Targeting of epigenetic mechanisms, such as the hydroxymethylation of DNA, has been intensively studied, with respect to the treatment of many serious pathologies, including oncological disorders. Recent studies demonstrated that promising therapeutic strategies could potentially be based on the inhibition of the TET1 protein (ten-eleven translocation methylcytosine dioxygenase 1) by specific iron chelators. Therefore, in the present work, we prepared a series of pyrrolopyrrole derivatives with hydrazide (1) or hydrazone (2-6) iron-binding groups. As a result, we determined that the basic pyrrolo[3,2-b]pyrrole derivative 1 was a strong inhibitor of the TET1 protein (IC50 = 1.33 μM), supported by microscale thermophoresis and molecular docking. Pyrrolo[3,2-b]pyrroles 2-6, bearing substituted 2-hydroxybenzylidene moieties, displayed no significant inhibitory activity. In addition, in vitro studies demonstrated that derivative 1 exhibits potent anticancer activity and an exclusive mitochondrial localization, confirmed by Pearson's correlation coefficient of 0.92.

• Klíčová slova
• TET1 protein inhibitor, hydrazone, mitochondria, pyrrolo[3,2-b]pyrrole,
• MeSH
• chelátory železa MeSH
• dioxygenasy * metabolismus   MeSH
• DNA MeSH
• hydrazony chemie   MeSH
• mitochondriální proteiny MeSH
• pyrroly * chemie   farmakologie   MeSH
• simulace molekulového dockingu MeSH
• železo MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chelátory železa MeSH
• dioxygenasy * MeSH
• DNA MeSH
• hydrazony MeSH
• mitochondriální proteiny MeSH
• pyrroly * MeSH
• železo MeSH

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.

• MeSH
• HeLa buňky MeSH
• histony chemie   metabolismus   MeSH
• lidé MeSH
• peptidy metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• proteasy metabolismus   MeSH
• proteomika metody   MeSH
• rekombinantní proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histony MeSH
• peptidy MeSH
• proteasy MeSH
• rekombinantní proteiny MeSH