Contemporary Applications of Thioamides and Methods for Their Synthesis.

Thioamides are naturally occurring isosteres of amide bonds in which the chalcogen atom of the carbonyl is changed from oxygen to sulfur. This substitution gives rise to altered nucleophilicity and hydrogen bonding properties with importance for both chemical reactivity and non-covalent interactions. As such, thioamides have been introduced into biologically active compounds to achieve improved target affinity and/or stability towards hydrolytic enzymes but have also been applied as probes of protein and peptide folding and dynamics. Recently, a series of new methods have been developed for the synthesis of thioamides as well as their utilization in peptide chemistry. Further, novel strategies for the incorporation of thioamides into proteins have been developed, enabling both structural and functional studies to be performed. In this Review, we highlight the recent developments in the preparation of thioamides and their applications for peptide modification and study of protein function.

Chiral Posttranslational Modification to Lysine ε-Amino Groups.

The sophistication of proteomic analysis has revealed that protein lysine residues are posttranslationally modified by a variety of acyl groups. Protein lysine acetylation regulates metabolism, gene expression, and microtubule formation and has been extensively studied; however, the understanding of the biological significance of other acyl posttranslational modifications (PTMs) is still in its infancy. The acylation of lysine residues is mediated either by acyltransferase "writer" enzymes or by nonenzymatic mechanisms and hydrolase enzymes, termed "erasers", that cleave various acyl PTMs to reverse the modified state. We have studied the human lysine deacylase enzymes, comprising the 11 Zn2+-dependent histone deacetylases (HDACs) and the 7 NAD+-consuming sirtuins (SIRTs), over the past decade. We have thus developed selective inhibitors and molecular probes and have studied the acyl substrate scope of each enzyme using chemically synthesized peptide substrates and photo-cross-linking probes. Recently, we have turned our attention to PTMs containing a stereogenic center, such as ε-N-ß-hydroxybutyryllysine (Kbhb) and ε-N-lactyllysine (Kla), that each comprise a pair of mirror image stereoisomers as modifications. Both modifications are found on histones, where they affect gene transcription in response to specific metabolic states, and they are found on cytosolic and mitochondrial enzymes involved in fatty acid oxidation (Kbhb) and glycolysis (Kla), respectively. Thus, chiral modifications to lysine side chains give rise to two distinct diastereomeric products, with separate metabolic origins and potentially different activities exhibited by writer and eraser enzymes. Lysine l-lactylation originates from l-lactate, a major energy carrier produced from pyruvate after glycolysis, and it is highly induced by metabolic states such as the Warburg effect. l-Lactate can possibly be activated by acyl-coenzyme A (CoA) synthetases and transferred to lysine residues by histone acetyltransferases such as p300. d-Lactylation, on the other hand, arises primarily from a nonenzymatic reaction with d-lactylglutathione, an intermediate in the glyoxalase pathway. In addition to their distinct origin, we found that both K(l-la) and K(d-la) modifications are erased by HDACs with different catalytic efficiencies. Also, K(l-bhb) and K(d-bhb) arise from different metabolites but depend on interconnected metabolic pathways, and the two stereoisomers of ε-N-3-hydroxy-3-methylglutaryllysine (Khmg) originate from a single precursor that may then be regulated differently by eraser enzymes. Distinguishing between the individual stereoisomers of PTMs is therefore of crucial importance. In the present Account, we will (1) revisit the long-standing evidence for the distinct production and dynamics of enantiomeric forms of chiral metabolites that serve as ε-N-acyllysine PTMs and (2) highlight the outstanding questions that arise from the recent literature on chiral lysine PTMs resulting from these metabolites.

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7.

The sirtuins are NAD+ -dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.

Investigation of Carboxylic Acid Isosteres and Prodrugs for Inhibition of the Human SIRT5 Lysine Deacylase Enzyme.

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing ϵ-N-carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism-based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high-affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5-dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked isosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

Aryl Fluorosulfate Based Inhibitors That Covalently Target the SIRT5 Lysine Deacylase.

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Finding the gas pedal on a slow sirtuin.

The class III histone deacetylase sirtuin 6 (SIRT6) modulates numerous functions in the cell by deacetylating histone lysine residues. Interestingly, SIRT6's efficiency in in vitro experiments is far greater against substrates carrying long-chain fatty acyl modifications such as myristoylated lysine compared with acetylated counterparts, but the deacetylase activity can be stimulated by fatty acids and small-molecule allosteric modulators. A new study helps to explain this puzzling activation using a novel activator, thorough kinetic investigation, and mutagenesis studies. These data help elucidate the molecular requirements for activation of SIRT6 and provide a foundation for development of activators for therapeutic purposes.

Rearrangement of Thiodepsipeptides by S → N Acyl Shift Delivers Homodetic Autoinducing Peptides.

Group behavior in many bacteria relies on chemically induced communication called quorum sensing (QS), which plays important roles in the regulation of colonization, biofilm formation, and virulence. In Gram-positive bacteria, QS is often mediated by cyclic ribosomally synthesized and posttranslationally modified peptides (RiPPs). In staphylococci, for example, most of these so-called autoinducing peptides (AIPs) contain a conserved thiolactone functionality, which has also been predicted to constitute a structural feature of AIPs from other genera. Here, we show that pentameric AIPs from Lactiplantibacillus plantarum, Clostridium perfringens, and Listeria monocytogenes that were previously presumed to be thiolactone-containing structures readily rearrange to become homodetic cyclopeptides. This finding has implications for the developing understanding of cross-species and potential cross-genus communication of bacteria and may help guide the discovery of peptide ligands to perturb their function.

The Chemical Biology-Medicinal Chemistry Continuum: EFMC's Vision.

The European Federation for Medicinal chemistry and Chemical biology (EFMC) is a federation of learned societies. It groups organizations of European scientists working in a dynamic field spanning chemical biology and medicinal chemistry. New ideas, tools, and technologies emerging from a wide array of scientific disciplines continuously energize this rapidly evolving area. Medicinal chemistry is the design, synthesis, and optimization of biologically active molecules aimed at discovering new drug candidates - a mission that in many ways overlaps with the scope of chemical biology. Chemical biology is by now a mature field of science for which a more precise definition of what it encompasses, in the frame of EFMC, is timely. This article discusses chemical biology as currently understood by EFMC, including all activities dealing with the design and synthesis of biologically active chemical tools and their use to probe, characterize, or influence biological systems.

Photo Cross-Linking Probes Containing ϵ-N-Thioacyllysine and ϵ-N-Acyl-(δ-aza)lysine Residues.

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. This work describes the development of peptide activity/affinity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of ϵ-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to the effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the ϵ-N-glutaryllysine and ϵ-N-myristoyllysine analogues, were successfully applied for the enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

Increasing the Functional Group Diversity in Helical ß-Peptoids: Achievement of Solvent- and pH-Dependent Folding.

We report the synthesis of a series of bis-functionalized ß-peptoid oligomers of the hexamer length. This was achieved by synthesizing and incorporating protected amino- or azido-functionalized chiral building blocks into precursor oligomers by a trimer segment coupling strategy. The resulting hexamers were readily elaborated to provide target compounds displaying amino groups, carboxy groups, hydroxy groups, or triazolo-pyridines, which should enable metal ion binding. Analysis of the novel hexamers by circular dichroism (CD) spectroscopy and 1H-13C heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy revealed robust helical folding propensity in acetonitrile. CD analysis showed a solvent-dependent degree of helical content in the structural ensembles when adding different ratios of protic solvents including an aqueous buffer. These studies were enabled by a substantial increase in solubility compared to previously analyzed ß-peptoid oligomers. This also allowed for the investigation of the effect of pH on the folding propensity of the amino- and carboxy-functionalized oligomers, respectively. Interestingly, we could show a reversible effect of sequentially adding acid and base, resulting in a switching between compositions of folded ensembles with varying helical content. We envision that the present discoveries can form the basis for the development of functional peptidomimetic materials responsive to external stimuli.

Functionalized Helical ß-Peptoids.

Peptidomimetic foldamers adopting well-defined three-dimensional structures while being stable toward proteolysis are of interest in biomedical research, chemical biology, and biomimetic materials science. Despite their backbone flexibility, ß-peptoids containing N-( S)-1-(1-naphthyl)ethyl ( Ns1npe) side chains can fold into unique triangular prism-shaped helices. We report herein the successful introduction of amino groups onto robustly folded ß-peptoid helices by construction and incorporation of novel chiral building blocks. This is the first example of an X-ray crystal structure of a linear ß-peptoid containing more than one type of side chain. We thus present a unique foldamer design comprising a robustly folded core with functionalized side chains protruding perpendicular to the helical axis to provide a highly predictable display of functional groups. This work paves the way for development of ß-peptoid foldamers with a desired function, such as catalytic properties or as scaffolds enabling polyvalent display.

Parameter subset selection techniques for problems in mathematical biology.

Patient-specific models for diagnostics and treatment planning require reliable parameter estimation and model predictions. Mathematical models of physiological systems are often formulated as systems of nonlinear ordinary differential equations with many parameters and few options for measuring all state variables. Consequently, it can be difficult to determine which parameters can reliably be estimated from available data. This investigation highlights pitfalls associated with practical parameter identifiability and subset selection. The latter refer to the process associated with selecting a subset of parameters that can be identified uniquely by parameter estimation protocols. The methods will be demonstrated using five examples of increasing complexity, as well as with patient-specific model predicting arterial blood pressure. This study demonstrates that methods based on local sensitivities are preferable in terms of computational cost and model fit when good initial parameter values are available, but that global methods should be considered when initial parameter value is not known or poorly understood. For global sensitivity analysis, Morris screening provides results in terms of parameter sensitivity ranking at a much lower computational cost.

Arylfluorosulfate-Based Electrophiles for Covalent Protein Labeling: A New Addition to the Arsenal.

Selective covalent modification of a targeted protein is a powerful tool in chemical biology and drug discovery, with applications ranging from identification and characterization of proteins and their functions to the development of targeted covalent inhibitors. Most covalent ligands contain an affinity motif and an electrophilic warhead that reacts with a nucleophilic residue of the targeted protein. Because the electrophilic warhead is prone to react and modify off-target nucleophiles, its reactivity should be balanced carefully to maximize target selectivity. Arylfluorosulfates have recently emerged as latent electrophiles for selective labeling of context-specific tyrosine and lysine residues in protein pockets. Here, we review the recent but intense introduction of arylfluorosulfates into the arsenal of available warheads for selective covalent modification of proteins. We highlight the untapped potential of this functional group for use in chemical biology and drug discovery.

An NAD+-Dependent Sirtuin Depropionylase and Deacetylase (Sir2La) from the Probiotic Bacterium Lactobacillus acidophilus 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- N-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 Lactobacillus acidophilus 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 bona fide 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 KM (280 nM vs 700 nM, for Kpro and Kac, respectively), whereas kcat was similar (21 × 10-3 s-1). Moreover, while NAD+ is a prerequisite for Sir2La-mediated deacylation, Sir2La has a very high KM for NAD+ compared to the expected levels of the dinucleotide in L. acidophilus. 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.

A Robust Proton Flux (pHlux) Assay for Studying the Function and Inhibition of the Influenza A M2 Proton Channel.

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.

Random Mutagenesis Analysis of the Influenza A M2 Proton Channel Reveals Novel Resistance Mutants.

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

Direct Peptide Cyclization and One-Pot Modification Using the MeDbz Linker.

The one-pot synthesis and modification of cyclic peptides through a self-cleaving on-resin protocol is described. We apply Dawson's MeDbz linker to achieve direct intramolecular peptide cyclization by thioesterification followed by S → N acyl shift. This native chemical ligation approach requires no activating additive and allows direct modification of the crude cyclic peptides in one-pot. The strategy was applied to synthesize 5 cyclic peptide natural products of varying ring size. Finally, one-pot modifications include desulfurization, fluorophore conjugation, and intramolecular disulfide formation.

Chemical Editing of Macrocyclic Natural Products and Kinetic Profiling Reveal Slow, Tight-Binding Histone Deacetylase Inhibitors with Picomolar Affinities.

Histone deacetylases (HDACs) are validated targets for treatment of certain cancer types and play numerous regulatory roles in biology, ranging from epigenetics to metabolism. Small molecules are highly important as tool compounds for probing these mechanisms as well as for the development of new medicines. Therefore, detailed mechanistic information and precise characterization of the chemical probes used to investigate the effects of HDAC enzymes are vital. We interrogated Nature's arsenal of macrocyclic nonribosomal peptide HDAC inhibitors by chemical synthesis and evaluation of more than 30 natural products and analogues. This furnished surprising trends in binding affinities for the various macrocycles, which were then exploited for the design of highly potent class I and IIb HDAC inhibitors. Furthermore, thorough kinetic investigation revealed unexpected inhibitory mechanisms of important tool compounds as well as the approved drug Istodax (romidepsin). This work provides novel inhibitors with varying potencies, selectivity profiles, and mechanisms of inhibition and, importantly, affords insight into known tool compounds that will improve the interpretation of their effects in biology and medicine.

Metabolic control by sirtuins and other enzymes that sense NAD+, NADH, or their ratio.

NAD+ is a dinucleotide cofactor with the potential to accept electrons in a variety of cellular reduction-oxidation (redox) reactions. In its reduced form, NADH is a ubiquitous cellular electron donor. NAD+, NADH, and the NAD+/NADH ratio have long been known to control the activity of several oxidoreductase enzymes. More recently, enzymes outside those participating directly in redox control have been identified that sense these dinucleotides, including the sirtuin family of NAD+-dependent protein deacylases. In this review, we highlight examples of non-redox enzymes that are controlled by NAD+, NADH, or NAD+/NADH. In particular, we focus on the sirtuin family and assess the current evidence that the sirtuin enzymes sense these dinucleotides and discuss the biological conditions under which this might occur; we conclude that sirtuins sense NAD+, but neither NADH nor the ratio. Finally, we identify future studies that might be informative to further interrogate physiological and pathophysiological changes in NAD+ and NADH, as well as enzymes like sirtuins that sense and respond to redox changes in the cell.