Potent dual MAGL/FAAH inhibitor AKU-005 engages endocannabinoids to diminish meningeal nociception implicated in migraine pain.

BACKGROUND: Engaging the endocannabinoid system through inhibition of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), degrading endocannabinoids (endoCBs) 2-arachidonoylglycerol (2-AG) and anandamide (AEA), was proposed as a promising approach to ameliorate migraine pain. However, the activity of MAGL and FAAH and action of endoCB on spiking activity of meningeal afferents, from which migraine pain originates, has not been explored thus far. Therefore, we here explored the analgesic effects of endoCB enhancement in rat and human meningeal tissues. METHODS: Both MAGL and FAAH activity and local 2-AG and AEA levels were measured by activity-based protein profiling (ABPP) and LC-MS/MS, respectively, in rat meninges obtained from hemiskulls of P38-P40 Wistar rats and human meninges from elderly patients undergoing non-migraine related neurosurgery. The action on endoCBs upon administration of novel dual MAGL/FAAH inhibitor AKU-005 on meningeal afferents excitability was tested by investigating paired KCl-induced spiking and validation with local (co-)application of either AEA or 2-AG. Finally, the specific TRPV1 agonist capsaicin and blocker capsazepine were tested. RESULTS: The basal level of 2-AG exceeded that of AEA in rat and human meninges. KCl-induced depolarization doubled the level of AEA. AKU-005 slightly increased spontaneous spiking activity whereas the dual MAGL/FAAH inhibitor significantly decreased excitation of nerve fibres induced by KCl. Similar inhibitory effects on meningeal afferents were observed with local applications of 2-AG or AEA. The action of AKU-005 was reversed by CB1 antagonist AM-251, implying CB1 receptor involvement in the anti-nociceptive effect. The inhibitory action of AEA was also reversed by AM-251, but not with the TRPV1 antagonist capsazepine. Data cluster analysis revealed that both AKU-005 and AEA largely increased long-term depression-like meningeal spiking activity upon paired KCl-induced spiking. CONCLUSIONS: In the meninges, high anti-nociceptive 2-AG levels can tonically counteract meningeal signalling, whereas AEA can be engaged on demand by local depolarization. AEA-mediated anti-nociceptive effects through CB1 receptors have therapeutic potential. Together with previously detected MAGL activity in trigeminal ganglia, dual MAGL/FAAH inhibitor AKU-005 appears promising as migraine treatment.

Inhibiting Endocannabinoid Hydrolysis as Emerging Analgesic Strategy Targeting a Spectrum of Ion Channels Implicated in Migraine Pain.

Migraine is a disabling neurovascular disorder characterized by severe pain with still limited efficient treatments. Endocannabinoids, the endogenous painkillers, emerged, alternative to plant cannabis, as promising analgesics against migraine pain. In this thematic review, we discuss how inhibition of the main endocannabinoid-degrading enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), could raise the level of endocannabinoids (endoCBs) such as 2-AG and anandamide in order to alleviate migraine pain. We describe here: (i) migraine pain signaling pathways, which could serve as specific targets for antinociception; (ii) a divergent distribution of MAGL and FAAH activities in the key regions of the PNS and CNS implicated in migraine pain signaling; (iii) a complexity of anti-nociceptive effects of endoCBs mediated by cannabinoid receptors and through a direct modulation of ion channels in nociceptive neurons; and (iv) the spectrum of emerging potent MAGL and FAAH inhibitors which efficiently increase endoCBs levels. The specific distribution and homeostasis of endoCBs in the main regions of the nociceptive system and their generation 'on demand', along with recent availability of MAGL and FAAH inhibitors suggest new perspectives for endoCBs-mediated analgesia in migraine pain.

Distinct Activity of Endocannabinoid-Hydrolyzing Enzymes MAGL and FAAH in Key Regions of Peripheral and Central Nervous System Implicated in Migraine.

In migraine pain, cannabis has a promising analgesic action, which, however, is associated with side psychotropic effects. To overcome these adverse effects of exogenous cannabinoids, we propose migraine pain relief via activation of the endogenous cannabinoid system (ECS) by inhibiting enzymes degrading endocannabinoids. To provide a functional platform for such purpose in the peripheral and central parts of the rat nociceptive system relevant to migraine, we measured by activity-based protein profiling (ABPP) the activity of the main endocannabinoid-hydrolases, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). We found that in trigeminal ganglia, the MAGL activity was nine-fold higher than that of FAAH. MAGL activity exceeded FAAH activity also in DRG, spinal cord and brainstem. However, activities of MAGL and FAAH were comparably high in the cerebellum and cerebral cortex implicated in migraine aura. MAGL and FAAH activities were identified and blocked by the selective and potent inhibitors JJKK-048/KML29 and JZP327A, respectively. The high MAGL activity in trigeminal ganglia implicated in the generation of nociceptive signals suggests this part of ECS as a priority target for blocking peripheral mechanisms of migraine pain. In the CNS, both MAGL and FAAH represent potential targets for attenuation of migraine-related enhanced cortical excitability and pain transmission.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

BACKGROUND: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. RESULTS: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. CONCLUSIONS: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

In Vivo Characterization of the Ultrapotent Monoacylglycerol Lipase Inhibitor {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048).

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

Loratadine analogues as MAGL inhibitors.

Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAGL, IC50=46 nM), and was found to have almost 150-fold higher selectivity over human recombinant fatty acid amide hydrolase (hFAAH, IC50=7.24 µM) and 35-fold higher selectivity over human α/ß-hydrolase-6 (hABHD6, IC50=1.79 µM). Additionally, compound 12a retained H1 antagonistic affinity (pA2=6.81) but did not show cannabinoid receptor activity, when tested at concentrations ⩽ 10 µM. Hence, compound 12a represents a novel dual-acting pharmacological tool possessing both MAGL-inhibitory and antihistaminergic activities.

Revisiting 1,3,4-Oxadiazol-2-ones: Utilization in the Development of ABHD6 Inhibitors.

This article describes our systematic approach to exploring the utility of the 1,3,4-oxadiazol-2-one scaffold in the development of ABHD6 inhibitors. Compound 3-(3-aminobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (JZP-169, 52) was identified as a potent inhibitor of hABHD6, with an IC50 value of 216 nM. This compound at 10 µM concentration did not inhibit any other endocannabinoid hydrolases, such as FAAH, MAGL and ABHD12, or bind to the cannabinoid receptors (CB1 and CB2). Moreover, in competitive activity-based protein profiling (ABPP), compound 52 (JZP-169) at 10 µM selectively targeted ABHD6 of the serine hydrolases of mouse brain membrane proteome. Reversibility studies indicated that compound 52 inhibited hABHD6 in an irreversible manner. Finally, homology modelling and molecular docking studies were used to gain insights into the binding of compound 52 to the active site of hABHD6.

Mutation of Cys242 of human monoacylglycerol lipase disrupts balanced hydrolysis of 1- and 2-monoacylglycerols and selectively impairs inhibitor potency.

Considerable progress has been made in recent years in developing selective, potent monoacylglycerol lipase (MAGL) inhibitors. In the investigations of measures to inhibit this enzyme, less attention has been paid to improving our understanding of its catalytic mechanisms or substrate preferences. In our study, we used site-directed mutagenesis, and we show via versatile activity assays combined with molecular modeling that Cys242 and Tyr194, the two opposing amino acid residues in the catalytic cavity of MAGL, play important roles in determining the rate and the isomer preferences of monoacylglycerol hydrolysis. In contrast to wild-type enzymes that hydrolyzed 1- and 2-monoacylglycerols at similar rates, mutation of Cys242 to alanine caused a significant reduction in overall activity (maximal velocity, Vmax), particularly skewing the balanced hydrolysis of isomers to favor the 2-isomer. Molecular modeling studies indicate that this was caused by structural features unfavorable toward 1-isomers as well as impaired recognition of OH-groups in the glycerol moiety. Direct functional involvement of Cys242 in the catalysis was found unlikely due to the remote distance from the catalytic serine. Unlike C242A, mutation of Tyr194 did not bias the hydrolysis of 1- and 2-monoacylglycerols but significantly compromised overall activity. Finally, mutation of Cys242 was also found to impair inhibition of MAGL, especially that by fluorophosphonate derivatives (13- to 63-fold reduction in potency). Taken together, this study provides new experimental and modeling insights into the molecular mechanisms of MAGL-catalyzed hydrolysis of the primary endocannabinoid 2-arachidonoylglycerol and related monoacylglycerols.

Robust hydrolysis of prostaglandin glycerol esters by human monoacylglycerol lipase (MAGL).

The primary route of inactivation of the endocannabinoid 2-arachidonoylglycerol in the central nervous system is through enzymatic hydrolysis, mainly carried out by monoacylglycerol lipase (MAGL), along with a small contribution by the α/ß-hydrolase domain (ABHD) proteins ABHD6 and ABHD12. Recent methodological progress allowing kinetic monitoring of glycerol liberation has facilitated substrate profiling of the human endocannabinoid hydrolases, and these studies have revealed that the three enzymes have distinct monoacylglycerol substrate and isomer preferences. Here, we have extended this substrate profiling to cover four prostaglandin glycerol esters, namely, 15-deoxy-Δ(12,14)-prostaglandin J2-2-glycerol (15d-PGJ2-G), PGD2-G, PGE2-G, and PGF2 α-G. We found that the three enzymes hydrolyzed the tested substrates, albeit with distinct rates and preferences. Although human ABHD12 (hABHD12) showed only marginal activity toward PGE2-G, hABHD6 preferentially hydrolyzed PGD2-G, and human MAGL (hMAGL) robustly hydrolyzed all four. This was particularly intriguing for MAGL activity toward 15d-PGJ2-G whose hydrolysis rate rivaled that of the best monoacylglycerol substrates. Molecular modeling studies combined with kinetic analysis supported favorable interaction with the hMAGL active site. Long and short MAGL isoforms shared a similar substrate profile, and hMAGL hydrolyzed 15d-PGJ2-G also in living cells. The ability of 15d-PGJ2-G to activate the canonical nuclear factor erythroid 2-related factor (Nrf2) signaling pathway used by 15d-PGJ2 was assessed, and these studies revealed for the first time that 15d-PGJ2 and 15d-PGJ2-G similarly activated Nrf2 signaling as well as transcription of target genes of this pathway. Our study challenges previous claims regarding the ability of MAGL to catalyze PG-G hydrolysis and extend the MAGL substrate profile beyond the classic monoacylglycerols.

Piperazine and piperidine carboxamides and carbamates as inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), are potential targets for various therapeutic applications. In this paper, we present more extensively the results of our previous work on piperazine and piperidine carboxamides and carbamates as FAAH and MAGL inhibitors. The best compounds of these series function as potent and selective MAGL/FAAH inhibitors or as dual FAAH/MAGL inhibitors at nanomolar concentrations. This study revealed that MAGL inhibitors should comprise leaving-groups with a conjugate acid pKa of 8-10, while diverse leaving groups are tolerated for FAAH inhibitors.

5-Aminothiazoles Reveal a New Ligand-Binding Site on Prolyl Oligopeptidase Which is Important for Modulation of Its Protein-Protein Interaction-Derived Functions.

A series of novel 5-aminothiazole-based ligands for prolyl oligopeptidase (PREP) comprise selective, potent modulators of the protein-protein interaction (PPI)-mediated functions of PREP, although they are only weak inhibitors of the proteolytic activity of PREP. The disconnected structure-activity relationships are significantly more pronounced for the 5-aminothiazole-based ligands than for the earlier published 5-aminooxazole-based ligands. Furthermore, the stability of the 5-aminothiazole scaffold allowed exploration of wider substitution patterns than that was possible with the 5-aminooxazole scaffold. The intriguing structure-activity relationships for the modulation of the proteolytic activity and PPI-derived functions of PREP were elaborated by presenting a new binding site for PPI modulating PREP ligands, which was initially discovered using molecular modeling and later confirmed through point mutation studies. Our results suggest that this new binding site on PREP is clearly more important than the active site of PREP for the modulation of its PPI-mediated functions.

Utilizing PET and MALDI Imaging for Discovery of a Targeted Probe for Brain Endocannabinoid α/ß-Hydrolase Domain 6 (ABHD6).

Multimodal imaging provides rich biological information, which can be exploited to study drug activity, disease associated phenotypes, and pharmacological responses. Here we show discovery and validation of a new probe targeting the endocannabinoid α/ß-hydrolase domain 6 (ABHD6) enzyme by utilizing positron emission tomography (PET) and matrix-assisted laser desorption/ionization (MALDI) imaging. [18F]JZP-MA-11 as the first PET ligand for in vivo imaging of the ABHD6 is reported and specific uptake in ABHD6-rich peripheral tissues and major brain regions was demonstrated using PET. A proof-of-concept study in nonhuman primate confirmed brain uptake. In vivo pharmacological response upon ABHD6 inhibition was observed by MALDI imaging. These synergistic imaging efforts used to identify biological information cannot be obtained by a single imaging modality and hold promise for improving the understanding of ABHD6-mediated endocannabinoid metabolism in peripheral and central nervous system disorders.

A prolyl oligopeptidase inhibitor reduces tau pathology in cellular models and in mice with tauopathy.

Tauopathies are neurodegenerative diseases that are characterized by accumulation of hyperphosphorylated tau protein, higher-order aggregates, and tau filaments. Protein phosphatase 2A (PP2A) is a major tau dephosphorylating phosphatase, and a decrease in its activity has been demonstrated in tauopathies, including Alzheimer's disease. Prolyl oligopeptidase is a serine protease that is associated with neurodegeneration, and its inhibition normalizes PP2A activity without toxicity under pathological conditions. Here, we assessed whether prolyl oligopeptidase inhibition could protect against tau-mediated toxicity in cellular models in vitro and in the PS19 transgenic mouse model of tauopathy carrying the human tau-P301S mutation. We show that inhibition of prolyl oligopeptidase with the inhibitor KYP-2047 reduced tau aggregation in tau-transfected HEK-293 cells and N2A cells as well as in human iPSC-derived neurons carrying either the P301L or tau-A152T mutation. Treatment with KYP-2047 resulted in increased PP2A activity and activation of autophagic flux in HEK-293 cells and N2A cells and in patient-derived iNeurons, as indicated by changes in autophagosome and autophagy receptor markers; this contributed to clearance of insoluble tau. Furthermore, treatment of PS19 transgenic mice for 1 month with KYP-2047 reduced tau burden in the brain and cerebrospinal fluid and slowed cognitive decline according to several behavioral tests. In addition, a reduction in an oxidative stress marker was seen in mouse brains after KYP-2047 treatment. This study suggests that inhibition of prolyl oligopeptidase could help to ameliorate tau-dependent neurodegeneration.

Biochemical and pharmacological characterization of human α/ß-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12).

In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins α/ß-hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)- and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors.

Metabolomics of Synovial Fluid and Infrapatellar Fat Pad in Patients with Osteoarthritis or Rheumatoid Arthritis.

Osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA) are inflammatory joint diseases with complex and insufficiently understood pathogeneses. Our objective was to characterize the metabolic fingerprints of synovial fluid (SF) and its adjacent infrapatellar fat pad (IFP) obtained during the same surgical operation from OA and RA knees. Non-targeted metabolite profiling was performed for 5 non-inflammatory trauma controls, 10 primary OA (pOA) patients, and 10 seropositive RA patients with high-resolution mass spectrometry-based techniques, and metabolites were matched with known metabolite identities. Groupwise differences in metabolic features were analyzed with the univariate Welch's t-test and the multivariate linear discriminant analysis (LDA) and principal component analysis (PCA). Significant discrimination of metabolite profiles was discovered by LDA for both SF and IFP and by PCA for SF based on diagnosis. In addition to a few drug-derived substances, there were 16 and 13 identified metabolites with significant differences between the diagnoses in SF and IFP, respectively. The pathways downregulated in RA included androgen, bile acid, amino acid, and histamine metabolism, and those upregulated included biotin metabolism in pOA and purine metabolism in RA and pOA. The RA-induced downregulation of androgen and bile acid metabolism was observed for both SF and IFP. The levels of 11 lipid metabolites, mostly glycerophospholipids and fatty acid amides, were also altered by these inflammatory conditions. The identified metabolic pathways could be utilized in the future to deepen our understanding of the pathogeneses of OA and RA and to develop not only biomarkers for their early diagnosis but also therapeutic targets.

Characterization of binding properties of monoglyceride lipase inhibitors by a versatile fluorescence-based technique.

Monoglyceride lipase (MGL) is a serine hydrolase that terminates the signaling of the primary endocannabinoid, 2-arachidonoyl glycerol (2-AG). Versatile high-throughput screening methods allowing the testing of MGL inhibitors are rare, thereby limiting the development and analysis of novel inhibitors. Here we describe an improved fluorescence-based technique that is capable of determining time- and dose-dependent inhibition of MGL with one or multiple binding sites and, at the same time, is capable of revealing the reversibility of inhibitor binding in a simple kinetic assay format. Known reference compounds as well as novel inhibitors, such as JZL184 and CAY10499, were evaluated for their MGL-binding properties and potency.

Regulating membrane lipid levels at the synapse by small-molecule inhibitors of monoacylglycerol lipase: new developments in therapeutic and PET imaging applications.

Monoacylglycerol lipase (MAGL) is a major endocannabinoid hydrolyzing enzyme and can be regulated to control endogenous lipid levels in the brain. This review highlights the pharmacological roles and in vivo PET imaging of MAGL in brain.

Evaluation of FASN inhibitors by a versatile toolkit reveals differences in pharmacology between human and rodent FASN preparations and in antiproliferative efficacy in vitro vs. in situ in human cancer cells.

De novo synthesis of fatty acids is essential to maintain intensive proliferation of cancer cells. Unlike normal cells that utilize food-derived circulating lipids for their fuel, cancer cells rely on heightened lipogenesis irrespective of exogenous lipid availability. Overexpression and activity of the multidomain enzyme fatty acid synthase (FASN) is crucial in supplying palmitate for protumorigenic activity. Therefore, FASN has been proposed as an attractive target for drug development. As an effort to set up an effective toolkit to study FASN inhibitors in human and rodent tissues, we validated activity-based protein profiling (ABPP) as a viable approach to unveil inhibitors targeting FASN thioesterase domain (FASN-TE). ABPP was combined with multi-well plate-assays designed for classical substrate-based FASN activity analysis together with powerful monitoring of cancer cell proliferation using IncuCyte® Live Cell Analyzing System. FASN-TE inhibitors were identified by competitive ABPP using HEK293 cell lysates in a screen of in-house compounds (200+) designed to target serine hydrolase (SH) family. The identified compounds were tested for their inhibitor potencies in vitro using a substrate-based activity assay monitoring FASN-dependent NADPH consumption in LNCaP prostate cancer cell preparation, in parallel with selected reference inhibitors, including orlistat (THL), GSK2194069, GSK837149A, platensimycin and BI-99179. LNCaP lysate supernatant was validated as a reliable native preparation to monitor FASN-dependent NADPH consumption as opposed to human glioma GAMG cells, whereas FASN enrichment was a prerequisite for accurate assays. While inhibitor pharmacology was identical between human prostate and glioma cancer cell FASN preparations, notable differences were revealed between human and rodent FASN preparations, especially for inhibitors targeting FASN-TE. ABPP combined with substrate-based assays facilitated identification of pan thiol-reactive inhibitor scaffolds, exemplified by the 1,2,4-thiadiazole moiety. Finally, selected compounds were evaluated for their antiproliferative efficacy in situ using GAMG cells. These studies revealed that while the tested compounds acted as potent FASN inhibitors in vitro, only a few showed antiproliferative efficacy in situ. To conclude, we describe a versatile toolkit to study FASN inhibitors in vitro and in situ using human cancer cells and reveal dramatic pharmacological differences between human and rodent FASN preparations.

Discovery of 12-Thiazole Abietanes as Selective Inhibitors of the Human Metabolic Serine Hydrolase hABHD16A.

Screening of an in-house library of compounds identified 12-thiazole abietanes as a new class of reversible inhibitors of the human metabolic serine hydrolase. Further optimization of the first hit compound lead to the 2-methylthiazole derivative 18, with an IC50 value of 3.4 ± 0.2 µM and promising selectivity. ABHD16A has been highlighted as a new target for inflammation-mediated pain, although selective inhibitors of hABHD16A (human ABHD16A) have not yet been reported. Our study presents abietane-type diterpenoids as an attractive starting point for the design of selective ABHD16A inhibitors, which will contribute toward understanding the significance of hABHD16A inhibition in vivo.

Design, synthesis, and biological evaluation of 2,4-dihydropyrano[2,3-c]pyrazole derivatives as autotaxin inhibitors.

Inhibition of Autotaxin (ATX) is a potential treatment strategy for several diseases, including tumors with elevated ATX-lysophosphatidic acid (LPA) signaling. Combining structure-based virtual screening together with hen egg-white Autotaxin (ewATX) activity assays enabled the discovery of novel small-molecule ATX inhibitors with a 2,4-dihydropyrano[2,3-c]pyrazole scaffold. These compounds are suggested to bind to the lipophilic pocket, leaving the active site unrestrained. Our most potent compound, (S)-6-amino-4-(3,4-dichlorophenyl)-3-(4-[(4-fluorobenzyl)oxy]phenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile [(S)-25], inhibited human ATX (hATX) with an IC50-value of 134nM. It also blocked ATX-evoked but not LPA-mediated A2058 melanoma cell migration. Noteworthy, molecular modeling correctly predicted the biologically active enantiomer of 2,4-dihydropyrano[2,3-c]pyrazoles, as verified by compound crystallization and activity assays. Our study established the ewATX activity assay as a valid and affordable tool in ATX inhibitor discovery. Overall, our study offers novel insights and approaches into design of novel ATX inhibitors targeting the hydrophobic pocket instead of the active site.