Title: Comprehensive in-silico analysis of deleterious SNPs in APOC2 and APOA5 and their differential expression in cancer and cardiovascular diseases conditions Deng H, Li J, Shah AA, Ge L, Ouyang W Ref: Genomics, 115:110567, 2023 : PubMed
Genetic variations in APOC2 and APOA5 genes involve activating lipoprotein lipase (LPL), responsible for the hydrolysis of triglycerides (TG) in blood and whose impaired functions affect the TG metabolism and are associated with metabolic diseases. In this study, we investigate the biological significance of genetic variations at the DNA sequence and structural level using various computational tools. Subsequently, 8 (APOC2) and 17 (APOA5) non-synonymous SNPs (nsSNPs) were identified as high-confidence deleterious SNPs based on the effects of the mutations on protein conservation, stability, and solvent accessibility. Furthermore, based on our docking results, the interaction of native and mutant forms of the corresponding proteins with LPL depicts differences in root mean square deviation (RMSD), and binding affinities suggest that these mutations may affect their function. Furthermore, in vivo, and in vitro studies have shown that differential expression of these genes in disease conditions due to the influence of nsSNPs abundance may be associated with promoting the development of cancer and cardiovascular diseases. Preliminary screening using computational methods can be a helpful start in understanding the effects of mutations in APOC2 and APOA5 on lipid metabolism; however, further wet-lab experiments would further strengthen the conclusions drawn from the computational study.
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.
Title: Nanoclusterzyme for Dual Colorimetric Sensings: A Case Study on [Au(14) (Dppp)(5) I(4) ](2) Zhao H, You Q, Zhu W, Li J, Deng H, Li MB, Zhao Y, Wu Z Ref: Small, :e2207936, 2023 : PubMed
The enzymatic activity of atomically precise metal nanoclusters has recently been recognized; however, the number of nanoclusterzymes is very small. Besides, the applications of nanoclusterzyme wait to be explored. Herein, a novel nanoclusterzyme is synthesized and its structure is majorly resolved by single-crystal X-ray diffraction and mass spectrometry, which reveal that the nanocluster consists of an Au(13) icosahedron capped by an exterior shell including four I, three Dppp (1,3-bis(diphenylphosphino) propane) ligands, and a rarely reported Dppp-Au-Dppp handle staple, which contributes a lot to the enzyme activity of [Au(14) (Dppp)(5) I(4) ](2+) nanocluster. The as-obtained nanocluster can catalyze oxygen to O(2) (-) under visible light irradiation with a specific activity up to 0.182 U.mg(-1) and lead to the blue color of 3,3',5,5'-tetramethylbenzidine (TMB) in both solution and solid states. With the addition of acetylcholinesterase (AChE), the blue color of (Au(14) + TMB) solution system disappears due to the nanoclusterzyme activity inhibition, but the further addition of organophosphorus pesticides (OPs) into the above mixture can restore the nanoclusterzyme and recover the blue color. Based on the color turn-off and on, the various nanoclusterzyme-containing systems are used to colorimetrically sense AChE and OPs with the detection limits reaching 0.04 mU.mL(-1) and 0.02 ng.mL(-1) , respectively.
Title: Discovering monoacylglycerol lipase inhibitors by a combination of fluorogenic substrate assay and activity-based protein profiling Deng H, Zhang Q, Lei Q, Yang N, Yang K, Jiang J, Yu Z Ref: Front Pharmacol, 13:941522, 2022 : PubMed
The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly metabolized by monoacylglycerol lipase (MAGL) in the brain. Selective inhibitors of MAGL provide valuable insights into the role of 2-AG in a variety of (patho)physiological processes and are potential therapeutics for the treatment of diseases such as neurodegenerative disease and inflammation, pain, as well as cancer. Despite a number of MAGL inhibitors been reported, inhibitors with new chemotypes are still required. Here, we developed a substrate-based fluorescence assay by using a new fluorogenic probe AA-HNA and successfully screened a focused library containing 320 natural organic compounds. Furthermore, we applied activity-based protein profiling (ABPP) as an orthogonal method to confirm the inhibitory activity against MAGL in the primary substrate-based screening. Our investigations culminated in the identification of two major compound classes, including quinoid diterpene (23, cryptotanshinone) and beta-carbolines (82 and 93, cis- and trans-isomers), with significant potency towards MAGL and good selectivity over other 2-AG hydrolases (ABHD6 and ABHD12). Moreover, these compounds also showed antiproliferative activities against multiple cancer cells, including A431, H1975, B16-F10, OVCAR-3, and A549. Remarkably, 23 achieved complete inhibition towards endogenous MAGL in most cancer cells determined by ABPP. Our results demonstrate the potential utility of the substrate-based fluorescence assay in combination with ABPP for rapidly discovering MAGL inhibitors, as well as providing an effective approach to identify potential targets for compounds with significant biological activities.
BACKGROUND: GPIHBP1, a glycolipid-anchored protein of capillary endothelial cells, is a crucial partner for lipoprotein lipase (LPL) in plasma triglyceride metabolism. GPIHBP1 autoantibodies block LPL binding to GPIHBP1 and lead to severe hypertriglyceridemia (HTG) and HTG-induced acute pancreatitis (HTG-AP). We sought to define the incidence of GPIHBP1 autoantibodies in patients with HTG-AP. OBJECTIVE: We determined the incidence of GPIHBP1 autoantibody in HTG-AP patients, and compared the clinical features and long-term outcomes between GPIHBP1 autoantibody-positive and negative groups. METHODS: An enzyme-linked immunosorbent assay was used to screen for GPIHBP1 autoantibody in 116 HTG-AP patients hospitalized from Jan 1, 2015 to Aug 31, 2019. All patients were followed up for 24 months. The primary outcome was the recurrence rate of HTG-AP during the two-year follow-up period. The incidence of recurrent episodes was analyzed by the Kaplan-Meier method and multivariable Cox regression was used to identify risk factors. RESULTS: GPIHBP1 autoantibodies were present in 17 of 116 study patients (14.66%). The 2-year recurrence rate of HTG-AP was much higher in the GPIHBP1 autoantibody-positive group (35%, 6 in 17) than in the negative group (4%, 4 in 99). The multivariable Cox regression analysis showed that GPIHBP1 autoantibody was an independent risk factor for HTG-AP recurrence in two years. CONCLUSIONS: The presence of GPIHBP1 autoantibody is common in patients with HTG-AP, and is an independent risk factor for two-year recurrence of HTG-AP.
The aim of this study was to investigate the effects of different doses of selenium (Se) on oxidative damage and neurotransmitter-related parameters in arsenic (As)-induced broiler brain tissue damage. Two hundred 1-day-old avian broilers were randomly divided into five groups and fed the following diets: control group (As 0.1 mg/kg + Se 0.2 mg/kg), As group (As 3 mg/kg + Se 0.2 mg/kg), low-Se group (As 3 mg/kg + Se 5 mg/kg), medium-Se group (As 3 mg/kg + Se 10 mg/kg), and high-Se group (As 3 mg/kg + Se 15 mg/kg). Glutathione (GSH), glutathione peroxidase (GSH-PX), nitric oxide (NO), nitric oxide synthase (NOS) activity, glutamate (Glu) concentration, glutamine synthetase (GS) activity, acetylcholinesterase (TchE) activity, and the apoptosis rate of brain cells were measured. The results showed that 3 mg/kg dietary As could induce oxidative damage and neurotransmitter disorder of brain tissue, increase the apoptosis rate of brain cells and cause damage to brain tissue, decrease activities of GSH and GSH-PX, decrease the contents of NO, decrease the activities of iNOS and tNOS, increase contents of Glu, and decrease activities of Gs and TchE. Compared with the As group, the Se addition of the low-Se and medium-Se groups protected against As-induced oxidative damage, neurotransmitter disorders, and the apoptosis rate of brain cells, with the addition of 10 mg/kg Se having the best effect. However, 15 mg/kg Se not only did not produce a protective effect against As damage but actually caused similar or severe damage.
Title: Monoacylglycerol lipase inhibitors: modulators for lipid metabolism in cancer malignancy, neurological and metabolic disorders Deng H, Li W Ref: Acta Pharm Sin B, 10:582, 2020 : PubMed
Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays a crucial role catalysing the hydrolysis of monoglycerides into glycerol and fatty acids. It links the endocannabinoid and eicosanoid systems together by degradation of the abundant endocannabinoid 2-arachidaoylglycerol into arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. MAGL inhibitors have been considered as important agents in many therapeutic fields, including anti-nociceptive, anxiolytic, anti-inflammatory, and even anti-cancer. Currently, ABX-1431, a first-in-class inhibitor of MAGL, is entering clinical phase 2 studies for neurological disorders and other diseases. This review summarizes the diverse (patho)physiological roles of MAGL and will provide an overview on the development of MAGL inhibitors. Although a large number of MAGL inhibitors have been reported, novel inhibitors are still required, particularly reversible ones.
Title: Therapeutic potential of targeting alpha/beta-Hydrolase domain-containing 6 (ABHD6) Deng H, Li W Ref: Eur Journal of Medicinal Chemistry, 198:112353, 2020 : PubMed
alpha/beta-Hydrolase domain 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes monoacylglycerol (MAG) lipids, particularly the endogenous cannabinoid 2-arachidonoylglycerol (2-AG), in both central and peripheral tissues. ABHD6 and its substrates have been shown to be involved in the modulation of various (patho)physiological processes, including neurotransmission, inflammation, insulin secretion, adipose browning, food intake, autoimmune disorders, as well as neurological and metabolic diseases, making this enzyme a promising therapeutic target to treat several diseases. This review will focus on the molecular mechanism, biological functions and pathological roles of ABHD6, as well as recent efforts to develop ABHD6 inhibitors, providing a strong basis for the development of small molecules by targeting ABHD6 to treat diverse diseases.
Title: Characterization of a novel carboxylesterase from Bacillus velezensis SYBC H47 and its application in degradation of phthalate esters Huang L, Meng D, Tian Q, Yang S, Deng H, Guan Z, Cai Y, Liao X Ref: J Biosci Bioeng, 129:588, 2020 : PubMed
Recently, residual plasticizer phthalate esters (PAEs) in the different environments pose a serious health threat to humans and mammals. Biodegradation has been considered a promising and eco-friendly way to eliminate PAEs. In this study, a gene (baces04) encoding the novel PAEs hydrolase, carboxylesterase (BaCEs04), was screened from the genome of Bacillus velezensis SYBC H47 via bioinformatics analysis. Then, baces04 was cloned and expressed in Escherichia coli BL21 (DE3). BaCEs04 belonged to the esterase family VI. It contained a conserved domain (Gly159-His160-Ser161-Leu162-Gly163) and a typical serine hydrolase catalytic site (Ser161-Asp204-His261). The characterization of BaCEs04 showed that the activity was optimal at 60 degrees C and pH 7.5. This enzyme also displayed high resistance to metal ions, organic solvents, and detergents. After treatment with BaCEs04 for 5 h, the degradation ratio of four different 1 mM PAEs, including dimethyl phthalate, diethyl phthalate, dipropyl phthalate, and dibutyl phthalate, was 32.4%, 50.5%, 77.9%, and 86.8%, respectively. The degradation products of four PAEs were identified as their corresponding monoalkyl phthalates. This is the first report that family VI esterase displaying PAE-hydrolysis activity. This study also proved that BaCEs04 could be used as an ideal candidate for the application in bioremediation and industry.
A novel feruloyl esterase (BpFae12) with rosmarinic acid (RA) hydrolysis activity was isolated from Bacillus pumilus W3 and expressed in Escherichia coli BL21 (DE3). With RA as a substrate, the optimal pH and temperature of BpFae12 were pH 8.0 and 50 degreesC, respectively. The specific enzyme activity was 12.8 U.mg(-1). BpFae12 showed the highest activity and substrate affinity toward RA (V(max) of 13.13 U.mg(-1), K(m) of 0.41 mM). Moreover, it also presented strong hydrolysis performance against chlorogenic acid (190.17 U.mg(-1)). RA was effectively Hydrolyzed into more bioactive caffeic acid and 3,4-dihydroxyphenyllactic acid by BpFae12, which have potential applications in the food industry.
Objective: DBPR108, a novel dipeptidyl-peptidase-4 inhibitor, has shown great antihyperglycemic effect in animal models. This study was to evaluate the efficacy and safety of DBPR108 monotherapy in type 2 diabetes mellitus (T2DM).Methods: This was a 12-week, double-blind, placebo-controlled phase II clinical trial. The newly diagnosed or inadequately controlled untreated T2DM patients were randomized to receive 50, 100, 200 mg DBPR108 or placebo in a ratio of 1:1:1:1. The primary efficacy outcome was HbA1c change from baseline to week 12. Relevant secondary efficacy parameters and safety were assessed. The clinical trial registration is NCT04124484.Results: Overall, 271 of the 276 randomized patients, who received 50 mg (n = 68), 100 mg (n = 67), 200 mg (n = 69) DBPR108 or placebo (n = 67), were included in full analysis set. At week 12, HbA1c change from baseline was -0.04 +/- 0.77 in placebo group, -0.51 +/- 0.71, -0.75 +/- 0.73, and -0.57 +/- 0.78 (%, p < .001 vs. placebo) in 50, 100, and 200 mg DBPR108 groups, respectively. Since week 4, DBPR108 monotherapy resulted in significant improvements in secondary efficacy parameters. At end of 12-week treatment, the goal of HbA1c >=7% was achieved in 29.85, 58.82, 55.22, and 47.83% of the patients in placebo, 50, 100, and 200 mg DBPR108 groups, respectively. The incidence of adverse events did not show significant difference between DBPR108 and placebo except mild hypoglycemia in DBPR108 200 mg group.Conclusions: The study results support DBPR108 100 mg once daily as the primary dosing regimen for T2DM patients in phase III development program.
Title: Expression and characterisation of feruloyl esterases from Lactobacillus fermentum JN248 and release of ferulic acid from wheat bran Deng H, Jia P, Jiang J, Bai Y, Fan TP, Zheng X, Cai Y Ref: Int J Biol Macromol, 138:272, 2019 : PubMed
Genes encoding six feruloyl esterases (FAEs; lbff0997, lbff0272, lbff1432, lbff1695, lbff1849, lbff0153) from Lactobacillus fermentum JN248 were cloned, overexpressed and characterised. Maximum enzyme activity was observed at 35 degrees C for recombinant FAEs LFFae0997, LFFae0272 and LFFae0153, at 30 degrees C for LFFae1695, and at 40 degrees C for LFFae1432and LFFae1849. For five of the enzymes, optimal activity was observed at pH7.0 or pH8.0, and high thermostability was measured up to 55 degrees C. By contrast, LFFae1432 lost less than 10.0% activity after incubation at 40 degrees C for 2h, and pH stability was highest between pH7.0 and pH9.0. In addition, LFFae1432 was the most robust esterase, with a higher affinity and hydrolytic activity against synthetic esters. The enzymes released ferulic acids (FAs) from de-starched wheat bran (DSWB), and 60.7% of the total alkali-extractable FAs were released when LFFae1432 was added alone, compared with less than 10% for the other enzymes. The amount of FAs released by FAEs increased when combined with xylanase. These FAEs could serve as promising biocatalysts for biodegradation, and LFFae1432 may hold promise for potential industrial applications.
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) alpha, alpha/beta-hydrolase domain-containing protein 4, alpha/beta-hydrolase domain-containing protein 6, alpha/beta-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL alpha. 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.
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.
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.
Inhibitors of diacylglycerol lipases and alpha,beta-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 DAGLalpha 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 DAGLalpha and ABHD6, as established using biochemical assays and competitive activity-based protein profiling on mouse brain extracts.
Title: Genetic analysis of the RIC3 gene in Han Chinese patients with Parkinson's disease He D, Hu P, Deng X, Song Z, Yuan L, Yuan X, Deng H Ref: Neuroscience Letters, 653:351, 2017 : PubMed
Parkinson's disease (PD) is the second-most common etiologically complex neurodegenerative disease. Genetic abnormalities are thought to play an important role in the development of PD. Recently, mutations in the resistance to inhibitors of cholinesterase 3 gene (RIC3) have been reported to cause autosomal-dominant PD in Indian population. To determine whether RIC3 gene coding variant(s) are associated with PD in Han Chinese population, the RIC3 gene coding region in 218 mainland Han Chinese patients with PD and the identified variants in 242 normal controls were examined using direct sequencing analysis. Four known single nucleotide variants (c.354C>A, p.L118L, rs10839976; c.389G>A, p.C130Y, rs55990541; c.403C>T, p.P135S, rs73411617; and c.1054G>A, p.D352N, rs11826236) were identified in the RIC3 gene coding region. No significant differences were observed in either genotypic or allelic distributions between the PD patients and the normal controls (all P>0.05) for these four variants. Haplotype analysis showed that the presence of haplotype A-G-C-G (rs10839976-rs55990541-rs73411617-rs11826236) was associated with a 0.764-fold decreased risk (P=0.049, OR=0.764, 95% CI=0.585-0.999) for PD, whereas the presence of haplotype C-A-C-A was associated with a 2.143-fold increased risk (P=0.039, OR=2.143, 95% CI=1.023-4.488) for PD. The findings indicate that four variants: rs10839976, rs55990541, rs73411617 and rs11826236 in the RIC3 gene coding region may play little or no role in the development of PD. Two RIC3 gene haplotypes of four variants: A-G-C-G, and C-A-C-A might relate to either protection against or increased susceptibility to PD in the Han Chinese population, respectively.
Title: miR27a promotes proliferation, migration, and invasion of colorectal cancer by targeting FAM172A and acts as a diagnostic and prognostic biomarker Liu W, Qian K, Wei X, Deng H, Zhao B, Chen Q, Zhang J, Liu H Ref: Oncol Rep, 37:3554, 2017 : PubMed
Accumulating evidence shows that mircroRNAs (miRNAs) play a crucial role in the development of colorectal cancer. In our previous study, FAM172A was demonstrated to be a novel tumor suppressor gene in CRC. Therefore, the aim of the present study was to identify whether the miR27a could be a diagnostic and prognostic marker and the regulatory relationships between miR27a and FAM172A. We demonstrated high levels of miR27a expression in tissues of patients with CRC as well as in CRC cell lines. There was a positive correlation between the levels of miR27a and the poor overall survival of patients with CRC. Furthermore, elevated levels of miR27a expression were associated with TNM stage and distant metastasis. Increased expression or inhibition of miR27a promoted or inhibited the metastasis of CRC cell lines, respectively. Moreover, we showed that miR27a directly targets the 3'-untranslated region of FAM172A mRNA by using a dual-luciferase assay. Increased or decreased expression of FAM172A expression was observed when miR27a expression was inhibited or elevated in the CRC cells, respectively. In summary, our study showed that miR27a expression is a diagnostic and prognostic marker and correlates with overall survival of patients with CRC. Therefore, it may be a therapeutic approach for preventing metastasis of CRC to inhibit expression of miR27a or increase expression of FAM172A.
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.
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.
Diacylglycerol lipases (DAGLalpha and DAGLbeta) 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 DAGLalpha 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.
Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The alpha/beta hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14-D3-ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone.
Orchids make up about 10% of all seed plant species, have great economical value, and are of specific scientific interest because of their renowned flowers and ecological adaptations. Here, we report the first draft genome sequence of a lithophytic orchid, Dendrobium catenatum. We predict 28,910 protein-coding genes, and find evidence of a whole genome duplication shared with Phalaenopsis. We observed the expansion of many resistance-related genes, suggesting a powerful immune system responsible for adaptation to a wide range of ecological niches. We also discovered extensive duplication of genes involved in glucomannan synthase activities, likely related to the synthesis of medicinal polysaccharides. Expansion of MADS-box gene clades ANR1, StMADS11, and MIKC(*), involved in the regulation of development and growth, suggests that these expansions are associated with the astonishing diversity of plant architecture in the genus Dendrobium. On the contrary, members of the type I MADS box gene family are missing, which might explain the loss of the endospermous seed. The findings reported here will be important for future studies into polysaccharide synthesis, adaptations to diverse environments and flower architecture of Orchidaceae.
Diacylglycerol lipase (DAGL)-alpha and -beta are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific beta-lactone-based probes led to the discovery of alpha-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-alpha/DAGL-beta inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.
Title: Resurfaced fluorescent protein as a sensing platform for label-free detection of copper(II) ion and acetylcholinesterase activity Lei C, Wang Z, Nie Z, Deng H, Hu H, Huang Y, Yao S Ref: Analytical Chemistry, 87:1974, 2015 : PubMed
Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H39GFP is a new variant of green fluorescent protein (GFP) containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H39GFP as the signal reporter, a label-free fluorometric sensor for Cu(2+) sensing was developed based on the unique multivalent metal ion-binding property of H39GFP and fluorescence quenching effect of Cu(2+) by electron transfer. The high affinity of H39GFP with Cu(2+) (Kd, 16.2 nM) leads to rapid detection of Cu(2+) in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H39GFP/Cu(2+) complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu(2+) and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL(-1)) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H39GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms.
Title: Soluble epoxide hydrolase inhibition ameliorates proteinuria-induced epithelial-mesenchymal transition by regulating the PI3K-Akt-GSK-3beta signaling pathway Liang Y, Jing Z, Deng H, Li Z, Zhuang Z, Wang S, Wang Y Ref: Biochemical & Biophysical Research Communications, 463:70, 2015 : PubMed
Soluble epoxide hydrolase (sEH) plays an essential role in chronic kidney disease by hydrolyzing renoprotective epoxyeicosatrienoic acids to the corresponding inactive dihydroxyeicosatrienoic acids. However, there have been few mechanistic studies elucidating the role of sEH in epithelial-mesenchymal transition (EMT). The present study investigated, in vitro and in vivo, the role of sEH in proteinuria-induced renal tubular EMT and the underlying signaling pathway. We report that urinary protein (UP) induced EMT in cultured NRK-52E cells, as evidenced by decreased E-cadherin expression, increased alpha-smooth muscle actin (alpha-SMA) expression, and the morphological conversion to a myofibroblast-like phenotype. UP incubation also resulted in upregulated sEH, activated phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling and increased phosphorylated glycogen synthase kinase-3beta (GSK-3beta). The PI3K inhibitor LY-294002 inhibited phosphorylation of Akt and GSK-3beta as well as blocking EMT. Importantly, pharmacological inhibition of sEH with 12-(3-adamantan-1-yl- ureido)-dodecanoic acid (AUDA) markedly suppressed PI3K-Akt activation and GSK-3beta phosphorylation. EMT associated E-cadherin suppression, alpha-SMA elevation and phenotypic transition were also attenuated by AUDA. Furthermore, in rats with chronic proteinuric renal disease, AUDA treatment inhibited PI3K-Akt activation and GSK-3beta phosphorylation, while attenuating levels of EMT markers. Overall, our findings suggest that sEH inhibition ameliorates proteinuria-induced renal tubular EMT by regulating the PI3K-Akt-GSK-3beta signaling pathway. Targeting sEH might be a potential strategy for the treatment of EMT and renal fibrosis.
A series of 5,6,7-trimethoxyflavone-6-chlorotacrine hybrids were designed, synthesized and evaluated as multifunctional agents for the treatment of Alzheimer's disease (AD). The results showed that the target compounds exhibited good acetylcholinesterase (AChE) inhibitory potencies, high selectivity toward AChE over butyrylcholinesterase (BCHE), potential antioxidant activities and significant inhibitory potencies of self-induced beta-amyloid peptide (Abeta) aggregation. In particular, compound 14c had the strongest AChE inhibitory activity with IC50 value of 12.8nM, potent inhibition of self-induced Abeta1-42 aggregation with inhibition ratio of 33.8% at 25muM. Moreover, compound 14c acted as an antioxidant, as well as a neuroprotectant. Furthermore, 14c could cross the blood-brain barrier (BBB) in vitro. The results showed that compound 14c might be a potential multifunctional candidate for the treatment of AD.
The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase alpha (DAGL-alpha) in the CNS. Selective inhibitors of DAGL-alpha will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-alpha, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-alpha activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-alpha activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-alpha. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-alpha inhibitors.
sn-1-Diacylglycerol lipase alpha (DAGL-alpha) is the main enzyme responsible for the production of the endocannabinoid 2-arachidonoylglycerol in the central nervous system. Glycine sulfonamides have recently been identified by a high throughput screening campaign as a novel class of inhibitors for this enzyme. Here, we report on the first structure-activity relationship study of glycine sulfonamide inhibitors and their brain membrane proteome-wide selectivity on serine hydrolases with activity-based protein profiling (ABPP). We found that (i) DAGL-alpha tolerates a variety of biaryl substituents, (ii) the sulfonamide is required for inducing a specific orientation of the 2,2-dimethylchroman substituent, and (iii) a carboxylic acid is essential for its activity. ABPP revealed that the sulfonamide glycine inhibitors have at least three off-targets, including alpha/beta-hydrolase domain 6 (ABHD6). Finally, we identified LEI-106 as a potent, dual DAGL-alpha/ABHD6 inhibitor, which makes this compound a potential lead for the discovery of new molecular therapies for diet-induced obesity and metabolic syndrome.
Title: Probing biochemical mechanisms of action of muscarinic M3 receptor antagonists with label-free whole cell assays Deng H, Wang C, Su M, Fang Y Ref: Analytical Chemistry, 84:8232, 2012 : PubMed
Binding kinetics of drugs is increasingly recognized to be important for their in vivo efficacy and safety profiles. However, little is known about the effect of drug binding kinetics on receptor signaling in native cells. Here we used label-free whole cell dynamic mass redistribution (DMR) assays under persistent and duration-controlled stimulation conditions to investigate the influence of the binding kinetics of four antagonists on the signaling of endogenous muscarinic M3 receptor in native HT-29 cells. Results showed that DMR assays under different conditions differentiated the biochemical mechanisms of action of distinct M3 antagonists. When co-stimulated with acetylcholine, tiotropium, a relatively slow binding antagonist, was found to selectively block the late signaling of the receptor, suggesting that acetylcholine attains its binding equilibrium faster than tiotropium does, thereby still being able to initiate its rapid response until the antagonist draws up and fully blocks the signaling. Furthermore, DMR assays under microfluidics allowed estimation of the residence times of these antagonists acting at the receptor in native cells, which were found to be the determining factor for the blockage efficiency of M3 receptor signaling under duration-controlled conditions. This study demonstrates that DMR assays can be used to elucidate the functional consequence of kinetics-driven antagonist occupancy in native cells.
Mycobacterium tuberculosis is one of most prevalent pathogens in the world. Drug-resistant strains of this pathogen caused by the excessive use of antibiotics have long posed serious threats to public health worldwide. A broader picture of drug resistance mechanisms at the genomic level can be obtained only with large-scale comparative genomic methodology. Two closely related Beijing family isolates, one resistant to four first-line drugs (CCDC5180) and one sensitive to them (CCDC5079), were completely sequenced. These sequences will serve as valuable references for further drug resistance site identification studies and could be of great importance for developing drugs targeting these sites.
PC12 cells, in the presence of nerve growth factor (NGF), support replication of the mouse-derived scrapie strains 139A and ME7, with the former yielding 100-1000-fold higher levels of infectivity. Infectivity remained cell-associated and cells did not show any gross morphological alterations, although changes were observed by electron microscopy in the form of an increased number of lipid droplets in 139A-infected cultures. Analysis of phospholipid metabolism in 139A infected cells indicated that scrapie replication did not change the inositol phosphate levels, but did stimulate phosphoinositide synthesis. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Since scrapie-infected cultures did not exhibit cell death or any gross changes, any scrapie-induced effects would probably be manifested in nonvital cellular functions. When compared to controls, infection with the 139A scrapie strain resulted in decreased activity of the cholinergic pathway-related enzymes, as well as the GABA synthetic pathway; however, the adrenergic pathway was unaffected by scrapie infection. The effects of the 139A scrapie strain on the cholinergic system appeared to be dose-dependent and were first detected prior to the detection of scrapie agent replication in these cells. No neurotransmitter-related enzymatic changes were detected in 263K- or 139R-infected PC12 cells. The enzymatic changes observed in ME7-infected PC12 cells and in Chandler agent-infected mouse neuroblastoma cells suggest that the significant changes in neurotransmitter levels in cultures exhibiting low infectivity titers must involve factors other than, but not excluding, replication of the agent. The role of additional factors is also suggested in studies of protein kinase C activity in 139A- and 139R-infected PC12 cells. These studies emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and, in addition, support the concept of variation among scrapie strains.
