A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluation

Sirtuins are important regulators of lysine acylation, which is implicated in cellular metabolism and transcriptional control. This makes the sirtuin class of enzymes interesting targets for development of small molecule probes with pharmaceutical potential. To achieve detailed profiling and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2 as a long chain deacylase enzyme. Novel enzymatic activities of SIRT2 were thus established in vitro, which warrant further investigation, and two known inhibitors, suramin and SirReal2, were profiled against substrates containing ε-<i>N</i>-acyllysine modifications of varying length

Correction to A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluation

Correction to A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluatio

An NAD⁺‑Dependent Sirtuin Depropionylase and Deacetylase (Sir2La) from the Probiotic Bacterium <i>Lactobacillus acidophilus</i> NCFM

Sirtuins, a group of NAD⁺-dependent deacylases, have emerged as the key connection between NAD⁺ metabolism and aging. This class of enzymes hydrolyzes a range of ε-<i>N</i>-acyllysine PTMs, and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacterium <i>Lactobacillus acidophilus</i> NCFM. Screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, demonstrated that one of the investigated proteins, Sir2La, is a <i>bona fide</i> NAD⁺-dependent sirtuin, catalyzing hydrolysis of acetyl-, propionyl-, and butyryllysine. Further substantiating the identity of Sir2La as a sirtuin, known sirtuin inhibitors, nicotinamide and suramin, as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. On the basis of steady-state kinetics, Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For nonfluorogenic peptide substrates, the preference is driven by a remarkably low <i>K</i>_M (280 nM vs 700 nM, for Kpro and Kac, respectively), whereas <i>k</i>_cat was similar (21 × 10^–3 s^–1). Moreover, while NAD⁺ is a prerequisite for Sir2La-mediated deacylation, Sir2La has a very high <i>K</i>_M for NAD⁺ compared to the expected levels of the dinucleotide in <i>L. acidophilus</i>. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl- and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs

Identification and Characterization of Second-Generation Invader Locked Nucleic Acids (LNAs) for Mixed-Sequence Recognition of Double-Stranded DNA

The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with “+1 interstrand zippers” of 2′-<i>N</i>-(pyren-1-yl)­methyl-2′-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2′-amino-α-l-LNA, 2′-<i>N</i>-methyl-2′-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2′-<i>O</i>-(pyren-1-yl)­methyl-RNA or 2′-<i>N</i>-methyl-2′-<i>N</i>-(pyren-1-yl)­methyl-2′-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics