Palmitoyl transferase ZDHHC20 promotes pancreatic cancer metastasis.

Metastasis is one of the defining features of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor prognosis. In this study, the palmitoyl transferase ZDHHC20 was identified in an in vivo short hairpin RNA (shRNA) screen as critical for metastatic outgrowth, with no effect on proliferation and migration in vitro or primary PDAC growth in mice. This phenotype is abrogated in immunocompromised animals and animals with depleted natural killer (NK) cells, indicating that ZDHHC20 affects the interaction of tumor cells and the innate immune system. Using a chemical genetics platform for ZDHHC20-specific substrate profiling, a number of substrates of this enzyme were identified. These results describe a role for palmitoylation in enabling distant metastasis that could not have been detected using in vitro screening approaches and identify potential effectors through which ZDHHC20 promotes metastasis of PDAC.

A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation.

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

Bi-allelic ACBD6 variants lead to a neurodevelopmental syndrome with progressive and complex movement disorders.

The acyl-CoA-binding domain-containing protein 6 (ACBD6) is ubiquitously expressed, plays a role in the acylation of lipids and proteins and regulates the N-myristoylation of proteins via N-myristoyltransferase enzymes (NMTs). However, its precise function in cells is still unclear, as is the consequence of ACBD6 defects on human pathophysiology. Using exome sequencing and extensive international data sharing efforts, we identified 45 affected individuals from 28 unrelated families (consanguinity 93%) with bi-allelic pathogenic, predominantly loss-of-function (18/20) variants in ACBD6. We generated zebrafish and Xenopus tropicalis acbd6 knockouts by CRISPR/Cas9 and characterized the role of ACBD6 on protein N-myristoylation with myristic acid alkyne (YnMyr) chemical proteomics in the model organisms and human cells, with the latter also being subjected further to ACBD6 peroxisomal localization studies. The affected individuals (23 males and 22 females), aged 1-50 years, typically present with a complex and progressive disease involving moderate-to-severe global developmental delay/intellectual disability (100%) with significant expressive language impairment (98%), movement disorders (97%), facial dysmorphism (95%) and mild cerebellar ataxia (85%) associated with gait impairment (94%), limb spasticity/hypertonia (76%), oculomotor (71%) and behavioural abnormalities (65%), overweight (59%), microcephaly (39%) and epilepsy (33%). The most conspicuous and common movement disorder was dystonia (94%), frequently leading to early-onset progressive postural deformities (97%), limb dystonia (55%) and cervical dystonia (31%). A jerky tremor in the upper limbs (63%), a mild head tremor (59%), parkinsonism/hypokinesia developing with advancing age (32%) and simple motor and vocal tics were among other frequent movement disorders. Midline brain malformations including corpus callosum abnormalities (70%), hypoplasia/agenesis of the anterior commissure (66%), short midbrain and small inferior cerebellar vermis (38% each) as well as hypertrophy of the clava (24%) were common neuroimaging findings. Acbd6-deficient zebrafish and Xenopus models effectively recapitulated many clinical phenotypes reported in patients including movement disorders, progressive neuromotor impairment, seizures, microcephaly, craniofacial dysmorphism and midbrain defects accompanied by developmental delay with increased mortality over time. Unlike ACBD5, ACBD6 did not show a peroxisomal localization and ACBD6-deficiency was not associated with altered peroxisomal parameters in patient fibroblasts. Significant differences in YnMyr-labelling were observed for 68 co- and 18 post-translationally N-myristoylated proteins in patient-derived fibroblasts. N-myristoylation was similarly affected in acbd6-deficient zebrafish and X. tropicalis models, including Fus, Marcks and Chchd-related proteins implicated in neurological diseases. The present study provides evidence that bi-allelic pathogenic variants in ACBD6 lead to a distinct neurodevelopmental syndrome accompanied by complex and progressive cognitive and movement disorders.

S-Palmitoylation during Retinoic Acid-Induced Neuronal Differentiation of SH-SY5Y Neuroblastoma Cells.

S-Palmitoylation is the covalent attachment of C14:0-C22:0 fatty acids (mainly C16:0 palmitate) to cysteines via thioester bonds. This lipid modification is highly abundant in neurons, where it plays a role in neuronal development and is implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. The knowledge of S-palmitoylation in neurodevelopment is limited due to technological challenges in analyzing this highly hydrophobic protein modification. Here, we used two orthogonal methods, acyl-biotin exchange (ABE) and lipid metabolic labeling (LML), to identify S-palmitoylated proteins and sites during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. We identified 2002 putative S-palmitoylated proteins in total, of which 650 were found with both methods. Significant changes in the abundance of S-palmitoylated proteins were detected, in particular for several processes and protein classes that are known to be important for neuronal differentiation, which include proto-oncogene tyrosine-protein kinase receptor (RET) signal transduction, SNARE protein-mediated exocytosis, and neural cell adhesion molecules. Overall, S-palmitoylation profiling by employing ABE and LML in parallel during RA-induced differentiation of SH-SY5Y cells revealed a subset of high confidence bona fide S-palmitoylated proteins and suggested an important role for S-palmitoylation in neuronal differentiation.

Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles.

Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID-ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID-ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID-ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID-ABPP providing the advantage of direct activity-based probe interaction site identification.

Recent advancements in mass spectrometry-based tools to investigate newly synthesized proteins.

Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry-based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging-based, and stable isotope labeling by amino acids in cell culture-based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.

ABHD2 Inhibitor Identified by Activity-Based Protein Profiling Reduces Acrosome Reaction.

ABHD2 is a serine hydrolase that belongs to the subgroup of the α,ß-hydrolase fold-containing proteins, which is involved in virus propagation, immune response, and fertilization. Chemical tools to selectively modulate the activity of ABHD2 in an acute setting are highly desired to investigate its biological role, but are currently lacking. Here, we report a library-versus-library screening using activity-based protein profiling (ABPP) to evaluate in parallel the selectivity and activity of a focused lipase inhibitor library against ABHD2 and a panel of closely related ABHD proteins. This screen resulted in the rapid identification of novel inhibitors for ABHD2. The selectivity of the inhibitor was further investigated in native mouse testis proteome by competitive ABPP, revealing a highly restricted off-target profile. The progesterone-induced acrosome reaction was reduced in a dose-dependent manner by the newly identified inhibitor, which provides further support for the key-role of ABHD2 in the P4-stimulated acrosome reaction. On this basis, the ABHD2 inhibitor is an excellent starting point for further optimization of ABHD2 inhibitors that can modulate sperm fertility and may lead to novel contraceptives.

Activity-Based Protein Profiling Identifies α-Ketoamides as Inhibitors for Phospholipase A2 Group XVI.

Phospholipase A2, group XVI (PLA2G16) is a thiol hydrolase from the HRASLS family that regulates lipolysis in adipose tissue and has been identified as a host factor enabling the cellular entry of picornaviruses. Chemical tools are essential to visualize and control PLA2G16 activity, but they have not been reported to date. Here, we show that MB064, which is a fluorescent lipase probe, also labels recombinant and endogenously expressed PLA2G16. Competitive activity-based protein profiling (ABPP) using MB064 enabled the discovery of α-ketoamides as the first selective PLA2G16 inhibitors. LEI110 was identified as a potent PLA2G16 inhibitor ( Ki = 20 nM) that reduces cellular arachidonic acid levels and oleic acid-induced lipolysis in human HepG2 cells. Gel-based ABPP and chemical proteomics showed that LEI110 is a selective pan-inhibitor of the HRASLS family of thiol hydrolases (i.e., PLA2G16, HRASLS2, RARRES3 and iNAT). Molecular dynamic simulations of LEI110 in the reported crystal structure of PLA2G16 provided insight in the potential ligand-protein interactions to explain its binding mode. In conclusion, we have developed the first selective inhibitor that can be used to study the cellular role of PLA2G16.

Chemical Proteomic Analysis of Serine Hydrolase Activity in Niemann-Pick Type C Mouse Brain.

The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/ß-hydrolase domain-containing protein 4, α/ß-hydrolase domain-containing protein 6, α/ß-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies.

2-Arachidonoylglycerol: A signaling lipid with manifold actions in the brain.

2-Arachidonoylglycerol (2-AG) is a signaling lipid in the central nervous system that is a key regulator of neurotransmitter release. 2-AG is an endocannabinoid that activates the cannabinoid CB1 receptor. It is involved in a wide array of (patho)physiological functions, such as emotion, cognition, energy balance, pain sensation and neuroinflammation. In this review, we describe the biosynthetic and metabolic pathways of 2-AG and how chemical and genetic perturbation of these pathways has led to insight in the biological role of this signaling lipid. Finally, we discuss the potential therapeutic benefits of modulating 2-AG levels in the brain.

Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification.

Activity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off-target activity. We recently used ABPP to determine the serine hydrolase interaction landscape of the experimental drug BIA 10-2474, thereby providing a potential explanation for the adverse side effects observed with this compound. ABPP allows mapping of protein interaction landscapes of inhibitors in cells, tissues and animal models. Whereas our previous protocol described quantification of proteasome activity using stable-isotope labeling, this protocol describes the procedures for identifying the in vivo selectivity profile of covalent inhibitors with label-free quantitative proteomics. The optimization of our protocol for label-free quantification methods results in high proteome coverage and allows the comparison of multiple biological samples. We demonstrate our protocol by assessing the protein interaction landscape of the diacylglycerol lipase inhibitor DH376 in mouse brain, liver, kidney and testes. The stages of the protocol include tissue lysis, probe incubation, target enrichment, sample preparation, liquid chromatography-mass spectrometry (LC-MS) measurement, data processing and analysis. This approach can be used to study target engagement in a native proteome and to identify potential off targets for the inhibitor under investigation. The entire protocol takes at least 4 d, depending on the number of samples.

Novel activity-based probes for N-acylethanolamine acid amidase.

The cysteine hydrolase, N-acylethanolamine acid amidase (NAAA) is a promising target for analgesic and anti-inflammatory drugs. Here, we describe the development of two unprecedented NAAA-reactive activity-based probes as research tools for application in the discovery of new inhibitors and for the in-depth characterization of NAAA in its cellular environment.

Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474.

A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system.

Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity.

The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

Chemical Proteomics Maps Brain Region Specific Activity of Endocannabinoid Hydrolases.

The biosynthetic and catabolic enzymes of the endocannabinoids tightly regulate endocannabinoid-mediated activation of the cannabinoid CB1 receptor. Monitoring the activities of these endocannabinoid hydrolases in different brain regions is, therefore, key to gaining insight into spatiotemporal control of CB1 receptor-mediated physiology. We have employed a comparative chemical proteomics approach to quantitatively map the activity profile of endocannabinoid hydrolases in various mouse brain regions at the same time. To this end, we used two different activity-based probes: fluorophosphonate-biotin (FP-biotin), which quantifies FAAH, ABHD6, and MAG-lipase activity, and MB108, which detects DAGL-α, ABHD4, ABHD6, and ABHD12. In total, 32 serine hydrolases were evaluated in the frontal cortex, hippocampus, striatum, and cerebellum. Comparison of endocannabinoid hydrolase activity in the four brain regions revealed that FAAH activity was highest in the hippocampus, and MAGL activity was most pronounced in the frontal cortex, whereas DAGL-α was most active in the cerebellum. Comparison of the activity profiles with a global proteomics data set revealed pronounced differences. This could indicate that post-translational modification of the endocannabinoid hydrolases is important to regulate their activity. Next, the effect of genetic deletion of the CB1 receptor was studied. No difference in the enzymatic activity was found in the cerebellum, striatum, frontal cortex, and hippocampus of CB1 receptor knockout animals compared to wild type mice. Our results are in line with previous reports and indicate that the CB1 receptor exerts no regulatory control over the basal production and degradation of endocannabinoids and that genetic deletion of the CB1 receptor does not induce compensatory mechanisms in endocannabinoid hydrolase activity.

Chiral disubstituted piperidinyl ureas: a class of dual diacylglycerol lipase-α and ABHD6 inhibitors.

Inhibitors of diacylglycerol lipases and α,ß-hydrolase domain containing protein 6 (ABHD6) are potential leads for the development of therapeutic agents for metabolic and neurodegenerative disorders. Here, we report the enantioselective synthesis and structure activity relationships of triazole ureas featuring chiral, hydroxylated 2-benzylpiperidines as dual inhibitors of DAGLα and ABHD6. The chirality of the carbon bearing the C2 substituent, as well as the position of the hydroxyl (tolerated at C5, but not at C3) has profound influence on the inhibitory activity of both DAGLα and ABHD6, as established using biochemical assays and competitive activity-based protein profiling on mouse brain extracts.

Competitive ABPP of Serine Hydrolases: A Case Study on DAGL-Alpha.

Competitive activity-based protein profiling is a highly efficient chemical biology technique to determine target engagement and selectivity profiles of enzyme inhibitors in complex proteomes. Fluorophosphonate-based fluorescent inhibitors are widely used as broad-spectrum probes for serine hydrolases. However, diacylglycerol lipase-α is not labeled by fluorophosphonate-based probes. To overcome this problem, we have developed a tailor-made activity-based probe that reacts with diacylglycerol lipase-α. Here we describe a case study in which we apply competitive activity-based protein profiling using a broad-spectrum and a tailor-made activity-based probe to establish selectivity and activity profiles of inhibitors targeting diacylglycerol lipase-α in the mouse brain proteome.

Triazole Ureas Act as Diacylglycerol Lipase Inhibitors and Prevent Fasting-Induced Refeeding.

Triazole ureas constitute a versatile class of irreversible inhibitors that target serine hydrolases in both cells and animal models. We have previously reported that triazole ureas can act as selective and CNS-active inhibitors for diacylglycerol lipases (DAGLs), enzymes responsible for the biosynthesis of 2-arachidonoylglycerol (2-AG) that activates cannabinoid CB1 receptor. Here, we report the enantio- and diastereoselective synthesis and structure-activity relationship studies. We found that 2,4-substituted triazole ureas with a biphenylmethanol group provided the most optimal scaffold. Introduction of a chiral ether substituent on the 5-position of the piperidine ring provided ultrapotent inhibitor 38 (DH376) with picomolar activity. Compound 38 temporarily reduces fasting-induced refeeding of mice, thereby emulating the effect of cannabinoid CB1-receptor inverse agonists. This was mirrored by 39 (DO34) but also by the negative control compound 40 (DO53) (which does not inhibit DAGL), which indicates the triazole ureas may affect the energy balance in mice through multiple molecular targets.

Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition.

Diacylglycerol lipases (DAGLα and DAGLß) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.