here, we describe the discovery of a bisubstrate NNMT inhibitor MS2734 (<b>6</b>) and characterization of this inhibitor in biochemical, biophysical, kinetic, and structural studies. Importantly, we obtained the first crystal structure of human NNMT in complex with a small-molecule inhibitor. The structure of the NNMT–<b>6</b> complex has unambiguously demonstrated that <b>6</b> occupied both substrate and cofactor binding sites. The findings paved the way for developing more potent and selective NNMT inhibitors in the future

VIVIT treatment reduces spontaneous secretion of colonic inflammatory cytokines.

<p>Explants from colons of VIVIT (n = 12 for <b>A</b>, <b>B</b>; n = 7 for <b>C</b>) and VEET (n = 9 for <b>A</b>, <b>B</b>; n = 6 for <b>C</b>) treated mice were incubated in RPMI medium for 24 h, released cytokines were then determined by ELISA. We noted a marked difference in the gut spontaneous secretion of TNF-α (<b>B</b>) in gut supernatants from VIVIT treated mice, with a statistically significant change in IL-12 p40 (<b>A</b>) and in interferon-γ (<b>C</b>) production compared to VEET treated mice. Values are normalized to weight of intestinal explants, and data are presented as mean ± SEM.</p

Discovery of Potent and Selective Inhibitors for G9a-Like Protein (GLP) Lysine Methyltransferase

G9a-like protein (GLP) and G9a are highly homologous protein lysine methyltransferases (PKMTs) sharing approximately 80% sequence identity in their catalytic domains. GLP and G9a form a heterodimer complex and catalyze mono- and dimethylation of histone H3 lysine 9 and nonhistone substrates. Although they are closely related, GLP and G9a possess distinct physiological and pathophysiological functions. Thus, GLP or G9a selective small-molecule inhibitors are useful tools to dissect their distinct biological functions. We previously reported potent and selective G9a/GLP dual inhibitors including UNC0638 and UNC0642. Here we report the discovery of potent and selective GLP inhibitors including <b>4</b> (MS0124) and <b>18</b> (MS012), which are >30-fold and 140-fold selective for GLP over G9a and other methyltransferases, respectively. The cocrystal structures of GLP and G9a in the complex with either <b>4</b> or <b>18</b> displayed virtually identical binding modes and interactions, highlighting the challenges in structure-based design of selective inhibitors for either enzyme