A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production

Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetyl-CoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.     

Enhancement of de novo fatty acid biosynthesis in hepatic cell line Huh7 expressing hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays important roles in the pathogeneses of liver steatosis as well as hepatocellular carcinomas due to HCV infection. In this study, we examined de novo fatty acid biosynthesis in hepatic cell line Huh7 cells expressing HCV core protein. The rate of metabolic labeling of cellular fatty acids with [(3)H]acetate in core-expressing (Uc39-6) cells was ca. 1.5-fold higher than that in non-expressing (Uc321) cells. The enzyme activities responsible for fatty acid biosynthesis were assayed in vitro. Cytosolic acetyl-CoA carboxylase activity in Uc39-6 cells was ca. 1.6-fold higher than that in Uc321 cells. On the other hand, cytosolic fatty acid synthase activity in Uc39-6 cells was only slightly higher than that in Uc321 cells. Immunoblot analysis of acetyl-CoA carboxylase 1 (ACC1), which is a rate-limiting enzyme for fatty acid biosynthesis, revealed a higher expression level of the protein in Uc39-6 cells than in Uc321 cells. The ACC1 mRNA content in Uc39-6 cells was 1.4-fold higher than that in Uc321 cells. These results strongly suggest that enhancement of fatty acid biosynthesis in core-expressing cells is caused by increased expression of fatty acid biosynthetic enzymes, especially ACC1. Up-regulation of de novo fatty acid biosynthesis by HCV core protein may affect cellular lipid metabolism, resulting in neutral lipid accumulation in HCV-infected cells.     

Inhibitors of mammalian acetyl-CoA carboxylase

Inhibition of acetyl-CoA carboxylase (ACC), with its resultant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation, has the potential to favorably affect, in a concerted manner, a multitude of the cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome. Studies in ACC2 knockout mice and in experimental animals treated with isozyme-specific antisense oligonucleotides or with isozyme-nonselective ACC inhibitors have demonstrated the potential for treating metabolic syndrome through this modality. Co-crystallization of the biotin carboxylase and carboxyltransferase domains of eukaryotic ACC in the presence of substrates and inhibitors has revealed characteristics of the catalytic center that can be exploited in drug discovery. A variety of structurally diverse, mechanistically distinct classes of ACC inhibitors have been disclosed in the scientific and patent literature. Isozyme-nonselective ACC inhibitors may provide the optimal therapeutic potential. However, demonstration of the full potential of isozyme-selective inhibitors, once identified, should reveal advantages and liabilities associated with single isozyme inhibition.     

High-throughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogeneous assay for protein kinases

Protein kinases are one of the most important target classes in high-throughput screening today. The use of generic assay technologies facilitates assay development for new targets and decreases the time needed for implementation of assays in robotic screening. For tyrosine kinases, several generic assay technology platforms are available. These technologies make use of high-affinity antibodies that discriminate between phosphorylated tyrosines and non-phosphorylated tyrosines. Similar generic antibodies specific for phosphoserine or phosphothreonine are lacking. Recently, a non-antibody-based fluorescence polarization assay for protein kinases has become available, called IMAP (Molecular Devices, Sunnyvale, CA). In this assay, a fluorescently labeled peptide substrate that is phosphorylated by kinase is captured on metal-derivatized nanoparticles. We have evaluated IMAP in high-throughput screening, and compared this technology with a competition fluorescence polarization immunoassay based on an antibody specific for a phosphorylated peptide substrate. A random collection of >250000 compounds was screened with the two assays. Fluorescent library compounds were identified by calculation of fluorescence intensity values from the screening data, and by assaying in the absence of fluorescent reagents. Fluorescence polarization artifacts were filtered out further by testing in an ELISA-based kinase assay. Our data show that IMAP is a robust technology for high-throughput screening of kinase targets, and suggest that it is less susceptible to fluorescence polarization artifacts than the competition fluorescence polarization immunoassay.     

Malonyl CoA, long chain fatty acyl CoA and insulin resistance in skeletal muscle

Malonyl CoA is an inhibitor of carnitine palmitoyl transferase 1 (CPT1), the enzyme that regulates the transfer of long chain fatty acyl CoA into mitochondria. By virtue of this effect, it is thought to play a key role in regulating fatty acid oxidation. Thus, when the supply of glucose to muscle is increased, malonyl CoA levels increase in keeping with a decreased need for fatty acid oxidation, and fatty acids are preferentially esterified to form diaglycerol and triglycerides. In contrast, during exercise, when the need for fatty acid oxidation is increased, malonyl CoA levels fall. Changes in glucose supply regulate malonyl CoA by modulating the concentration of cytosolic citrate, an allosteric activator of acetyl CoA carboxylase (ACC), the rate-limiting enzyme for malonyl CoA formation and a precursor of its substrate cytosolic acetyl CoA. Conversely, exercise lowers the concentration of malonyl CoA, by activating an AMP-activated protein kinase, which phosphorylates and inhibits ACC. A number of reports have linked sustained increases in the concentration of malonyl CoA in muscle to insulin resistance. In this paper, we review these reports, as well as the notion that changes in malonyl CoA contribute to the increases in long chain fatty acyl CoA, (LCFA CoA), diacylglycerol and triglyceride content and changes in protein kinase C activity and distribution observed in insulin-resistant muscle. We also review the implications of the malonyl CoA/LCFA CoA hypothesis to two other proposed mechanisms for insulin resistance, the glucose-fatty acid cycle and the hexosamine theory.     

Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1- methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors

A structurally novel acetyl-CoA carboxylase (ACC) inhibitor is identified from high-throughput screening. A preliminary structure-activity relationship study led to the discovery of potent dual ACC1/ACC2 and ACC2 selective inhibitors against human recombinant ACC1 and ACC2. Selective ACC2 inhibitors exhibited IC50<20 nM and >1000-fold selectivity against ACC1. (S)-Enantiomer 9p exhibited high ACC2 activity and lowered muscle malonyl-CoA dose-dependently in acute rodent studies, whereas (R)-enantiomer 9o was weak and had no effect on the malonyl-CoA level.     

Lipogenic enzymes as therapeutic targets for obesity and diabetes

Since storage of excess fat in peripheral tissues is a contributing factor leading to obesity and type II diabetes, many investigators are studying the key lipid metabolizing enzymes found in adipose tissue as drug targets to reduce excess fat. The availability of cultured cell lines and primary stem cells, preadipocyetes, and adipocytes has facilitated therapeutic approaches aimed at targeting fat storage. This includes developing inhibitors for enzymes regulating lipogenesis in these cells, such as acetyl-CoA carboxylase, fatty acid synthase, diacylgycerol acyl transferase, and stearoyl CoA desaturase. High level expression of each protein is often used to confirm stem cells have undergone adipogenesis. Inhibition of these enzymes often leads to reduced fat cell fat differentiation and lipid synthesis and may also contribute to increased fat oxidation and energy expenditure. This article reviews developments in pharmaceutical research on these enzymes, with particular emphasis on the role of the enzymes in adipose tissue metabolism.     

Targeting acetyl-CoA carboxylases: small molecular inhibitors and their therapeutic potential

Acetyl-CoA carboxylases (ACCs) play a rate-limiting role in fatty acid biosynthesis in plants, microbes, mammals and humans. ACCs have the activity of both biotin carboxylase (BC) and carboxyltransferase (CT), catalyzing carboxylation of Acetyl-CoA to malonyl-CoA. In the past years, ACCs have been used as targets for herbicides in agriculture and for drug discovery and development of human diseases, such as microbial infections, diabetes, obesity and cancer. A great number of small molecule ACC inhibitors have been developed, including natural and non-natural (artificial) products. These chemicals target BC reaction, CT reaction or ACC phosphorylation. This article provides a comprehensive review and updates of ACC inhibitors, with a focus on their therapeutic application in metabolic syndromes and malignant diseases. The patent status of common ACC inhibitors is discussed.     

Treating the metabolic syndrome: acetyl-CoA carboxylase inhibition

Metabolic syndrome is defined as a clustering of cardiovascular risk factors (abdominal obesity, hyperinsulinaemia, atherogenic dislipidaemia, hypertension, hypercoagulability) that together increase the risk of developing coronary heart disease and Type-2 diabetes. Inhibition of acetyl-CoA carboxylase (ACC), with its resultant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation, has the potential to favourably affect, in a concerted manner, a multitude of cardiovascular risk factors associated with metabolic syndrome. Studies in ACC2 knockout mice and in experimental animals treated with isozyme-nonselective ACC inhibitors have demonstrated the potential for treating metabolic syndrome through this modality. A variety of structurally diverse, mechanistically distinct classes of ACC inhibitors have been disclosed in the scientific and patent literature. Isozyme-nonselective ACC inhibitors may provide the optimal therapeutic potential for beneficially affecting metabolic syndrome. However, demonstration of the full potential of isozyme-selective inhibitors, once identified, should reveal advantages and liabilities associated with single isozyme inhibition. Whereas demonstrating clinical efficacy of an ACC inhibitor should be straightforward, the heterogeneity of the patient population and absence of established guidelines regarding approval end points for agents simultaneously affecting multiple aspects of metabolic syndrome will pose developmental challenges for initial market entries.     

细菌脂肪酸合成酶—抗菌药物的筛选靶点

目的对细菌脂肪酸生物合成所涉及到的酶及其抑制剂的筛选研究进行综述。方法通过查阅国内外相关文献21篇,对细菌脂肪酸生物合成所涉及到的酶及其抑制剂的研究现状进行整理和归纳。结果对细菌脂肪酸合成途径中几个重要的酶,即:β酮脂酰ACP还原酶(FabG)、羟脂酰ACP脱水酶(FabA/FabZ)、烯脂酰ACP还原酶(FabI/FabK/FabL)、β酮脂酰ACP合成酶(FabB/FabF/FabH)等酶的结构及其抑制剂进行了介绍。结论对参与细菌脂肪酸生物合成的酶的结构、催化机理和理化性质方面仍需深入研究,以期促进抗菌药物的筛选、设计工作。     

Acetyl-CoA carboxylase inhibition for the treatment of metabolic syndrome

Metabolic syndrome is defined as a clustering of cardiovascular risk factors (abdominal obesity, hyperinsulinemia, atherogenic dyslipidemia, hypertension and hypercoagulability) that together increase the risk of developing coronary heart disease and type 2 diabetes. Inhibition of acetyl-CoA carboxylase (ACC), which results in inhibition of fatty acid synthesis and stimulation of fatty acid oxidation, has the potential to favorably affect a multitude of cardiovascular risk factors associated with metabolic syndrome. ACC exists as two tissue-specific isozymes, ACC1 present in lipogenic tissues (liver and adipose) and ACC2 present in oxidative tissues (liver, heart and skeletal muscle). Studies in both ACC2 knockout mice and animals administered isozyme-nonselective ACC inhibitors have demonstrated the utility of treating metabolic syndrome through this modality. An isozyme-non-selective ACC inhibitor may potentially provide the optimal therapeutic for beneficially affecting metabolic syndrome. However, demonstration of the full potential of isozyme-selective inhibitors, once identified, should reveal advantages and liabilities associated with single isozyme inhibition. While demonstrating clinical efficacy of an ACC inhibitor should be relatively straightforward, the heterogeneity of the patient population and the absence of established guidelines regarding approval endpoints for agents simultaneously affecting multiple aspects of metabolic syndrome will pose developmental challenges for initial market entries.     

Identification and synthesis of novel inhibitors of acetyl-CoA carboxylase with in vitro and in vivo efficacy on fat oxidation

Acetyl CoA carboxylase isoforms 1 and 2 (ACC1/2) are key enzymes of fat utilization and their inhibition is considered to improve aspects of the metabolic syndrome. To identify pharmacological inhibitors of ACC1/2, a high throughput screen was performed which resulted in the identification of the lead compound 3 ( Gargazanli , G. ; Lardenois , P. ; Frost , J. ; George , P. Patent WO9855474 A1, 1998 ) as a moderate selective ACC2 inhibitor. Optimization of 3 led to 4m ( Zoller , G. ; Schmoll , D. ; Mueller , M. ; Haschke , G. ; Focken , I. Patent WO2010003624 A2, 2010 ) as a submicromolar dual ACC1/2 inhibitor of the rat and human isoforms. 4m possessed favorable pharmacokinetic parameters. This compound stimulated fat oxidation in vivo and reduced plasma triglyceride levels in a rodent model after subchronic administration. 4m is a suitable tool compound for the elucidation of the pharmacological potential of ACC1/2 inhibition.     

Short-term inhibition of fatty acid biosynthesis in isolated hepatocytes by mono-aromatic compounds

An overview is presented of a selected number of mono-aromatic derivatives and their short-term effects on hepatic fatty acid biosynthesis. The compounds discussed in this paper are ortho-hydroxybenzoate (salicylate), meta-hydroxybenzoate, para-hydroxybenzoate, benzoate, para-t-butylbenzoate, para-aminosalicylate, clofibrate, halofenate, alpha-cyano-4-hydroxycinnamate and benfluorex. All of these drugs inhibit fatty acid biosynthesis by isolated rat liver cells, albeit with different effectiveness. In contrast, the compounds have differential effects on fatty acid esterification and oxidation by isolated hepatocytes. An attempt is made to describe in molecular terms the underlying mechanisms of the acute inhibitory effects of the mono-aromatic derivatives on hepatic lipogenesis. It is proposed that all of the drugs exert an inhibitory action at the level of acetyl-CoA carboxylase, the enzyme generally considered to catalyse the rate-limiting step in hepatic fatty acid synthesis. This inhibitory effect may be either direct, i.e. by an alteration of the enzyme's structure as a result of interaction between drug and enzyme, or indirect, i.e. through a drug-induced change in the cellular levels of allosteric effectors of acetyl-CoA carboxylase.     

A continuous time-resolved fluorescence assay for identification of BACE1 inhibitors

The aspartic protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) mediates the production of the neurotoxic amyloid beta peptide and is therefore considered an important drug target for treatment of Alzheimer's disease. We describe a new homogeneous time-resolved fluorescence quenching assay for the identification of BACE1 inhibitors that is characterized by minimal compound interference and allows both kinetic and end-point measurements. A fluorescent Eu-chelate as fluorescent donor, coupled to the N-terminus of a peptide containing the amyloid precursor protein Swedish mutation with a quenching molecule at the C-terminus as acceptor, is used as substrate. Upon peptide cleavage by BACE1, the energy transfer between donor and acceptor molecules is interrupted, leading to increased fluorescence emission of the donor. Compound interference, a common problem in fluorescence assays, is minimized with this technology because of the large Stoke's shift and the time-resolved fluorescence emission of the Eu-chelate. The assay reproduced IC50 values of known inhibitors and detected them also as hits in a screening campaign. A high signal-to-noise ratio of 289 and a Z' factor of 0.76 make this assay suitable for high-throughput screening.     

Ganoderma lucidum polysaccharide peptide alleviates hepatoteatosis via modulating bile acid metabolism dependent on FXR-SHP/FGF

OBJECTIVE Ganoderma lucidum polysaccharide peptide(GLPP) is a group of extract from Ganoderma lucidum with a molecular mass of approximately 5×105, which ratio of polysaccharide to peptide is approximately 95%/5%. The purpose of this study was to determine whether GLPP has therapeutic effect on Non-alcoholic fatty liver disease(NAFLD). METHODS Ob/ob mouse model and Apo C3 transgenic mouse model were used for exploring the effect of GLPP on NAFLD. Key metabolic pathways and enzymes were identified by metabolomics combining with KEGG and PIUmet analyses and key enzymes were detected by Western blotting. Hepatosteatosis models of HepG 2 cells and primary hepatocytes were used to further confirm the therapeutic effect of GLPP on NAFLD. RESULTS GLPP administrated for a month alleviated hepatosteatosis, dyslipidemia, liver dysfunction and liver insulin resistance. Pathways of glycerophospholipid metabolism, fatty acid metabolism and primary bile acid biosynthesis were involved in the therapeutic effect of GLPP on NAFLD. Detection of key enzymes revealed that GLPP reversed low expression of CYP7 A1, CYP8 B1, FXR,SHP and high expression of FGFR4 in ob/ob mice and Apo C3 mice. Besides, GLPP inhibited fatty acid synthesis by reducing the expression of SREBP1 c, FAS and ACC via a FXR-SHP dependent mechanism. Additionally, GLPP reduced the accumulation of lipid droplets and the content of TG in HepG 2 cells and primary hepatocytes induced by oleic acid and palmitic acid. CONCLUSION GLPP significantly improves NAFLD via regulating bile acid synthesis dependent on FXR-SHP/FGF pathway, which finally inhibits fatty acid synthesis, indicating that GLPP might be developed as a therapeutic drug for NAFLD.     

Transcreener™: screening enzymes involved in covalent regulation

Enzymes that catalyse group transfer reactions comprise a significant fraction of the human proteome and are a rich source of drug targets because of their role in covalent regulatory cycles. Phosphorylation, glycosylation, sulfonation, methylation and acetylation represent some of the key types of group transfer reactions that modulate the function of diverse biomolecules through covalent modification. Development of high-throughput screening methods for these enzymes has been problematic because of the diversity of acceptor substrates. Recently, the authors developed a novel assay platform called Transcreener? that relies upon fluorescence detection of the invariant reaction product of a group transfer reaction, usually a nucleotide. This platform enables screening of any isoform in a family of group transfer enzymes, with any acceptor substrate, using the same assay reagents.     

Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation

Acetyl CoA carboxylase (ACC1 and ACC2) generates malonyl CoA, a substrate for de novo lipogenesis (DNL) and an inhibitor of mitochondrial fatty acid β-oxidation (FAO). Malonyl CoA is also a substrate for microsomal fatty acid elongation, an important pathway for saturated (SFA), mono- (MUFA) and polyunsaturated fatty acid (PUFA) synthesis. Despite the interest in ACC as a target for obesity and cancer therapy, little attention has been given to the role ACC plays in long chain fatty acid synthesis. This report examines the effect of pharmacological inhibition of ACC on DNL and palmitate (16:0) and linoleate (18:2, n − 6) metabolism in HepG2 and LnCap cells. The ACC inhibitor, soraphen A, lowers cellular malonyl CoA, attenuates DNL and the formation of fatty acid elongation products derived from exogenous fatty acids, i.e., 16:0 and 18:2, n − 6; IC₅₀ ∼ 5 nM. Elevated expression of fatty acid elongases (Elovl5, Elovl6) or desaturases (FADS1, FADS2) failed to override the soraphen A effect on SFA, MUFA or PUFA synthesis. Inhibition of fatty acid elongation leads to the accumulation of 16- and 18-carbon unsaturated fatty acids derived from 16:0 and 18:2, n − 6, respectively. Pharmacological inhibition of ACC activity will not only attenuate DNL and induce FAO, but will also attenuate the synthesis of very long chain saturated, mono- and polyunsaturated fatty acids.     

Multiparameter analysis of a screen for progesterone receptor ligands: comparing fluorescence lifetime and fluorescence polarization measurements

Direct measurement of the fluorescence lifetime (FLT) of a fluorescent label is an emerging method for high-throughput screening. Changes in the fluorescence lifetime can be correlated to changes in the non-radiative relaxation pathway(s) for the excited state of the label. These pathways can be environmentally sensitive, such as when a labeled analyte is free in solution versus bound to a receptor. Because lifetime is an intrinsic property of a fluorophore, it is not concentration dependent, and therefore has advantages similar to those of ratiometric fluorescent techniques such as fluorescence resonance energy transfer or fluorescence polarization. We have applied the FLT measurement technique to a screen of a small compound library in order to identify compounds that bind to the progesterone receptor, and compared the results to those obtained by performing the assay in fluorescence polarization mode. Each readout modality showed excellent Z'; values, with the FLT readout performing slightly better in this respect. Interfering compounds could be rapidly identified for either assay format by comparing the results between the two formats.     

Transcreener: screening enzymes involved in covalent regulation

Enzymes that catalyse group transfer reactions comprise a significant fraction of the human proteome and are a rich source of drug targets because of their role in covalent regulatory cycles. Phosphorylation, glycosylation, sulfonation, methylation and acetylation represent some of the key types of group transfer reactions that modulate the function of diverse biomolecules through covalent modification. Development of high-throughput screening methods for these enzymes has been problematic because of the diversity of acceptor substrates. Recently, the authors developed a novel assay platform called Transcreener that relies upon fluorescence detection of the invariant reaction product of a group transfer reaction, usually a nucleotide. This platform enables screening of any isoform in a family of group transfer enzymes, with any acceptor substrate, using the same assay reagents.     

Evaluation of a screening method by liquid chromatography-tandem mass spectrometry for estimating effect of drugs on the activation and β-oxidation of fatty acids in mitochondria

Objectives Fatty acid metabolism is controlled not only by the acyl‐coenzyme A (CoA) synthetases but by some enzymes in the β‐oxidation cycle. Medium‐chain and long‐chain acyl‐CoA esters are key metabolites in fatty acid metabolism. We have developed an enzymatic assay method for determining chain shortening of the acyl‐CoAs via β‐oxidation from palmitic and octanoic acids in liver mitochondria. We have evaluated the assay method for detecting whether drugs influence the activation or the β‐oxidation of fatty acids. Methods Liver mitochondria were used for investigating the effect of drugs on fatty acid metabolism. The drugs selected were salicylic acid, diclofenac, valproic acid and paracetamol. Each acyl‐CoA formed was analysed by liquid chromatography–tandem mass spectrometry. Key findings After less than 5 min of incubation, the levels of acyl‐CoAs reflected the acyl‐CoA synthetase activity, whereas after 60‐min incubation they reflected the activity of some enzymes in the β‐oxidation cycle. Salicylic acid, diclofenac and valproic acid inhibited the medium‐chain acyl‐CoA synthetases, whereas valproic acid only exhibited a weak inhibitory activity toward the β‐oxidation of the medium‐chain fatty acids. In the case of long‐chain fatty acid metabolism, salicylic acid and diclofenac inhibited both the activation and β‐oxidation, whereas valproic acid was a weak inhibitor for only the β‐oxidation activity. Paracetamol showed hardly any influence on the metabolism of medium‐chain and long‐chain fatty acids. Conclusions These findings suggest that salicylic acid, diclofenac, valproic acid and paracetamol exert a different influence on fatty acid metabolism depending on the length of the acyl chain. This assay allows sensitive and selective analysis for predicting the pathways by which drugs exert a greater influence over fatty acid metabolism.