Transcriptional activity of <i>Eip74EF</i> and <i>Eip75B</i> promoters during the third larval instar.

<p>Transcript levels from the <i>Eip74EF-RA</i> (A), <i>Eip74EF-RB</i> (B) and <i>Eip75B-RC</i> (C) promoters in <i>w¹¹¹⁸</i> larvae are shown. The charts show the average of relative RNA levels in percent of the maximum RNA quantity measured with the given primer pairs, the error bars indicate the standard error.</p

Ecdysone Induced Gene Expression Is Associated with Acetylation of Histone H3 Lysine 23 in <em>Drosophila melanogaster</em>

<div><p>Posttranslational modification of histones regulates transcription but the exact role that acetylation of specific lysine residues plays in biological processes <em>in vivo</em> is still not clearly understood. To assess the contribution of different histone modifications to transcriptional activation <em>in vivo</em>, we determined the acetylation patterns on the ecdysone induced <em>Eip74EF</em> and <em>Eip75B</em> genes in <em>Drosophila melanogaster</em> larvae by chromatin immunoprecipitation. We found that acetylation of histone H3 lysine 23 is localized to promoters and correlates with endogenous ecdysone induced gene activation. In contrast, acetylation of lysines 8, 12 and 16 of histone H4 and lysine 9 of histone H3 showed minor differences in their distribution on the regulatory and transcribed regions tested, and had limited or no correlation with ecdysone induced transcriptional activity. We found that dCBP, which is encoded by the <em>nejire</em> gene, acetylates H3 lysine 23 <em>in vivo</em>, and silencing of <em>nejire</em> leads to reduced expression of the <em>Eip74EF</em> and <em>Eip75B</em> genes. Our results suggest that acetylation of specific lysine residues of histones contribute specifically to the dynamic regulation of transcription. Furthermore, along with previous studies identify CBP dependent H3 lysine 23 acetylation as an evolutionarily conserved chromatin modification involved in steroid induced gene activation.</p> </div

The nej/dCBP protein is responsible for H3K23 specific acetylation in <i>Drosophila</i>.

<p>(A) The level of acetylated H3K23 is unchanged in L3 larvae homo- or hemizygous for loss of function mutant alleles of the <i>gcn5</i>, <i>mof</i> or <i>chm</i> histone acetyltransferase genes, and also in homozygous <i>Ada2a</i> or <i>Ada2b</i> mutants specific for the GCN5 containing ATAC and SAGA complexes, respectively. (B) In <i>nej³</i> mutant embryos the level of acetyl-H3K23 is significantly reduced compared to the wild-type as quantitated by immunostaining of whole mount embryos. Mean pixel intensity ± s.e.m. are shown, P<0.01. (C) ChIP analysis of chromatin samples from <i>gcn5^E333St</i> homozygous null mutant (gcn5) and heterozygous (cont) wandering L3 larvae using acetyl-H3K23 specific antibody detects the presence of the acetyl-H3K23 mark in <i>gcn5</i> mutants. In <i>engrailed-GAL4 UAS-GFP</i> embryos also carrying either an <i>UAS-dCBP</i> (D) or an <i>UAS-dCBP-FLAD</i> (E) transgene, the UAS transgenes are expressed in the posterior part of every segment, as visualized by GFP fluorescence. Immunostaining with anti-acetyl-H3K23 specific antibody reveals that the level of acetyl-H3K23 is dramatically increased in embryos overexpressing <i>UAS-dCBP</i> (F), while it is unchanged in embryos overexpressing the <i>UAS-dCBP-FLAD</i> construct (G), which is mutated in the acetyltransferase domain of CBP.</p

dCBP is required for larval development and ecdysone induced gene expression.

<p>(A) Heat-shock activated one-off expression of a <i>UAS-nejire-RNAi</i> construct in <i>hs-GAL4> UAS-nej-RNAi</i> animals (empty bars) reduces viability compared to non-heat-shocked controls (filled bars). Especially strong effect could be observed if the heat-shock was administered between days 3 and 6 after egg laying. Viability is expressed as the ratio of the number of eclosed <i>hs-GAL4> UAS-nej-RNAi</i> flies and <i>UAS-nej-RNAi</i> non-expressing control siblings. (B) The level of <i>Eip74EF</i> and <i>Eip75B</i> specific transcripts is decreased in <i>nej-RNAi</i> expressing <i>hs-GAL4> nej-RNAi</i> wandering L3 larvae (empty bars) compared to non-expressing control siblings (filled bars). Quantitative PCR analysis was performed with intronic primer pairs specific for the <i>Eip74EF-RA</i>, <i>-RB</i> or <i>Eip75B-RC</i> promoters (E74-RA, E74-RB and E75-RC, respectively), and with primer pairs located in downstream exons detecting mRNA products of the two genes (E74-ex8 and E75-ex8). The chart shows the relative RNA quantities normalized to transcript levels in non-expressing controls ± s.d.</p

Specific histone acetylations show different spatial and temporal patterns on ecdysone induced genes.

<p>Chromatin samples from middle-stage L3 larvae (mid-L3, empty bars), from wandering L3 larvae (w-L3, grey bars) or from late L3 larvae prior pupariation (spev-L3, black bars) were immunoprecipitated with antibodies against (A) histone H3, (B) acetyl-H3K9, (C) acetyl-H3K23, (D) acetyl-H4K8, (E) acetyl-H4K12 or (F) acetyl-H4K16, then quantitated by real-time PCR. In case of histone H3 (A), the amount of precipitated DNA is expressed in the percent of the total input control (TIC). Charts B-F shows the relative quantities of the DNA precipitated by the specific antibodies normalized to the amount of the H3 precipitate, the average ± s.e.m. of three biological replicates are show. (Percentage values do not compare between different antibodies.) The primers detected the following regions: RA-promoter, intron 1, RB-promoter, intron 5 and exon 8 of <i>Eip74EF</i>; RC-promoter, intron 2 and exon 8 of <i>Eip75B</i>, the promoter of <i>Rpl32</i> and a euchromatic intergenic control region.</p

Image5.pdf

<p>The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.</p

Image3.pdf

<p>The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.</p

Image1.pdf

<p>The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.</p

Image6.pdf

<p>The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.</p

Image4.pdf

<p>The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.</p