Substrates for Efficient Fluorometric Screening Employing the NAD-Dependent Sirtuin 5 Lysine Deacylase (KDAC) Enzyme

The class III lysine deacylases (KDACs), also known as the sirtuins, have emerged as interesting drug targets for therapeutic intervention in a variety of diseases. To gain a deeper understanding of the processes affected by sirtuins, the development of selective small molecule modulators of individual isozymes has been a longstanding goal. Essential for the discovery of novel modulators, however, are good screening protocols and mechanistic insights with regard to the targets in question. We therefore evaluated the activities of the seven human sirtuin hydrolases against a panel of fluorogenic substrates. Both commonly used, commercially available substrates and novel chemotypes designed to address recent developments in the field of lysine post-translational modification were evaluated. Our investigations led to the discovery of two new fluorogenic ε-<i>N</i>-succinyllysine-containing substrates that enable highly efficient and enzyme-economical screening employing sirtuin 5 (SIRT5). Furthermore, optimized protocols for facile kinetic investigations were developed, which should be valuable for enzyme kinetic investigations. Finally, these protocols were applied to a kinetic analysis of the inhibition of SIRT5 by suramin, a potent sirtuin inhibitor previously shown by X-ray crystallography to bind the substrate pocket of the human SIRT5 KDAC enzyme

Backbone-Fluorinated 1,2,3-Triazole-Containing Dipeptide Surrogates

The 1,2,3-triazole moiety can be incorporated as a peptide bond bioisostere to provide protease resistance in peptidomimetics. Herein, we report the synthesis of peptidomimetic building blocks containing backbone-fluorinated 1,4-disubstituted 1,2,3-triazole moieties. Synthetic protocols for the preparation of various Xaa-Gly dipeptide surrogates in the form of Xaa-ψ­[triazole]-F₂Gly building blocks were established, and selected examples were introduced into the endogenous peptide opioid receptor ligand Leu-enkephalin as a model compound

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

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

Discovery of HDAC Inhibitors That Lack an Active Site Zn²⁺-Binding Functional Group

Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn²⁺ ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure–activity characterizations of cyclic α₃β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn²⁺-binding group. The lead compounds (e.g., <b>15</b> and <b>26</b>) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of <b>26</b> indicates that the cyclic α₃β-tetrapeptide is a fast-on/off competitive inhibitor of HDACs 1–3 with <i>K</i>_i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the active-site opening, but not with the active site Zn²⁺, can have potential in drug design

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

<i>Cis</i>–<i>Trans</i> Amide Bond Rotamers in β‑Peptoids and Peptoids: Evaluation of Stereoelectronic Effects in Backbone and Side Chains

Non-natural peptide analogs have significant potential for the development of new materials and pharmacologically active ligands. One such architecture, the β-peptoids (N-alkyl-β-alanines), has found use in a variety of biologically active compounds but has been sparsely studied with respect to folding propensity. Thus, we here report an investigation of the effect of structural variations on the <i>cis</i>–<i>trans</i> amide bond rotamer equilibria in a selection of monomer model systems. In addition to various side chain effects, which correlated well with previous studies of α-peptoids, we present the synthesis and investigation of <i>cis</i>–<i>trans</i> isomerism in the first examples of peptoids and β-peptoids containing thioamide bonds as well as trifluoroacetylated peptoids and β-peptoids. These systems revealed an increase in the preference for <i>cis</i>-amides as compared to their parent compounds and thus provide novel strategies for affecting the folding of peptoid constructs. By using NMR spectroscopy, X-ray crystallographic analysis, and density functional theory calculations, we present evidence for the presence of thioamide–aromatic interactions through C_sp²–H···S_amide hydrogen bonding, which stabilize certain peptoid conformations

<i>Cis</i>–<i>Trans</i> Amide Bond Rotamers in β‑Peptoids and Peptoids: Evaluation of Stereoelectronic Effects in Backbone and Side Chains

Non-natural peptide analogs have significant potential for the development of new materials and pharmacologically active ligands. One such architecture, the β-peptoids (N-alkyl-β-alanines), has found use in a variety of biologically active compounds but has been sparsely studied with respect to folding propensity. Thus, we here report an investigation of the effect of structural variations on the <i>cis</i>–<i>trans</i> amide bond rotamer equilibria in a selection of monomer model systems. In addition to various side chain effects, which correlated well with previous studies of α-peptoids, we present the synthesis and investigation of <i>cis</i>–<i>trans</i> isomerism in the first examples of peptoids and β-peptoids containing thioamide bonds as well as trifluoroacetylated peptoids and β-peptoids. These systems revealed an increase in the preference for <i>cis</i>-amides as compared to their parent compounds and thus provide novel strategies for affecting the folding of peptoid constructs. By using NMR spectroscopy, X-ray crystallographic analysis, and density functional theory calculations, we present evidence for the presence of thioamide–aromatic interactions through C_sp²–H···S_amide hydrogen bonding, which stabilize certain peptoid conformations

Total Synthesis and Full Histone Deacetylase Inhibitory Profiling of Azumamides A–E as Well as β²- <i>epi</i>-Azumamide E and β³-<i>epi</i>-Azumamide E

Cyclic tetrapeptide and depsipeptide natural products have proven useful as biological probes and drug candidates due to their potent activities as histone deacetylase (HDAC) inhibitors. Here, we present the syntheses of a class of cyclic tetrapeptide HDAC inhibitors, the azumamides, by a concise route in which the key step in preparation of the noncanonical disubstituted β-amino acid building block was an Ellman-type Mannich reaction. By tweaking the reaction conditions during this transformation, we gained access to the natural products as well as two epimeric homologues. Thus, the first total syntheses of azumamides B–D corroborated the originally assigned structures, and the synthetic efforts enabled the first full profiling of HDAC inhibitory properties of the entire selection of azumamides A–E. This revealed unexpected differences in the relative potencies within the class and showed that azumamides C and E are both potent inhibitors of HDAC10 and HDAC11

Structure–Activity Relationship Study Based on Autoinducing Peptide (AIP) from Dog Pathogen <i>S. schleiferi</i>

Herein, an effective protocol for solid-phase synthesis of peptide thiolactones by concomitant ring closure and cleavage from the solid support is reported. The strategy was applied for mapping the importance of the structural features in <i>S. schleiferi</i> AIP (<b>5</b>) by performing an alanine scan and truncation of this natural compound. This furnished some of the most potent inhibitors of accessory gene regulator (<i>agr</i>)-I in the human pathogen <i>S. aureus</i> reported to date