Christian A. Olsen

Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5

We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.

Probing the bioactive conformation of an archetypal natural product HDAC inhibitor with conformationally homogeneous triazole-modified cyclic tetrapeptides.

Fooling enzymes with mock amides: Analogues of apicidin, a cyclic-tetrapeptide inhibitor of histone deacetylase (HDAC), were designed with a 1,4- or 1,5-disubstituted 1,2,3-triazole in place of a backbone amide bond to fix the bond in question in either a trans-like or a cis-like configuration. Thus, the binding affinity of distinct peptide conformations (see picture) could be probed. One analogue proved in some cases to be superior to apicidin as an HDAC inhibitor.

Antimicrobial, Hemolytic, and Cytotoxic Activities of β-Peptoid–Peptide Hybrid Oligomers: Improved Properties Compared to Natural AMPs

While natural host-defense antimicrobial peptides (AMPs) and analogues thereof have been investigated intensely in the last two decades with the purpose of combating the still increasing threat from emerging multiresistant pathogenic microbes, 2] compounds with peptidomimetic backbones have received considerable attention due to their superior stability against proteolytic enzymes. 4] Typically, studies of peptidomimetic AMP analogues have involved a brief microbiological evaluation of an array of oligomers, and only occasionally has testing been performed across a broader range of microorganisms or involved systematic structure–activity relationship (SAR) studies. Such investigations have proven fruitful for a-peptidic AMPs, 18] and might reveal unexpected lead structures and selectivity profiles when applied to peptidomimetics as well. Therefore, we have performed a more rigorous microbiological evaluation as well as toxicity profiling of a series of oligomers based on our b-peptoid–peptide hybrid backbone architecture. 20] Antimicrobial activities were determined alongside the archetypal cationic AMP magainin-2 and its clinically tested derivative pexiganan against a series of five important pathogens belonging to different classes. The obtained SAR data were subsequently correlated with various measurements of toxicity towards mammalian cells. Thereby we were able to derive useful trends for the future design of antibacterial and antifungal peptidomimetic constructs with potential for enhanced selectivity. Three subclasses 1 a–3 d (Scheme 1) were originally designed to address the general effects of length, type of cationic side chains, and presence of a-chirality in the b-peptoid residues. These series had previously been confirmed to possess membrane activity, as indicated by testing for hemolytic and prehemolytic effects, as well as by calcein release experiments with model liposomes, albeit the interaction of these compounds with intracellular targets cannot be ruled out based on our data. The all-aliphatic compound 4 and the mixed aromatic–aliphatic chimera 5 were included to address the importance of lipophilicity and type of cationic residue. Finally, we included three 5/6-carboxyfluorescein-labeled oligomers (6–8) to assess the influence of the presence of this widely used fluorophore on the antimicrobial activity, which might have important implications for confocal fluorescence microscopy studies of the interaction of labeled compounds with live bacteria. The chimeras 4–8 were of dodecamer length to minimize undesired mammalian cell toxicity that might be observed with increasing length. This compound collection was tested against a variety of clinically relevant pathogens and human red blood cells (Table 1). Scheme 1. Chemical structures of the examined hybrid oligomers. The abbreviations used for the b-peptoid units were adapted from the abbreviations commonly used for peptoids (i.e. , N-alkylglycines), 7] by adding the b-prefix. bNspe = N-(S)-1-phenylethyl-balanine, bNphe =b-N-phenylalanine, bNsce = N-(S)-1-cyclohexylethyl-b-alanine, hArg = homoarginine, CF = 5/6-carboxyfluoresceinoyl.

Cis−Trans 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 cis-trans 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 cis-trans 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 cis-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(sp(2))-H···S(amide) hydrogen bonding, which stabilize certain peptoid conformations.

Discovery of Potent and Selective Histone Deacetylase Inhibitors via Focused Combinatorial Libraries of Cyclic α3β-Tetrapeptides

Histone deacetylase (HDAC) inhibitors are powerful tools in understanding epigenetic regulation and have proven especially promising for the treatment of various cancers, but the discovery of potent, isoform-selective HDAC inhibitors has been a major challenge. We recently developed a cyclic alpha(3)beta-tetrapeptide scaffold for the preparation of HDAC inhibitors with novel selectivity profiles ( J. Am. Chem. Soc. 2009 , 131 , 3033 ). In this study, we elaborate this scaffold with respect to side chain diversity by synthesizing one-bead-one-compound combinatorial libraries of cyclic tetrapeptide analogues and applying two generations of these focused libraries to the discovery of potent HDAC ligands using a convenient screening platform. Our studies led to the first HDAC6-selective cyclic tetrapeptide analogue, which extends the use of cyclic tetrapeptides to the class II HDAC isoforms. These findings highlight the persistent potential of cyclic tetrapeptides as epigenetic modulators and possible anticancer drug lead compounds.

Peptoid–Peptide Hybrid Backbone Architectures

Peptidomimetic oligomers and foldamers have received considerable attention for over a decade, with β‐peptides and the so‐called peptoids (N‐alkylglycine oligomers) representing prominent examples of such architectures. Lately, hybrid or mixed backbones consisting of both α‐ and β‐amino acids (α/β‐peptides) have been investigated in some detail as well. The present Minireview is a survey of the literature concerning hybrid structures of α‐amino acids and peptoids, including β‐peptoids (N‐alkyl‐β‐alanine oligomers), and is intended to give an overview of this area of research within the field of peptidomimetic science.

Design, Synthesis, Biological Evaluation, and Structural Characterization of Potent Histone Deacetylase Inhibitors Based on Cyclic α/β-Tetrapeptide Architectures

Histone deacetylases (HDACs) are a family of enzymes found in bacteria, fungi, plants, and animals that profoundly affect cellular function by catalyzing the removal of acetyl groups from -N-acetylated lysine residues of various protein substrates including histones, transcription factors, alpha-tubulin, and nuclear importers. Although the precise roles of HDAC isoforms in cellular function are not yet completely understood, inhibition of HDAC activity has emerged as a promising approach for reversing the aberrant epigenetic states associated with cancer and other chronic diseases. Potent new isoform-selective HDAC inhibitors would therefore help expand our understanding of the HDAC enzymes and represent attractive lead compounds for drug design, especially if combined with high-resolution structural analyses of such inhibitors to shed light on the three-dimensional pharmacophoric features necessary for the future design of more potent and selective compounds. Here we present structural and functional analyses of a series of beta-amino-acid-containing HDAC inhibitors inspired by cyclic tetrapeptide natural products. To survey a diverse ensemble of pharmacophoric configurations, we systematically varied the position of the beta-amino acid, amino acid chirality, functionalization of the Zn(2+)-coordinating amino acid side chain, and alkylation of the backbone amide nitrogen atoms around the macrocycle. In many cases, the compounds were a single conformation in solution and exhibited potent activities against a number of HDAC isoforms as well as effective antiproliferative and cytotoxic activities against human tumor cells. High-resolution NMR solution structures were determined for a selection of the inhibitors, providing a useful means of correlating detailed structural information with potency. The structure-based approach described here is expected to furnish valuable insights toward the future design of more selective HDAC inhibitors.

Expansion of the Lysine Acylation Landscape

Leaving marks: the number of known posttranslational modifications for lysine has been expanded considerably. In addition to acetylation of side-chain amino functionalities of lysine residues in proteins, crotonylation, succinylation, and malonylation have now been identified as posttranslational modifications in histone and in non-histone proteins.

Cellular uptake and membrane-destabilising properties of α-peptide/β-peptoid chimeras: lessons for the design of new cell-penetrating peptides

Novel peptidomimetic backbone designs with stability towards proteases are of interest for several pharmaceutical applications including intracellular delivery. The present study concerns the cellular uptake and membrane-destabilising effects of various cationic chimeras comprised of alternating N-alkylated beta-alanine and alpha-amino acid residues. For comparison, homomeric peptides displaying octacationic functionalities as well as the Tat(47-57) sequence were included as reference compounds. Cellular uptake studies with fluorescently labelled compounds showed that guanidinylated chimeras were taken up four times more efficiently than Tat(47-57). After internalisation, the chimeras were localised primarily in vesicular compartments and diffusively in the cytoplasm. In murine NIH3T3 fibroblasts, the chimeras showed immediate plasma membrane permeabilising properties, which proved highly dependent on the chimera chain length, and were remarkably different from the effects induced by Tat(47-57). Finally, biophysical studies on model membranes showed that the chimeras in general increase the permeability of fluid phase and gel phase phosphatidylcholine (PC) vesicles without affecting membrane acyl chain packing, which suggests that they restrict lateral diffusion of the membrane lipids by interaction with phospholipid head groups. The alpha-peptide/beta-peptoid chimeras described herein exhibit promising cellular uptake properties, and thus represent proteolytically stable alternatives to currently known cell-penetrating peptides.

Profiling of Substrates for Zinc‐dependent Lysine Deacylase Enzymes: HDAC3 Exhibits Decrotonylase Activity In Vitro

Systematic screening of the activities of the eleven human zinc-dependent lysine deacylases against a series of fluorogenic substrates as well as kinetic evaluation revealed substrates for screenings of histone deacetylases HDAC10 and HDAC11 at reasonably low enzyme concentrations. Furthermore, HDAC3 in complex with nuclear receptor corepressor 1 (HDAC3-NCoR1) was shown to harbor decrotonylase activity in vitro.