Serine Hydrolase Research Articles

Crystal Structure and Functional Characterization of an S-Formylglutathione Hydrolase (BuSFGH) from Burkholderiaceae sp.

S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of Burkholderiaceae sp. SFGH (BuSFGH) at 1.73 Å resolution. Structural analysis showed that the overall structure of BuSFGH has a typical α/β hydrolase fold, with a central β-sheet surrounded by α-helices. Analytical ultracentrifugation analysis showed that BuSFGH formed a stable dimer in solution. The enzyme activity assay indicated that BuSFGH has a high preference for short-chain p-nitrophenyl esters, such as p-nitrophenyl acetate. The activity of BuSFGH toward p-nitrophenyl acetate was five times higher than that of p-nitrophenyl butylate. Molecular modeling studies on the p-nitrophenyl acetate-bound BuSFGH structure indicate that Gly52, Leu53, Trp96, His147, Ser148, Trp182, Phe228, and His259 residues may be crucial for substrate binding. Collectively, these results are useful for understanding the substrate-binding mechanism and substrate specificity of BuSFGH. They can also provide useful insights for designing modified BuSFGHs with different substrate specificities.

Abstract 5302: Unmasking aggressive tumor profiles in lung adenocarcinomas through the molecular abundances and catalytic action of serine hydrolases

Abstract (a) Current proteomic methods quantify solely the catalytically active portion of enzymes without normalizing the active portion to the total enzyme amount, thus compromising comparison of the results. We take advantage of a SWATH/DIA-MS workflow and describe a method based on depletion-dependent ABPP (dd-ABPP) able to concurrently determine catalytically active enzymes and their total molecular abundances, as well as quantify the contextual sample proteomes. (b) We monitored high quality peptides of more than 190 SH activities and molecular abundances of around 4000 contextual tissue proteins in advanced lung adenocarcinomas (LUAD). While we found that LUAD tumors display enhanced proteolytic activities compared with control lung tissue, the most prominent characteristic of an aggressive tumor phenotype was enhanced lipolysis of metabolic SHs emphasized by IAH1, ABHD12, LYPLA2 and ABHD10. (c) Molecular signatures of the activity profiles of 23 SHs and 59 contextual tissue proteins discriminate aggressive tumors at the time of diagnosis. Enhanced detection of S-palmitoylated proteins correlating by active-enzyme capture uncovers their functional links with the metabolic SHs related to lipoprotein depalmitoylation via enzymes displaying increased de-palmitoylase activities and enhanced fatty acid metabolism in aggressive LUAD. To further validate our findings, we analysed LUAD tumors for panels of saturated and unsaturated fatty acid (C8-C22) levels. Strikingly, we detected a statistically significant increase in monounsaturated palmitoleic acid (C16:1) in the aggressive compared with less-aggressive tumors. Notably, the primary source of palmitoleic acid in the cellular environments results from an excess of free palmitic acid and its conversion by rate-limiting enzyme Stearoyl-CoA desaturase 1 (SCD) that is enhanced in LUAD cancer. (d) We detected a significant increase in palmitoleic acid levels in aggressive tumors. To a certain extent our data support previous findings on the active process of lipid desaturation in lung adenocarcinoma cells as the main source of energy for cell proliferation. Our approach also revealed that changes in the active lipase fractions between subtypes do not follow changes in enzyme protein quantities, suggesting that lipase regulations are related rather to peculiarities of the enzyme protein sequence, a distorted enzyme-inhibitor equilibrium, or a distorted molecular composition of the tissue itself. Since the inhibitors of serine lipases are promising targets in other RAS-mutant carcinomas, and the inhibitors of a rate-limiting SCD1 enzyme prevent palmitic acid conversion to a monounsaturated form, these metabolic vulnerabilities detected in aggressive LUAD will be helpful for future therapeutic strategies in lung cancer. Citation Format: Tatjana Sajic, Stephan Arni, Rudolf Aebersold, Sven Hillinger. Unmasking aggressive tumor profiles in lung adenocarcinomas through the molecular abundances and catalytic action of serine hydrolases. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5302.

Sequence and Structural Motifs Controlling the Broad Substrate Specificity of the Mycobacterial Hormone-Sensitive Lipase LipN.

Mycobacterium tuberculosis has a complex life cycle transitioning between active and dormant growth states depending on environmental conditions. LipN (Rv2970c) is a conserved mycobacterial serine hydrolase with regulated catalytic activity at the interface between active and dormant growth conditions. LipN also catalyzes the xenobiotic degradation of a tertiary ester substrate and contains multiple conserved motifs connected with the ability to catalyze the hydrolysis of difficult tertiary ester substrates. Herein, we expanded a library of fluorogenic ester substrates to include more tertiary and constrained esters and screened 33 fluorogenic substrates for activation by LipN, identifying its unique substrate signature. LipN preferred short, unbranched ester substrates, but had its second highest activity against a heteroaromatic five-membered oxazole ester. Oxazole esters are present in multiple mycobacterial serine hydrolase inhibitors but have not been tested widely as ester substrates. Combined structural modeling, kinetic measurements, and substitutional analysis of LipN showcased a fairly rigid binding pocket preorganized for catalysis of short ester substrates. Substitution of diverse amino acids across the binding pocket significantly impacted the folded stability and catalytic activity of LipN with two conserved motifs (HGGGW and GDSAG) playing interconnected, multidimensional roles in regulating its substrate specificity. Together this detailed substrate specificity profile of LipN illustrates the complex interplay between structure and function in mycobacterial hormone-sensitive lipase homologues and indicates oxazole esters as promising inhibitor and substrate scaffolds for mycobacterial hydrolases.

Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant-pathogen interface.

The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae. Using activity-based proteomics with a cocktail of biotinylated probes, we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). The activity of 82 of these hydrolases (mostly SHs) increases during infection, while the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active β-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis-related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity and contains an E112Q active site substitution that is essential for antibacterial activity and is present only in Nicotiana species. This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalised Nicotiana-specific antibacterial NbPR3.

The serine hydrolase ABHD6 controls survival and thermally induced seizures in a mouse model of Dravet syndrome

Evidence suggests that inhibition of α/β hydrolase-domain containing 6 (ABHD6) reduces seizures; however, the molecular mechanism of this therapeutic response remains unknown. We discovered that heterozygous expression of Abhd6 (Abhd6+/−) significantly reduced the premature lethality of Scn1a+/− mouse pups, a genetic mouse model of Dravet Syndrome (DS). Both Abhd6+/− mutation and pharmacological inhibition of ABHD6 reduced the duration and incidence of thermally induced seizures in Scn1a+/− pups. Mechanistically, the in vivo anti-seizure response resulting from ABHD6 inhibition is mediated by potentiation of gamma-aminobutyric acid receptors Type-A (GABAAR). Brain slice electrophysiology showed that blocking ABHD6 potentiates extrasynaptic (tonic) GABAAR currents that reduce dentate granule cell excitatory output without affecting synaptic (phasic) GABAAR currents. Our results unravel an unexpected mechanistic link between ABHD6 activity and extrasynaptic GABAAR currents that controls hippocampal hyperexcitability in a genetic mouse model of DS. Brief summaryThis study provides the first evidence for a mechanistic link between ABHD6 activity and the control of extrasynaptic GABAAR currents that controls hippocampal hyperexcitability in a genetic mouse model of Dravet Syndrome and can be targeted to dampened seizures.

Rational Construction of a Novel Bioluminescent Substrate for Sensing the Tumor-Associated Hydrolase Notum.

Notum, one of the key serine hydrolases in mammals, hydrolyzes the palmitoleoyl moieties of many important proteins and modulates multiple signaling pathways including Wnt/β-catenin signaling. Notum is tightly associated with multiple human diseases, but the reliable and practical tools for sensing Notum activities in complex biological systems are rarely reported. Herein, an efficient strategy was used to rationally construct a specific bioluminescent substrate for Notum. Following computer-aided molecular design and experimental verification, octanoyl luciferin (OL) was selected as the optimum substrate for human Notum, with excellent specificity, high detection sensitivity and high signal-to-noise ratio. Under physiological conditions, OL was readily hydrolyzed by Notum or Notum-containing biological specimens to release d-luciferin that could be easily detected by various fluorescence devices in the presence of luciferase. The applicability of OL for real-time sensing native Notum was examined in living cells, extracellular matrix, and tissue preparations. OL was also used for constructing a high-throughput assay for screening of Notum inhibitors, while a natural compound (bergapten) was newly identified as a potent Notum inhibitor. Collectively, this study devises a reliable and easy-to-use tool for sensing Notum activities in biological systems, which will strongly facilitate hNotum-associated fundamental studies, disease diagnosis, and drug discovery.

WFDC12-overexpressing contributes to the development of atopic dermatitis via accelerating ALOX12/15 metabolism and PAF accumulation

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by eczema-like skin lesions, dry skin, severe itching, and recurrent recurrence. The whey acidic protein four-disulfide core domain gene WFDC12 is highly expressed in skin tissue and up-regulated in the skin lesions of AD patients, but its role and relevant mechanism in AD pathogenesis have not been studied yet. In this study, we found that the expression of WFDC12 was closely related to clinical symptoms of AD and the severity of AD-like lesions induced by DNFB in transgenic mice. WFDC12-overexpressing in the epidermis might promote the migration of skin-presenting cells to lymph nodes and increase Th cell infiltration. Meanwhile, the number and ratio of immune cells and mRNA levels of cytokines were significantly upregulated in transgenic mice. In addition, we found that ALOX12/15 gene expression was upregulated in the arachidonic acid metabolism pathway, and the corresponding metabolite accumulation was increased. The activity of epidermal serine hydrolase decreased and the accumulation of platelet-activating factor (PAF) increased in the epidermis of transgenic mice. Collectively, our data demonstrate that WFDC12 may contribute to the exacerbation of AD-like symptoms in DNFB-induced mouse model by enhancing arachidonic acid metabolism and PAF accumulation and that WFDC12 may be a potential therapeutic target for human atopic dermatitis.

Activity‐Based Protein Profiling – Celebrating the Groundbreaking Contributions of Benjamin Cravatt

Activity‐Based Protein Profiling – Celebrating the Groundbreaking Contributions of Benjamin Cravatt

Lipases and their Applications in Biomedical Field

Abstract: Lipases are carboxylic ester hydrolase enzymes, constituting the class of serine hydrolases, requiring no cofactor for their action. They have various substrates and produce glycerol and free fatty acids through the hydrolysis of fats and oils. Owing to their wide applications and the simplicity with which they can be mass-produced, these are a significant group of biotechnologically important enzymes. In addition, lipases have the special characteristic of operating at a lipid/ water interface. Present review focuses on the medical and therapeutic use of lipases. These enzymes and their inhibitors have applications with remarkable success for managing or even treating diseases such as cancer, obesity, atherosclerosis, and Alzheimer’s. Lipases have also been used to produce healthier fatty acids and low-fat cheese. The characteristic physicochemical and catalytic properties of enzymes make them ideal for biosensors and digestive aids in individuals with exocrine pancreatic inefficiency. Newer avenues open as deeper and more relevant studies are being conducted on newer lipases.

Efficient Synthesis of an Alkyne Fluorophosphonate Activity‐Based Probe and Applications in Dual Colour Serine Hydrolase Labelling

AbstractTagged fluorophosphonates (FP‐probes) were the first activity‐based probes to be reported in 1999. More than 20 years later, FP‐probes are widely used tools for the profiling of serine hydrolase‐activity. However, their synthesis tends to be cumbersome and low‐yielding. In this paper, we report a short and high‐yielding route towards an FP‐alkyne derivative. This probe is suitable for bioorthogonal 2‐step detection of serine hydrolases. Alternatively, it can be converted to an array of fluorescent probes via click chemistry to azide‐functionalized fluorophores. Here, we constructed red‐ and green‐fluorescent FP‐probes and applied these in dual colour labelling of whole proteomes. Specifically, we illustrate application in inhibitor assessment in tissue lysates and in a ‘pulse‐chase ABPP’ experiment in whole cells. Overall, we think that these probes, due to their high yield and different colours, are attractive for novel applications within ABPP.

Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta.

Lipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase α and β (DAGLα, DAGLβ) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks. DAGLα and DAGLβ mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGLβ transcripts to different cell types of the placenta. DAGLβ activity was determined by in- gel and MS-based activity-based protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek™ lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 µM] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined. We demonstrate that mRNA expression of DAGLβ prevails in placental tissue, compared to DAGLα (p ≤ 0.0001) and that DAGLβ is mainly located to CK7 positive trophoblasts (p ≤ 0.0001). Although few DAGLα transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLβ is the principal DAGL in the placenta. DAGLβ dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLβ by DH376 led to reduced MAG tissue levels (p ≤ 0.01), including 2-AG (p≤0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta. Our results emphasize the role of DAGLβ activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies.

CRISPR-Cas9-Mediated Mutation of Methyltransferase METTL4 Results in Embryonic Defects in Silkworm Bombyx mori

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm.

Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae.

Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.

Abstract WP233: Sex Differences In The Impact Of Focal Ischemia On Cognitive Impairment In Middle-aged Rats And The Effect Of Iv Mir20a-3p Treatment

Introduction: Our previous studies show that the small non-coding RNA, mir20a-3p, is neuroprotective for stroke in the acute phase (Branyan et al., 2021). Here we used a battery of tests to assess the integrated functioning of affective and cognitive circuits in the chronic phase of stroke and the impact of mir20a-3p. Methods: Middle-aged (12-month-old) female and male Sprague-Dawley rats were subject to middle cerebral artery occlusion (MCAo) using endothelin-1. Mir20a-3p mimic or scrambled oligo was administered i.v. 4h, 24h, and 60d after stroke. Cognitive changes (30-100d after stroke) were assessed by contextual fear conditioning (CFC) and the novel object recognition test (NORT). Blood samples were collected at 60 and 100d to assess cytokines and short chain fatty acids. Results: MCAo impaired sensory motor performance in the acute phase, which was ameliorated by Mir20a-3p treatment. At 30, 60 and 100d after stroke, retrieval of fear memory was significantly reduced over time in sham and stroke males and females. However, the rate of decline of fear memory was significantly accelerated in the scramble treated female stroke group, but not in the scramble treated male stroke group. Similarly, declarative memory (NORT) declined in all female groups at 30 days post stroke and continued to worsen only in the stroke+ scrambled treated group. Male rats did not show any change in performance due to stroke or its treatment at comparable time points of testing. Hyperintensities in the forebrain of T2 weighted MRI images were decreased in mir20a-3p treated stroke females compared to males. Correlation analysis showed that in females at 60d post stroke, the extent of Remote Fear memory recall was highly positively correlated with the levels of the SCFA butyric acid (+0.81) and valeric acid (+0.61), as well as the pro-inflammatory cytokine IL-12p70 (+0.64), and the serine hydrolase, butyrylcholinesterase (+0.71). Conclusion: Although acute sensory-motor impairment is seen to a similar extent in males and females, stroke significantly affected cognitive function in females but not males, and this was associated with alterations in inflammatory mediators. Mir20a-3p, which is predicted to repress inflammatory analytes, abrogated stroke effects in females.

Machine learning analysis of SH-dipeptide dynamics from atomistic molecular dynamics simulations.

Serine-Histidine (SH-dipeptide) is a minimal peptide system with both hydrolase and peptide synthesis activity, and has been argued to be a potential candidate proto-enzyme in exobiological and/or origin-of-life settings. Here, we use a combination of machine learning and atomistic molecular dynamics to characterize the conformational landscape and dynamics of SH-dipeptide at ambient temperature, as a function of pH. We also compare the behavior of SH-dipeptide with Serine-Histidine-Aspartic Acid (SHD), a small peptide that emulates the most common serine hydrolase catalytic triad. We focus on identifying low-dimensional degrees of freedom that optimally impute the trajectory of the peptide as a whole, providing a framework that can be used for further studies of this system.

Acetylcholinesterase Activity Staining in Freshwater Planarians.

The serine hydrolase acetylcholinesterase (AChE) is an important neuronal enzyme which catalyzes the hydrolysis of the neurotransmitter acetylcholine and other choline esters. The breakdown of acetylcholine by AChE terminates synaptic transmission and regulates neuromuscular communication. AChE inhibition is a common mode of action of various insecticides, such as carbamates and organophosphorus pesticides. Freshwater planarians, especially the species Dugesia japonica, have been shown to possess AChE activity and to be a suitable alternative model for studying the effects of pesticides in vivo. AChE activity can be quantified in homogenates using the Ellman assay. However, this biochemical assay requires specialized equipment and large numbers of planarians. Here, we present a protocol for visualizing AChE activity in individual planarians. Activity staining can be completed in several hours and can be executed using standard laboratory equipment (a fume hood, nutator, and light microscope with imaging capability). We describe the steps for preparing the reagents, and the staining and imaging of the planarians. Planarians are treated with 10% acetic acid and fixed with 4% paraformaldehyde and then incubated in a staining solution containing the substrate acetylthiocholine. After incubation in the staining solution for 3.5 hr on a nutator at 4°C, or stationary on ice, planarians are washed and mounted for imaging. Using exposure to an organophosphorus pesticide as an example, we show how AChE inhibition leads to a loss of staining. Thus, this simple method can be used to qualitatively evaluate AChE inhibition due to chemical exposure or RNA interference, providing a new tool for mechanistic studies of effects on the cholinergic system. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparing the staining solution Basic Protocol 2: Fixing, staining, and imaging whole-mount planarian specimens for visualization of acetylcholinesterase activity.

Firefly luciferin methyl ester illuminates the activity of multiple serine hydrolases.

Firefly luciferin methyl ester is hydrolyzed by monoacylglycerol lipase MAGL, amidase FAAH, poorly-characterized hydrolase ABHD11, and hydrolases known for S-depalmitoylation (LYPLA1/2), not just esterase CES1. This enables activity-based bioluminescent assays for serine hydrolases and suggests that the 'esterase activity' responsible for hydrolyzing ester prodrugs is more diverse than previously supposed.

Abstract 1442: Shifted Activity of Serine Hydrolases in Mycobacterium smegmatis During the Dormant to Active Transition

Abstract 1442: Shifted Activity of Serine Hydrolases in Mycobacterium smegmatis During the Dormant to Active Transition

Synthesis, activity and metabolic stability of propan-2-one substituted tetrazolylalkanoic acids as dual inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase.

The serine hydrolases cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are interesting targets for the development of new anti-inflammatory and analgesic drugs. Structural modifications of a potent dual inhibitor with a propan-2-one substituted tetrazolylpropionic acid moiety led to compounds with also nanomolar activity against both enzymes but better physicochemical properties. The structure-activity relationships showed that the variations had partially divergent effects on the inhibitory activity of the compounds towards cPLA2α and FAAH reflecting differences in the binding mode to the enzymes. Furthermore, the metabolic stability of the target structures was investigated in vitro.

Chemical Proteomics Reveals Antibiotic Targets of Oxadiazolones in MRSA.

Phenotypic screening is a powerful approach to identify novel antibiotics, but elucidation of the targets responsible for the antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode of action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones identified in a phenotypic screen to have high antibacterial potency against multidrug-resistant Staphylococcus aureus. In situ competitive and comparative chemical proteomics with a tailor-made activity-based probe, in combination with transposon and resistance studies, revealed several cysteine and serine hydrolases as relevant targets. Our data showcase oxadiazolones as a novel antibacterial chemotype with a polypharmacological mode of action, in which FabH, FphC, and AdhE play a central role.