β-Mannanases are endo-acting glycoside hydrolases (GHs) that cleave β-1,4 glycosidic linkages in mannan-rich plant cell wall polysaccharides. They find application in the food and paper industries. Activity-based probes (ABPs) are powerful tools for GH profiling in complex biological samples, yet to date, bespoke ABPs reporting on mannanases have not been reported. Here, we describe the synthesis of cyclophellitol-inspired ABPs based on mannobiose, mannotriose, and glucomannose, and their use in reporting mannanase activities in secretomes from saprophytic bacteria and fungi grown on mannan-containing biomass polysaccharides. In addition to mannanases, our ABPs also labelled cellulases in secretomes from both Aspergillus niger and Cellvibrio japonicus, which may indicate broader ("negative-subsite") substrate specificity in these enzymes. Mechanistic proof of active-site nucleophile labelling by our ABPs was obtained for both AnManA and CjMan26C by X-ray crystallography and for both AnManA and AnMan26A by mass spectrometry. Together, our results establish mannanase-targeted ABPs that may find use alongside existing reagents that report on retaining GHs that process other bulk polysaccharides.

The proteasome inhibitors bortezomib, carfilzomib, and ixazomib all act by inhibiting multiple active sites of both constitutive proteasomes and immunoproteasomes. These clinical anticancer drugs are effective, but also display side effects, and evidence is amassing that their toxicity arises from constitutive proteasome inhibition. In this work, we describe the structure-guided discovery of a new class of pan-immunoproteasome-selective inhibitors. We identified the peptide epoxyketone BocPip-Ser (8), which targets all three human immunoproteasome active sites potently and with excellent selectivity over constitutive proteasome active sites (IC50 values for i-subunits ≤ 0.92 μM; IC50 ratio β1c/β1i: 13, β2c/β2i: 14, β5c/β5i: 18; Table 1 and Figure 3). We propose compound 8 (BocPip-Ser), which is of a similar size and general properties as carfilzomib, as a lead compound for the development of improved drugs targeting hematological cancers, and possibly also autoimmune diseases, driven by immunoproteasome but not constitutive proteasome activities.

Bacterial persisters are a subpopulation of cells that exhibit a transient non-susceptible phenotype in the presence of bactericidal antibiotic concentrations. This phenotype can lead to the survival and regrowth of bacteria after treatment, resulting in relapse of infections. It is also a contributing factor to antibacterial resistance. Multiple processes are believed to cause persister formation; however, identifying the proteins expressed during the induction of persistence is challenging because the persister state is rare, transient, and does not result in genetic changes. In this study, we used Bio-Orthogonal Non-Canonical Amino Acid Tagging (BONCAT) to label and retrieve the proteome expressed during persistence and recovery for two strains of Staphylococcus aureus exposed to β-lactam and fluoroquinolone antibiotics. After incubating antibiotic-exposed bacteria with the methionine ortholog L-azidohomoalanine to label the proteins of persister cells, we retrieved labeled proteins using click chemistry-pulldown methodology. Analysis of the retrieved proteome of persisters with Label-Free Quantification-Liquid chromatography mass spectrometry (LFQ-LCMS)-based proteomics revealed widespread changes in translation. Our analysis uncovered previously identified persister genes, including, for example, relA/spot-system, changes in purine and amino acid metabolism, the upregulation and downregulation of transcription factors, and changes to influx and efflux pumps, thus validating our methodology. In addition, we also identified numerous novel persister-associated proteins. Few changes were conserved across the two strains and both antibiotics. Instead, results suggest that the mechanisms of persister formation vary across genotypes and the drugs to which strains are exposed. These findings provide evidence that the entry into persistence is an active process that dramatically alters the translational behavior of cells and suggest that downregulation of metabolism, by diverse but functionally similar processes, in persister cells enables cells to survive antibiotic pressure.IMPORTANCEIn this study, we have applied a technique called "Bioorthogonal Non-Canonical Amino Acid-Tagging," or BONCAT, to identify which proteins are expressed when bacteria are in the persister state. Our work makes novel contributions to our understanding of persister cells, a bacterial sub-population that gives rise to recurrent infections, and establishes BONCAT as a valuable tool to study phenotypic heterogeneity in bacterial populations.

The human genome encodes 518 protein kinases that are pivotal for drug discovery in various therapeutic areas such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, making it difficult to develop selective inhibitors. To characterize and prioritize kinase-inhibiting drug candidates, efficient methods are desired to determine target engagement across the cellular kinome. In this study, we present CellEKT (Cellular Endogenous Kinase Targeting), an optimized and robust chemical proteomics platform for investigating cellular target engagement of endogenously expressed kinases using the sulfonyl fluoride-based probe XO44 and two new probes ALX005 and ALX011. The optimized workflow enabled the determination of the kinome interaction landscape of covalent and non-covalent drugs across over 300 kinases, expressed as half maximum inhibitory concentration (IC50), which were validated using distinct platforms like phosphoproteomics and NanoBRET. With CellEKT, target engagement profiles were linked to their substrate space. CellEKT has the ability to decrypt drug actions and to guide the discovery and development of drugs.

The key role of monoacylglycerol lipase (MAGL) in signaling pathways involving endocannabinoids and eicosanoids makes it a promising therapeutic target for numerous diseases, such as (neuro)inflammatory and neuropsychiatric disorders, along with acute tissue damage and cancer. From a mechanistic point of view, MAGL metabolizes the most common endocannabinoid, 2-arachidonylglycerol (2-AG), and additionally influences inflammatory signaling pathways by releasing arachidonic acid (AA). As of now, the therapeutic potential of MAGL has primarily been evaluated through the use of inhibitors and genetic knockout models. Herein, we describe the development of the first reversible MAGL proteolysis targeting chimeras (PROTACs). PROTAC degradation efficiency was dependent on the nature of the linker and E3-ligase system used. Cell permeability was proposed as a main limiting factor for their target engagement and degradation efficacy due to the excessive size and polarity of PROTAC conjugates. Molecular dynamics (MD) simulations revealed a chameleon-like behavior allowing the molecule to hide polar groups by forming intramolecular hydrogen bonds, showing environment-dependent lipophilicity. The best-performing reversible MAGL-PROTAC 4 is permeable, highly efficient with a DC50 < 1.0 nM, and specific in a relevant glioblastoma cell line, which was demonstrated by lipidomic and proteomic analysis. This PROTAC platform enables pharmacological silencing of both MAGLs’ catalytic and potentially elusive structural functions without impacting other proteins. We show that sole modulation of the endocannabinoid system via MAGL degradation using reversible PROTACs potently modulates the endocannabinoid 2-arachidonoyl (2-AG) and therefore holds great therapeutic potential.

Epistasis, the non-additive effect of mutations, substantially undermines our ability to predict the evolutionary trajectories of enzymes. Epistatic effects are evident in the evolution of serine β-lactamases, where synergistic mutations can enhance antimicrobial resistance. We recently demonstrated that positive epistasis drives clavulanic acid resistance in double-mutant libraries of the β-lactamase BlaC. Here, we employed various biochemical and structural approaches to investigate molecular mechanisms underlying epistasis in the fitness of two double mutant variants, I105Y-S130G and I105G-G132N. For the latter enzyme, epistatic compensation of catalytic activity was detected for multiple substrates and proved to be highly buffer-dependent. Non-additive effects were also evident in the thermostability profile of the I105G-G132N variant. The interplay between the reduced clavulanic acid sensitivity of the S130G and G132N variants and active-site modifications induced by Ile105 substitutions is discussed. The results demonstrate that the origins of epistasis can be rooted in multiple enzyme traits, highlighting its important role in the evolution of antimicrobial resistance.

Compared to bortezomib treatment, multiple myeloma (MM) treatment with the proteasome inhibitor carfilzomib is associated with a higher incidence of cardiovascular adverse events. However, the mechanism underlying such cardiopathogenic side effects in MM patients remains elusive. Here, we show that carfilzomib-specific proteasome inhibition profoundly impairs cardiomyocyte contractility. Using an unbiased multiomics approach in vitro and in vivo, followed by in vitro validation, we elucidated carfilzomib-related changes in contractility proteins and cellular translation, retinol oxidative metabolism, and the angiotensin II derivative, angiotensin A. Subsequently, all-trans retinoic acid and angiotensin II type 1 receptor inhibitor prevented cardiomyocytes from experiencing carfilzomib-induced toxicity in human and murine in vitro and in vivo models through stabilization of protein and metabolic homeostasis. Our data reveal a mechanism underlying carfilzomib-induced cardiotoxicity that closely mirrors clinical observations and may open new avenues for management of such potentially lethal side effects in patients with MM.

Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells. Using this approach, we identified six compounds with PI properties ((-)-kinetin-riboside, manumycin-A, puromycin dihydrochloride, resistomycin, tegaserod maleate, and thapsigargin). Although the docking scores pinpointed their ability to bind to the β5 subunit, our in vitro study revealed that these compounds inhibited the β1, β2, and β5 catalytic sites to some extent. As shown with bortezomib, only manumycin-A, puromycin dihydrochloride, and tegaserod maleate resulted in excessive accumulation of ubiquitinated proteins and elevated HMOX1 expression. Taken together, our integrated drug repositioning approach and subsequent in vitro validation studies identified six compounds demonstrating properties similar to proteasome inhibitors.

Kidney filtration is ensured by the interaction of podocytes, endothelial and mesangial cells. Immunoglobulin accumulation at the filtration barrier is pathognomonic for glomerular injury. The mechanisms that regulate filter permeability are unknown. Here, we identify a pivotal role for the proteasome in a specific cell type. Combining genetic and inhibitor-based human, pig, mouse, and Drosophila models we demonstrate that the proteasome maintains filtration barrier integrity, with podocytes requiring the constitutive and glomerular endothelial cells the immunoproteasomal activity. Endothelial immunoproteasome deficiency as well as proteasome inhibition disrupt the filtration barrier in mice, resulting in pathologic immunoglobulin deposition. Mechanistically, we observe reduced endocytic activity, which leads to altered membrane recycling and endocytic receptor turnover. This work expands the concept of the (immuno)proteasome as a control protease orchestrating protein degradation and antigen presentation and endocytosis, providing new therapeutic targets to treat disease-associated glomerular protein accumulations.

This study explores the use of activity-based protein profiling to study protein tyrosine phosphatases. With the discovery of allosteric SHP2 inhibitors, this enzyme family has resurfaced as interesting drug targets. Therefore, we envisioned that previously described direct electrophiles and quinone methide-based traps targeting phosphatases could be applied in competitive activity-based protein profiling assays. This study evaluates three direct electrophiles, specifically, a vinyl sulfonate, a vinyl sulfone, and an alpha-bromobenzylphosphonate as well as three quinone methide-based traps as activity-based probes. For all these moieties it was previously shown that they could selectively engage in assays with purified or overexpressed phosphatases in bacterial lysates. However, this study demonstrates that probes based on these moieties all suffer from unspecific labelling. Direct electrophiles were either unspecific or not activity-based, while quinone methide-based traps showed dependence on phosphatase activity but also resulted in unspecific labelling due to diffusion after activation. This phenomenon, termed ' bystander' labelling, occurred even with catalytically inactive SHP2 mutants. We concluded that alternative strategies or chemistries are needed to apply activity-based protein profiling in phosphatase research. Moreover, this study shows that quinone methide-based designs have limited potential in probe and inhibitor development strategies due to their intrinsic reactivity.

Proteasome degradation is an integral part of cellular growth and function. Proteasomal intervention may mitigate adverse myocardial remodeling, but is associated with the onset of heart failure. Previously, we have demonstrated that increasing abundance of cardiac Lmp2 and its incorporation into proteasome complexes is an endogenous mechanism for proteasome regulation during hypertrophic remodeling of the heart induced by chronic ß-adrenoreceptor stimulation. Here, we investigated whether Lmp2 is required for myocardial remodeling not driven by inflammation and show that Lmp2 is a tipping element for growth and function in the heart but not for proteasome insufficiency. While it has no apparent impact under unchallenged conditions, myocardial remodeling without Lmp2 exacerbates hypertrophy and restricts cardiac function. Under chronic ß-adrenoreceptor stimulation, as seen in the development of cardiovascular disease and the manifestation of heart failure, genetic ablation of Lmp2 in mice caused augmented concentric hypertrophy of the left ventricle. While the heart rate was similarly elevated as in wildtype, myocardial contractility was not maintained without Lmp2, and apparently uncoupled of the ß-adrenergic response. Normalized to the exacerbated myocardial mass, contractility was reduced by 41% of the pretreatment level, but would appear preserved at absolute level. The lack of Lmp2 interfered with elevated 26S proteasome activities during early cardiac remodeling reported previously, but did not cause bulk proteasome insufficiency, suggesting the Lmp2 containing proteasome subpopulation is required for a selected group of proteins to be degraded. In the myocardial interstitium, augmented collagen deposition suggested matrix stiffening in the absence of Lmp2. Indeed, echocardiography of left ventricular peak relaxation velocity (circumferential strain rate) was reduced in this treatment group. Overall, targeting Lmp2 in a condition mimicking chronic ß-adrenoreceptor stimulation exhibited the onset of heart failure. Anticancer therapy inhibiting proteasome activity, including Lmp2, is associated with adverse cardiac events, in particular heart failure. Sparing Lmp2 may be an avenue to reduce adverse cardiac events when chronic sympathetic nervous system activation cannot be excluded.

ObjectiveTo decipher the mechanisms by which the major human milk oligosaccharide (HMO), 2’-fucosyllactose (2’FL), can affect body weight and fat mass gain on high-fat diet (HFD) feeding in mice. We wanted to elucidate whether 2’FL metabolic effects are linked with changes in intestinal mucus production and secretion, mucin glycosylation and degradation, as well as with the modulation of the gut microbiota, faecal proteome and endocannabinoid (eCB) system.Results2’FL supplementation reduced HFD-induced obesity and glucose intolerance. These effects were accompanied by several changes in the intestinal mucus layer, including mucus production and composition, and gene expression of secreted and transmembrane mucins, glycosyltransferases and genes involved in mucus secretion. In addition, 2’FL increased bacterial glycosyl hydrolases involved in mucin glycan degradation. These changes were linked to a significant increase and predominance of bacterial generaAkkermansiaandBacteroides, different faecal proteome profile (with an upregulation of proteins involved in carbon, amino acids and fat metabolism and a downregulation of proteins involved in protein digestion and absorption) and, finally, to changes in the eCB system. We also investigated faecal proteomes from lean and obese humans and found similar changes observed comparing lean and obese mice.ConclusionOur results show that the HMO 2’FL influences host metabolism by modulating the mucus layer, gut microbiota and eCB system and propose the mucus layer as a new potential target for the prevention of obesity and related disorders.

Affinity-based probes are valuable tools for detecting binding interactions between small molecules and proteins in complex biological environments. Metalloproteins are a class of therapeutically significant biomolecules which bind metal ions as part of key structural or catalytic domains and are compelling targets for study. However, there is currently a limited range of chemical tools suitable for profiling the metalloproteome. Here, we describe the preparation and application of a novel, photoactivatable affinity-based probe for detection of a subset of previously challenging to engage metalloproteins. The probe, bearing an 8-mercaptoquinoline metal chelator, was anticipated to engage several zinc metalloproteins, including the 26S-proteasome subunit Rpn11. Upon translation of the labelling experiment to mammalian cell lysate and live cell experiments, proteomic analysis revealed that several metalloproteins were competitively enriched. The diazirine probe SMK-24 was found to effectively enrich multiple components of the minichromosome maintenance complex, a zinc metalloprotein assembly with helicase activity essential to DNA replication. Cell cycle analysis experiments revealed that HEK293 cells treated with SMK-24 experienced stalling in G0/G1 phase, consistent with inactivation of the DNA helicase complex. This work represents an important contribution to the library of cell-permeable chemical tools for studying a collection of metalloproteins for which no previous probe existed.

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr –/– and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Little is known about the mechanistic significance of the ubiquitin proteasome system (UPS) in a kidney autoimmune environment. In membranous nephropathy (MN), autoantibodies target podocytes of the glomerular filter resulting in proteinuria. Converging biochemical, structural, mouse pathomechanistic, and clinical information we report that the deubiquitinase Ubiquitin C-terminal hydrolase L1 (UCH-L1) is induced by oxidative stress in podocytes and is directly involved in proteasome substrate accumulation. Mechanistically, this toxic gain-of-function is mediated by non-functional UCH-L1, which interacts with and thereby impairs proteasomes. In experimental MN, UCH-L1 becomes non-functional and MN patients with poor outcome exhibit autoantibodies with preferential reactivity to non-functional UCH-L1. Podocyte-specific deletion of UCH-L1 protects from experimental MN, whereas overexpression of non-functional UCH-L1 impairs podocyte proteostasis and drives injury in mice. In conclusion, the UPS is pathomechanistically linked to podocyte disease by aberrant proteasomal interactions of non-functional UCH-L1.

Supplementary Methods, figures and tables

Cells pre-treated for 3h with 20 µM nelfinavir.

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl 4 -induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB 1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB 2 , but not CB 1 , antagonists. The CB 1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.

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.

Control cells, treated only with biotin