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Thioesters are considered to be “energy-rich” functional groups that are susceptible to attack by thiolate and amine nucleophiles while remaining hydrolytically stable at neutral pH, which enables thioester chemistry to take place in an aqueous medium. Thus, the inherent reactivity of thioesters enables their fundamental roles in biology and unique applications in chemical synthesis. Here, we investigate the reactivity of thioesters that mimic acyl-coenzyme A (CoA) species and S-acylcysteine modifications as well as aryl thioesters applied in chemical protein synthesis by native chemical ligation (NCL). We developed a fluorogenic assay format for the direct and continuous investigation of the rate of reaction between thioesters and nucleophiles (hydroxide, thiolate, and amines) under various conditions and were able to recapitulate previously reported reactivity of thioesters. Further, chromatography-based analyses of acetyl- and succinyl-CoA mimics revealed striking differences in their ability to acylate lysine side chains, providing insight into nonenzymatic protein acylation. Finally, we investigated key aspects of native chemical ligation reaction conditions. Our data revealed a profound effect of the tris-(2-carboxyethyl)phosphine (TCEP) commonly used in systems where thiol-thioester exchange occurs, including a potentially harmful hydrolysis side reaction. These data provide insight into the potential optimization of native chemical ligation chemistry.

The 1,287 genes targeted by the shRNA of the leukemia library.

Standard hematologic parameters in SIRT5-/- mice and SIRT5+/+ littermates.

Plasmids.

Patient sample information.

Antibodies.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.Significance:Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML.See related commentary by Li and Melnick, p. 198.

All supplemental figures and legends.

Next generation sequencing for 52 myeloid malignancies-related genes.

Oligonucleotide sequences.

Ranked list of 1,287 genes from shRNA library screen in primary AML cells. The 1,287 genes assessed with an shRNA library screen were sorted by the second highest percentile fold change present in 2 shRNA and across 2 samples, with the 34 genes showing a fold change in the top 2 percent in more than 2 samples listed first.

The sulfur–carbonyl bond in a thioester is an “energy-rich” functionality but is hydrolytically stable, while susceptible to attack by thiolate and amine nucleophiles, enabling thioester chemistry to take place in aqueous medium. In this work, we developed a fluorogenic assay format for the direct and continuous investigation of the rate of reaction between thioesters and nucleophiles under various conditions. We designed three different substrates, mimicking the chemical environment in acyl-CoA, S-acylcysteine, and an activated thioester commonly used in chemical protein synthesis by native chemical ligation. The data resulting from monitoring of the reaction of these substrates with a series of nucleophiles at varying pH values generally recapitulated previously reported reactivity of thioesters. We investigated key aspects of native chemical ligation reaction conditions. Our data revealed a profound effect of the tris-(2-carboxyethyl)phosphine (TCEP) commonly used in systems where thiol–thioester exchange occurs, including a potentially harmful hydrolysis side reaction. These data provide insight for potential optimization of native chemical ligation chemistry.

The aim of this work is to clarify whether electric-powered aviation can comply with requirements from the Danish government for a zero-emission carbon dioxide footprint by 2030 on domestic flight routes. The battery types are presented, in an attempt to determine whether electric air transportation can become commercially viable. The gap in evaluating the feasibility of moving by electric aircraft by 2030 is met through this research, by analysing the feasibility, through current developments, of delivering enough energy in terms of Wh/kg as compared to conventional fuel for a combustion-driven aircraft. The calculations, based on lithium (Li)-ion batteries with an energy density of 260 Wh/kg, showed that the goals will be met only with the development of new battery technology. The results indicate that there will be a reduction in carbon dioxide emissions by approximately 17%. According to aircraft manufacturers, it will be possible to accommodate up to 186 passengers in a fully electric-driven aircraft, which can meet the demand for domestic flights in Denmark. Other types of batteries, such as lithium–sulfur (Li–S) and lithium–oxygen (Li–O2) are relevant to investigate because of their higher energy density and usage of natural minerals other than just lithium.

Depression is common in old age and is associated with both disability, increased mortality and increased impairment from physical diseases.To estimate the prevalence of depression among old patients in Norwegian general practice, to evaluate the extent they talk about it during their consultation, if it was previously known or suspected by their general practitioner (GP), and how frequent the depressed patients visit their GP.Cross-sectional study among patients and GPs at 18 primary care clinics in the south of Norway.Patients ≥65 years who visited their GP were asked to complete the Patient Health Questionaire-9 (PHQ9). The GPs reported what kind of issues the patient presented at the consultation, if a current depression were known, and the consultation frequency.44 (11%) of 383 patients reported moderate or severe depressive symptoms (PHQ9 ≥10). Among the cases with data from both patient and GP (369), 38 patients (10%) reported moderately depressive symptoms. Of these only 12 (32%) mentioned psychological problems to their GP during their consultation, 12 (32%) were neither known to the GP with previous depression nor suspected by the GP of having a current one, and 68% of them visit their GP ≥5 times a year.Elderly patients tend to speak little of their depression to the GP. Almost 1 of 3 elderly patients with moderate depressive symptoms were unrecognised by their GP. GPs should more often suspect potential mental health problems in old patients who come frequently to visits.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. Significance: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML. See related commentary by Li and Melnick, p. 198.

Imbalance in the autonomic nervous system can lead to orthostatic intolerance manifested by dizziness, lightheadedness, and a sudden loss of consciousness (syncope); these are common conditions, but they are challenging to diagnose correctly. Uncertainties about the triggering mechanisms and the underlying pathophysiology have led to variations in their classification. This study uses machine learning to categorize patients with orthostatic intolerance. We use random forest classification trees to identify a small number of markers in blood pressure, and heart rate time-series data measured during head-up tilt to (a) distinguish patients with a single pathology and (b) examine data from patients with a mixed pathophysiology. Next, we use Kmeans to cluster the markers representing the time-series data. We apply the proposed method analyzing clinical data from 186 subjects identified as control or suffering from one of four conditions: postural orthostatic tachycardia (POTS), cardioinhibition, vasodepression, and mixed cardioinhibition and vasodepression. Classification results confirm the use of supervised machine learning. We were able to categorize more than 95% of patients with a single condition and were able to subgroup all patients with mixed cardioinhibitory and vasodepressor syncope. Clustering results confirm the disease groups and identify two distinct subgroups within the control and mixed groups. The proposed study demonstrates how to use machine learning to discover structure in blood pressure and heart rate time-series data. The methodology is used in classification of patients with orthostatic intolerance. Diagnosing orthostatic intolerance is challenging, and full characterization of the pathophysiological mechanisms remains a topic of ongoing research. This study provides a step toward leveraging machine learning to assist clinicians and researchers in addressing these challenges. Graphical abstract Machine learning tools utilized to analyze heart rate (HR) and blood pressure (BP) time-series data from syncope and control patients. Results show that machine learning can provide accurate classification of disease groups for 98% of patients and we identified two subgroups within the control patients differentiated by their BP response.

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 either mediated by acyltransferase ‘writer’ enzymes or through non-enzymatic mechanisms and hydrolase enzymes, termed ‘erasers’, 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 last decade. We have thus developed selective inhibitors and molecular probes as well as studied the acyl substrate scope of each enzyme using chemically synthesized peptide substrates and photo-crosslinking probes. Recently, we have turned our attention to lysine PTMs containing a stereogenic center, such as -N--hydroxybutyryllysine (Kbhb) and -N-lactyllysine (Kla), that each comprise a pair of mirror image stereoisomers. 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 derives 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 non-enzymatic 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, while the two stereoisomers of -N-3-hydroxy-3-methylglutaryllysine (Khmg) derive 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 distinct production and dynamics of enantiomeric forms of chiral metabolites that serve as epsilon-N-acyllysine PTMs and (2) highlight the outstanding questions that arise from the recent literature on chiral lysine PTMs resulting from these metabolites.

Bacterial neonatal meningitis results in high mortality and morbidity rates for those affected. Although improvements in diagnosis and treatment have led to a decline in mortality rates, morbidity rates have remained relatively unchanged. Bacterial resistance to antibiotics in this clinical setting further underlines the need for developing other technologies, such as phage therapy. We exploited an in vitro phage therapy model for studying bacterial neonatal meningitis based on Escherichia coli (E. coli) EV36, bacteriophage (phage) K1F and human cerebral microvascular endothelial cells (hCMECs). We show that phage K1F is phagocytosed and degraded by constitutive- and PAMP-dependent LC3-assisted phagocytosis and does not induce expression of inflammatory cytokines TNFα, IL-6, IL-8 or IFNβ. Additionally, we observed that phage K1F temporarily decreases the barrier resistance of hCMEC cultures, a property that influences the barrier permeability, which could facilitate the transition of immune cells across the endothelial vessel in vivo. Collectively, we demonstrate that phage K1F can infect intracellular E. coli EV36 within hCMECs without themselves eliciting an inflammatory or defensive response. This study illustrates the potential of phage therapy targeting infections such as bacterial neonatal meningitis and is an important step for the continued development of phage therapy targeting antibiotic-resistant bacterial infections generally.

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl epsilon‐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 has 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 SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compond. 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 provide a key reference for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

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.

Histone deacetylases (HDACs) 1–3 regulate chromatin structure and gene expression. These three enzymes are targets for cancer chemotherapy and are studied for the treatment of immune disorders and neurodegeneration, but there is a lack of selective pharmacological tool compounds to unravel their individual roles. Potent inhibitors of HDACs 1–3 often display slow-binding kinetics, which causes a delay in inhibitor–enzyme equilibration and may affect assay readout. Here, we compare the potency and selectivity of slow-binding inhibitors measured by discontinuous and continuous assays. We find that entinostat, a clinical candidate, inhibits HDACs 1–3 by a two-step, slow-binding mechanism with lower potencies than previously reported. In addition, we show that RGFP966, commercialized as HDAC3-selective probe, is a slow-binding inhibitor with inhibitor constants of 57 nM, 31 nM, and 13 nM against HDACs 1–3, respectively. These data highlight a need for thorough kinetic investigation in the development of selective HDAC probes.Table of Contents artwork

BackgroundDepression is common in old age and is associated with disability, increased mortality, and impairment from physical diseases.AimTo estimate the prevalence of depression among older patients in Norwegian general practice, to evaluate the extent they talk about it during their consultation, whether it was previously known or suspected by their GP, and how frequently patients with depression visit their GP.Design & settingCross-sectional study among patients and GPs at 18 primary care clinics in the south of Norway.MethodPatients aged ≥65 years who visited their GP were asked to complete the Patient Health Questionnaire-9 (PHQ-9). The GPs reported what kind of issues the patient presented at the consultation, if a current depression was known, and the consultation frequency.ResultsForty-four (11.4%) of 383 patients reported moderate or severe depressive symptoms (PHQ-9 ≥10). Among the cases with data from both patient and GP (n= 369), 38 patients (10.3%) reported moderately depressive symptoms. Of these, only 12 (31.6%) mentioned psychological problems to their GP during their consultation; 12 (31.6%) with previous depression were neither known to the GP nor suspected of currently having depression; and 67.6% of them visited their GP ≥5 times a year.ConclusionOlder patients tend to speak little of their depression to the GP. Almost one in three older patients with moderate depressive symptoms were unrecognised by their GP. Older patients who frequently visit the GP should be suspected of potentially having mental health problems.

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing -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 biosisosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

Cyclic peptides are becoming increasingly important in drug discovery due to their specific binding properties, larger surface area compared to small molecules, and their ready and modular synthetic accessibility. In this protocol, we describe an on-resin, cleavage-inducing cyclization methodology for the synthesis of cyclic thiodepsipeptides and cyclic homodetic peptides using the 3-amino-4-(methylamino)benzoic acid (MeDbz) linker. We further describe three post-cyclization one-pot procedures, which include desulfurization, disulfide bond formation, and S-alkylation of cysteine residues.

International audience; 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 candidatesa 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 englobes, 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.

Cyclic peptides are becoming increasingly important in drug discovery due to their specific binding properties, larger surface area compared to small molecules, and their ready and modular synthetic accessibility. In this protocol, we describe an on-resin, cleavage-inducing cyclization methodology for the synthesis of cyclic thiodepsipeptides and cyclic homodetic peptides using the 3-amino-4-(methylamino)benzoic acid (MeDbz) linker. We further describe three post-cyclization one-pot procedures, which include desulfurization, disulfide bond formation, and S-alkylation of cysteine residues.

Group behavior in many bacteria relies on chemically induced communication called quorum sensing (QS), which plays important roles in 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 been predicted to constitute a structural feature of AIPs from other species as well. Here, we show that pentameric AIPs from Lactobacillus 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 the cross-species and cross-genus communication of bacteria and may help guide the discovery of peptide ligands to perturb their function.

34 Pags.- 1 Tabls.- 4 Figs.- 1 Glossary. The definitive version is available at: https://www.sciencedirect.com/science/journal/13601385, Zn2+-dependent histone deacetylases are widely distributed in archaea, bacteria, and eukaryotes. Through deacetylation of histones and other biomolecules, these enzymes regulate mammalian gene expression, microtubule stability, and polyamine metabolism. In plants, they play essential roles in development and stress response, but little is known about their biochemistry. We provide here a holistic revision of plant histone deacetylase (HDA) phylogeny and translate recent lessons from other organisms. HDA evolution correlates with a gain of structural ductility/disorder, as observed for other proteins. We also highlight two recently identified Brassicaceae-specific HDAs, as well as unprecedented key mutations that would affect the catalytic activity of individual HDAs. This revised phylogeny will contextualize future studies and illuminate research on plant development and adaptation., This work was supported by the Government of Aragón-European Regional Development Fund (FEDER; grant E35_17R to I. Y.).

Histone deacetylases (HDACs) are enzymes involved in the epigenetic control of gene expression. A handful of HDAC inhibitors have been approved for the treatment of cancer, and HDAC inhibition has also been proposed as a novel therapeutic strategy for neurodegenerative disorders. These disorders include progranulin (PGRN)-deficient forms of frontotemporal dementia caused by mutations in the GRN gene that lead to haploinsufficiency. Hydroxamic acid-based inhibitors of HDACs 1–3, reported to have fast-on/fast-off binding kinetics, induce increased expression of PGRN in human neuronal models, while the benzamide class of slow-binding HDAC inhibitors does not produce this effect. These observations indicate that the kinetics of HDAC inhibitor binding can be tuned for optimal induction of human PGRN expression in neurons. Here, we further expand on these findings using human cortical-like, glutamatergic neurons. We provide evidence that two prototypical, potent hydroxamic acid HDAC inhibitors that induce PGRN (panobinostat and trichostatin A) exhibit an initial fast-binding step followed by a second, slower step, referred to as mechanism B of slow binding, rather than simpler fast-on/fast-off binding kinetics. In addition, we show that trapoxin A, a macrocyclic, epoxyketone-containing class I HDAC inhibitor, exhibits slow binding with high, picomolar potency and also induces PGRN expression in human neurons. Finally, we demonstrate induction of PGRN expression by fast-on/fast-off, highly potent, macrocyclic HDAC inhibitors with ethyl ketone or ethyl ester Zn(2+) binding groups. Taken together, these data expand our understanding of HDAC1–3 inhibitor binding kinetics, and further delineate the specific combinations of structural and kinetic features of HDAC inhibitors that are optimal for upregulating PGRN expression in human neurons and thus may have translational relevance in neurodegenerative disease.

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non-S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus. Bacteria communicate through excretion of minute amounts of chemical signalling molecules that affect virulence, biofilm formation and colonization. In staphylococci, these molecules, called autoinducing peptides, are macrocyclic thiolactone-containing peptides. Now, a simple enrichment method, based on chemoselective capture on polymer beads, has been developed that enables the identification of previously unknown autoinducing peptides.

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.

Staphylococci utilize secreted autoinducing peptides (AIPs) to regulate group behaviour through a process called quorum sensing (QS). Here, we survey the QS interaction landscape within the Staphylococcus genus by assembling a unique compound collection, comprising all the currently known AIPs. These ribosomally synthesized and posttranslationally modified peptides (RiPPs) were obtained by chemical synthesis and mapping of their ability to modulate QS was evaluated using reporter strains of common human and animal colonizing pathogens (S. aureus, S. epidermidis, S. lugdunensis). The resulting map of >200 native QS interactions provides a holistic view of nodes that contribute to the complex signalling network within the Staphylococcus genus. This overview reveals surprising cross-species QS induction and identify the first pan-inhibitory AIP, which is then shown to attenuate MRSA induced skin infection in a mouse model. Our results expose a complex universe of possible staphylococcal interactions and provide further impetus for development of therapeutics based on QS modulators targeting antibiotic resistant pathogens.

Lysine l-lactylation [K(l-la)] is a newly discovered histone mark that can be stimulated under conditions of high glycolysis, such as the Warburg effect. K(l-la) is associated with functions that are different from the widely studied histone acetylation. While K(l-la) can be introduced by the acetyltransferase p300, histone delactylase enzymes remain unknown. Here, we report the systematic evaluation of zinc- and NAD+-dependent HDACs for their ability to cleave ε-N-l-lactyllysine marks. Our screens identified HDACs 1–3 and SIRT1–3 as delactylases in vitro. HDACs 1–3 show robust activity toward not only K(l-la) but also K(d-la) and diverse short-chain acyl modifications. We further confirmed the de-l-lactylase activity of HDACs 1 and 3 in cells. Identification of p300 and HDAC3 as regulatory enzymes suggests that histone lactylation is installed and removed by enzymes as opposed to spontaneous chemical reactivity. Our results therefore represent an important step toward full characterization of this pathway’s regulatory elements.

Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes., Current histone microarrays cannot be used to directly study the transient interactions of histone deacetylases (HDACs). Here, the authors show that hydroxamic acid-modified microarrays can capture HDACs, provide insights into their substrate specificity, and serve to develop peptide inhibitors.

Summary Histone deacetylases (HDACs) are ubiquitous enzymes that cleave post-translational ε-N-acyllysine modifications. The continued identification of diverse acyl modifications at lysine residues in proteins has resulted in discovery of new insight into the biological roles of these enzymes. Here, we describe a fluorogenic high-throughput screening protocol to identify deacylase activities. We describe the careful optimization of continuous, coupled enzyme assays, which provide efficient determination of kinetic parameters. These techniques can facilitate inhibitor assay design and provide fundamental understanding of HDAC biochemistry. For complete details on the use and execution of this protocol, please refer to Moreno-Yruela et al. (2018)., Graphical abstract, Highlights • Histone deacetylases hydrolyze multiple different acyl modifications of lysine • Fluorogenic substrates enable high-throughput screening of deacylase activities • We develop continuous, coupled enzyme assays by careful optimization • Continuous assays allow accurate determination of enzyme kinetic parameters, Histone deacetylases (HDACs) are ubiquitous enzymes that cleave post-translational ε-N-acyllysine modifications. The continued identification of diverse acyl modifications at lysine residues in proteins has resulted in discovery of new insight into the biological roles of these enzymes. Here, we describe a fluorogenic high-throughput screening protocol to identify deacylase activities. We describe the careful optimization of continuous, coupled enzyme assays, which provide efficient determination of kinetic parameters. These techniques can facilitate inhibitor assay design and provide fundamental understanding of HDAC biochemistry.

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.

Journal of medicinal chemistry 63(22), 13709 - 13718 (2020). doi:10.1021/acs.jmedchem.0c01202, Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure–function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms., Published by ACS, Washington, DC

Zn