Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.

Nonpeptidic propargylamines as inhibitors of lysine specific demethylase 1 (LSD1) with cellular activity.

Lysine demethylases play an important role in epigenetic regulation and thus in the development of diseases like cancer or neurodegenerative disorders. As the lysine specific demethylase 1 (LSD1/KDM1) has been strongly connected to androgen and estrogen dependent gene expression, it serves as a promising target for the therapy of hormone dependent cancer. Here, we report on the discovery of new small molecule inhibitors of LSD1 containing a propargylamine warhead, starting out from lysine containing substrate analogues. On the basis of these substrate mimicking inhibitors, we were able to increase potency by a combination of similarity-based virtual screening and subsequent synthetic optimization resulting in more druglike LSD1 inhibitors that led to histone hypermethylation in breast cancer cells.

The role of histone demethylases in cancer therapy.

Reversible histone methylation has emerged in the last few years as an important mechanism of epigenetic regulation. Histone methyltransferases and demethylases have been identified as contributing factors in the development of several diseases, especially cancer. Therefore, they have been postulated to be new drug targets with high therapeutic potential. Here, we review histone demethylases with a special focus on their potential role in oncology drug discovery. We present an overview over the different classes of enzymes, their biochemistry, selected data on their role in physiology and already available inhibitors.

Inhibitors of the NAD(+)-Dependent Protein Desuccinylase and Demalonylase Sirt5.

NAD(+)-dependent histone deacetylases (sirtuins) play important roles in epigenetic regulation but also through nonhistone substrates for other key cellular events and have been linked to the pathogenesis of cancer, neurodegeneration, and metabolic diseases. The subtype Sirt5 has been shown recently to act as a desuccinylating and demalonylating enzyme. We have established an assay for biochemical testing of Sirt5 using a small labeled succinylated lysine derivative. We present a comparative study on the profiling of several established sirtuin inhibitors on Sirt1-3 as well as Sirt5 and also present initial results on a screening for new compounds that block Sirt5. Thiobarbiturates were identified as new Sirt5 inhibitors in the low micromolar range, which are selective over Sirt3 that can be found in the same cell compartment as Sirt5.

Thiobarbiturates as sirtuin inhibitors: virtual screening, free-energy calculations, and biological testing.

NAD+-dependent histone deacetylases (sirtuins) are enzymes that cleave acetyl groups from lysine residues in histones and other proteins. Potent selective sirtuin inhibitors are interesting tools for the investigation of the biological functions of these enzymes and may be future drugs for the treatment of cancer or neurodegenerative diseases. Herein we present the results from a protein-based virtual screen of a commercial database with subsequent biological testing of the most promising compounds. The combination of docking and in vitro experimental testing resulted in the identification of novel sirtuin inhibitors with thiobarbiturate structure. To rationalize the experimental results, free-energy calculations were carried out by molecular mechanics Poisson-Boltzmann/surface area (MM-PBSA) calculations. A significant correlation between calculated binding free energies and measured Sirt2 inhibitory activities was observed. The analyses suggested a molecular basis for the interaction of the identified thiobarbiturate derivatives with human Sirt2. Based on the docking and MM-PBSA calculations we synthesized and tested five further thiobarbiturates. The MM-PBSA method correctly predicted the activity of the novel thiobarbiturates. The identified compounds will be used to further explore the therapeutic potential of sirtuin inhibitors.