Staphylococci utilize secreted autoinducing peptides (AIPs) to regulate group behaviour through a process called quorum sensing (QS). For staphylococcal pathogens such as S. aureus, QS regulates expression of major virulence factors and QS inhibition has been proposed as an alternative to antibiotics for treatment of infections with methicillin resistant S. aureus (MRSA). Here, we surveyed the interaction map between QS systems of the pathogens Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis and the 36 currently known AIPs from 22 staphylococcal species. We identified seven of these ribosomally synthesized and post-translationally modified peptides (RiPPs) in this study and all synthetic peptides were assessed for their ability to modulate QS. The mapped interactions of >280 native QS pairings were divided into human- and animal-associated staphylococci showing substantial differences in inhibitory potencies between the groups. In particular, AIPs of the bovine-associated species S. simulans displayed potential as QS inhibitors in the strains investigated in this study and were therefore chosen as starting point for a structure-activity relationship study. This study provides insights into the requirements for QS interference, yielding the most potent inhibitors reported to date for S. epidermidis and S. lugdunensis. Further, we tested an S. simulans AIP as anti-virulence agent in an assay to assess risk of acquired suppression of the inhibitory effect, and we established an assay set-up to successfully monitor agr deactivation of virulent MRSA by the QS inhibitor. Finally, a peptide was shown to attenuate skin infection caused by MRSA in a mouse model. Our results reveal a complex network of staphylococcal interactions and provide further impetus for the development of therapeutic strategies, based on QS modulation to target antibiotic-resistant pathogens. ### Competing Interest Statement The authors have declared no competing interest.

Cysteine thioesters are involved in a myriad of central biological transformations due to their unique reactivity. Despite their well-studied properties, we discovered an unexpected transamidation reaction of cysteine thioesters that leads to peptide backbone cleavage. S-Acylcysteine-containing peptides were found to spontaneously fragment by cleavage of the amide bond in the i-1 position to the acylated cysteine residue at pH 8-10. We present compelling evidence of a mechanism involving a central reversible thioester-to-imide acyl transfer step. The discovered transamidation reaction was found to be highly sequence dependent and to occur in peptides containing post-translational modifications (PTMs) such as cysteine S-acetylation and S-palmitoylation as well as in peptide-peptide branched thioesters, mimicking class I intein splicing. Thus, the inherent reactivity of peptide backbones containing S-acylcysteine residues should represent a starting point for investigation of endogenous protein behavior and may serve as a foundation for the discovery of mild new peptide and protein transformations.

Histone deacetylase (HDAC) enzymes are epigenetic regulators that affect diverse protein function by removing acyl groups from lysine side chains throughout the proteome. The most recently discovered human isozyme, HDAC11, differs from other HDACs in substrate preference and tissue expression profile. Elucidation of the biological function of this enzyme has been scarce and only a few chemical probes to help advance this insight have been developed thus far. Here we discovered macrocyclic inhibitors that exhibit selectivity for HDAC11 and penetrate the cytoplasmic membrane in cultured cells as determined by the chloroalkane penetration assay. Our work establishes the combination of de novo macrocycle synthesis with incorporation of N-alkylated hydroxamic acid moieties as a viable strategy for targeting HDAC11. Further, this study demonstrates the potential of applying macrocyclic peptide-based library synthesis to directly furnish high-affinity, cell-permeating ligands. The discovered inhibitors comprise tool compounds for the investigation of the biological function of HDAC11.

Sirtuin 5 (SIRT5) is a lysine deacylase enzyme that cleaves negatively charged epsilon-N-acyllysine posttranslational modifications, arising from short dicarboxylic acids. Inhibition of SIRT5 has been suggested as a target for treatment of leukemia and breast cancer. In this work, we performed a focused structure-activity relationship study that identified highly potent inhibitors of SIRT5. Examples of these inhibitors were shown by kinetic evaluation to function as mechanism-based inactivators. Masking of a crucial carboxylate functionality in the inhibitors provided prodrugs, which were demonstrated to bind SIRT5 in cells. This work underscores the importance of kinetic characterization of enzyme inhibitors and provides insights for the further optimization of inhibitors of SIRT5 with potential for in vivo applications.

Since their introduction as a new strategy for synthesizing diverse chemotypes, sulfur(VI)–fluoride exchange (SuFEx) transformations have found applications ranging from polymer chemistry and covalent probe development to bioconjugation tools and chemistries for the synthesis of compound libraries. The collection of SuFEx reactions has expanded significantly since their introduction as a concept, comprising functionalities with varying reactivities towards different nucleophiles; thus, enabling the generation of a wide array of sulfur-containing functional groups for the linkage of structural elements in diverse chemotypes. In this review, we focus on the most recent developments in the use of SuFEx chemistry as a means for the preparation of compound libraries for biological screening as well as the introduction of SuFEx hubs into various biomolecules.

Staphylococci utilize secreted autoinducing peptides (AIPs) to regulate group behavior through a process called quorum sensing (QS). For pathogenic staphylococci, such as Staphylococcus aureus, QS regulates the expression of major virulence factors, and QS inhibition has been proposed as an alternative to antibiotics for the treatment of infections with methicillin-resistant S. aureus (MRSA). Here, we surveyed the interaction map between QS systems of the pathogens S. aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis and all the currently known staphylococcal AIPs, covering 21 different species. We identified six of these ribosomally synthesized and post-translationally modified peptides (RiPPs) in this study and compiled the full collection of AIPs by chemical synthesis. The resulting mapping provided 280 QS interactions that were divided into human- and animal-associated staphylococci, showing substantial differences in inhibitory potencies between the groups. AIPs of the bovine-associated species Staphylococcus simulans displayed potential as QS inhibitors toward the investigated strains and were therefore chosen as a starting point for a structure-activity relationship study. This study provides insights into the requirements for QS interference, yielding the most potent inhibitors reported to date for S. epidermidis and S. lugdunensis. Furthermore, we tested an S. simulans AIP as an anti-virulence agent in an assay to assess the risk of acquired suppression of the inhibitory effect, and we established an assay set-up to successfully monitor agr deactivation of virulent MRSA by the QS inhibitor. Finally, a peptide was shown to attenuate skin infection caused by MRSA in a mouse model. Our results reveal a complex network of staphylococcal interactions and provide further impetus for the investigation of QS modulation in the targeting of antibiotic-resistant pathogens.IMPORTANCEBacteria from the Staphylococcus genus produce macrocyclic peptides, called autoinducing peptides (AIPs), used in inter-cell communication with their kin. Differences in AIP sequence and length produced by different staphylococcal species can result in communication interference, altering the physiology of co-inhabiting staphylococci in complex microbiotas. Opportunistic pathogens like Staphylococcus aureus regulate the expression of toxins using this peptide-mediated communication, and its inhibition has, therefore, been proposed as a strategy to target infections caused by methicillin-resistant S. aureus (MRSA). The systematic mapping of AIP activities, structure-activity relationship studies, and evaluation of resistance development provided in this paper, therefore, serve as a resource for the future discovery of inhibitory peptides for the investigation of bacterial communication.

Despite recent FDA approvals, Alzheimer's disease (AD) still represents an unmet medical need. Among the different available therapeutic approaches, the development of multitarget molecules represents one of the most widely pursued. In this work, we present a second generation of dual ligands directed toward highly networked targets that are deeply involved in the development of the disease, namely, Histone Deacetylases (HDACs) and Glycogen Synthase Kinase 3β (GSK-3β). The synthesized compounds are highly potent GSK-3β, HDAC2, and HDAC6 inhibitors with IC50 values in the nanomolar range of concentrations. Among them, compound 4 inhibits histone H3 and tubulin acetylation at 0.1 μM concentration, blocks hyperphosphorylation of tau protein, and shows interesting immunomodulatory and neuroprotective properties. These features, together with its ability to cross the blood-brain barrier and its favorable physical-chemical properties, make compound 4 a promising hit for the development of innovative disease-modifying agents.

Quorum sensing (QS) is a mechanism that regulates group behavior in bacteria, and in Gram-positive bacteria, the communication molecules are often cyclic peptides, called autoinducing peptides (AIPs). We recently showed that pentameric thiolactone-containing AIPs from Listeria monocytogenes, and from other species, spontaneously undergo rapid rearrangement to homodetic cyclopeptides, which hampers our ability to study the activity of these short-lived compounds. Here, we developed chemically modified analogues that closely mimic the native AIPs while remaining structurally intact, by introducing N-methylation or thioester-to-thioether substitutions. The stabilized AIP analogues exhibit strong QS agonism in L. monocytogenes and allow structure-activity relationships to be studied. Our data provide evidence to suggest that the most potent AIP is in fact the very short-lived thiolactone-containing pentamer. Further, we find that the QS system in L. monocytogenes is more promiscuous with respect to the structural diversity allowed for agonistic AIPs than reported for the more extensively studied QS systems in Staphylococcus aureus and Staphylococcus epidermidis. The developed compounds will be important for uncovering the biology of L. monocytogenes, and the design principles should be broadly applicable to the study of AIPs in other species.

Multistep synthesis performed on solid support is a powerful means to generate small molecule libraries for the discovery of chemical probes to dissect biological mechanisms as well as for drug discovery. Therefore, expansion of the collection of robust chemical transformations amenable to solid-phase synthesis is desirable for achieving chemically diverse libraries for biological testing. Here we show that sulfur(VI) fluoride ex-change (SuFEx) chemistry, exemplified by pairing phenols with aryl fluorosulfates, can be used for solid-phase synthesis of biologically active compounds. As a case study, we designed and synthesized a library of 84 hy-droxamic acid containing small molecules, providing a rich source of inhibitors with diverse selectivity pro-files across the human histone deacetylase enzyme family, which is a validated drug target. Among other dis-coveries, we identified a scaffold that furnished inhibitors of HDAC11 with exquisite selectivity in vitro and a selective inhibitor of HDAC6 that was shown to bind this target enzyme selectively over HDAC8 in cells, using cellular thermal shift assays (CETSA). Our results encourage the further use of SuFEx chemistry for the syn-thesis of diverse small molecule libraries, provides insight for future design of selective HDAC inhibitors, and show that CETSA can be applied for evaluation of cellular target engagement of HDAC inhibitors.

Lysine l-lactylation (Kl-la) is a novel protein posttranslational modification (PTM) driven by l-lactate. This PTM has three isomers: Kl-la, N-ε-(carboxyethyl)-lysine (Kce) and d-lactyl-lysine (Kd-la), which are often confused in the context of the Warburg effect and nuclear presence. Here we introduce two methods to differentiate these isomers: a chemical derivatization and high-performance liquid chromatography analysis for efficient separation, and isomer-specific antibodies for high-selectivity identification. We demonstrated that Kl-la is the primary lactylation isomer on histones and dynamically regulated by glycolysis, not Kd-la or Kce, which are observed when the glyoxalase system was incomplete. The study also reveals that lactyl-coenzyme A, a precursor in l-lactylation, correlates positively with Kl-la levels. This work not only provides a methodology for distinguishing other PTM isomers, but also highlights Kl-la as the primary responder to glycolysis and the Warburg effect. Using a combination of antibody- and LC–MS/MS-based methods, Zhang et al. reveal lysine l-lactylation as the key lactylation isomer in cellular histones, responding dynamically to glycolysis and positively correlating with lactyl-CoA levels, providing insights into the Warburg effect.

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.

Classical Zn2+-dependent deac(et)ylases play fundamental regulatory roles in life and are well characterized in eukaryotes regarding their structures, substrates and physiological roles. In bacteria, however, classical deacylases are less well understood. We construct a Generalized Profile (GP) and identify thousands of uncharacterized classical deacylases in bacteria, which are grouped into five clusters. Systematic structural and functional characterization of representative enzymes from each cluster reveal high functional diversity, including polyamine deacylases and protein deacylases with various acyl-chain type preferences. These data are supported by multiple crystal structures of enzymes from different clusters. Through this extensive analysis, we define the structural requirements of substrate selectivity, and discovered bacterial de-d-/l-lactylases and long-chain deacylases. Importantly, bacterial deacylases are inhibited by archetypal HDAC inhibitors, as supported by co-crystal structures with the inhibitors SAHA and TSA, and setting the ground for drug repurposing strategies to fight bacterial infections. Thus, we provide a systematic structure-function analysis of classical deacylases in bacteria and reveal the basis of substrate specificity, acyl-chain preference and inhibition. In bacteria Zn2+-dependent deacylases are underexplored. Here, the authors identify bacterial deacylases, providing systemic structure-function analyses to reveal the basis of substrate specificity, acyl-chain preference and inhibition.

Posttranslational modifications alter the structure and function of proteins. Now, genetic code expansion enables encoding of epsilon-N-succinyllysine and epsilon-N-glutaryllysine residues to decipher the effects of these modifications on enzymatic activity, protein-protein interactions and protein-DNA interactions.

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

Next generation sequencing for 52 myeloid malignancies-related genes.

Patient sample information.

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.