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High-throughput screening for fatty acid uptake inhibitors in humanized yeast identifies atypical antipsychotic drugs that cause dyslipidemias

2007; Elsevier BV; Volume: 49; Issue: 1 Linguagem: Inglês

10.1194/jlr.d700015-jlr200

1539-7262

Hong Li, Paul N. Black, Aalap Chokshi, Angel Sandoval‐Alvarez, Ravi Vatsyayan, Whitney Sealls, Concetta Dirusso,

Receptor Mechanisms and Signaling

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12). The library used consisted of 2,080 compounds with known biological activities. Of these, ∼1.8% reduced cell-associated C1-BODIPY-C12 fluorescence and were selected as potential inhibitors of human FATP2-mediated fatty acid uptake. Based on secondary screens, 28 compounds were selected as potential fatty acid uptake inhibitors. Some compounds fell into four groups with similar structural features. The largest group was structurally related to a family of tricyclic, phenothiazine-derived drugs used to treat schizophrenia and related psychiatric disorders, which are also known to cause metabolic side effects, including hypertriglyceridemia. Potential hit compounds were studied for specificity of interaction with human FATP and efficacy in human Caco-2 cells. This study validates this screening system as useful to assess the impact of drugs in preclinical screening for fatty acid uptake. Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12). The library used consisted of 2,080 compounds with known biological activities. Of these, ∼1.8% reduced cell-associated C1-BODIPY-C12 fluorescence and were selected as potential inhibitors of human FATP2-mediated fatty acid uptake. Based on secondary screens, 28 compounds were selected as potential fatty acid uptake inhibitors. Some compounds fell into four groups with similar structural features. The largest group was structurally related to a family of tricyclic, phenothiazine-derived drugs used to treat schizophrenia and related psychiatric disorders, which are also known to cause metabolic side effects, including hypertriglyceridemia. Potential hit compounds were studied for specificity of interaction with human FATP and efficacy in human Caco-2 cells. This study validates this screening system as useful to assess the impact of drugs in preclinical screening for fatty acid uptake. During the past 20 years, obesity among adults and children has increased significantly in the United States. Being overweight or obese increases the risk for the development of adverse health conditions, including hypertension, dyslipidemia, diabetes, cardiovascular disease, and certain types of cancer, particularly colon and breast (1Bray G.A. Risks of obesity.Endocrinol. Metab. Clin. North Am. 2003; 32: 787-804Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 2Cook S. Hugli O. Egli M. Vollenweider P. Burcelin R. Nicod P. Thorens B. Scherrer U. Clustering of cardiovascular risk factors mimicking the human metabolic syndrome X in eNOS null mice.Swiss Med. Wkly. 2003; 133: 360-363PubMed Google Scholar, 3Lebovitz H.E. 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Fat1p and ACSL are both essential when cells are grown under hypoxic conditions, which render yeast auxotrophic for long-chain unsaturated fatty acids (30Faergeman N.J. DiRusso C.C. Elberger A. Knudsen J. Black P.N. Disruption of the Saccharomyces cerevisiae homologue to the murine fatty acid transport protein impairs uptake and growth on long-chain fatty acids.J. Biol. Chem. 1997; 272: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Likewise, specific interactions have also been demonstrated between FATP1 and ACSL1 to potentiate fatty acid uptake in murine adipocytes (28Richards M.R. Harp J.D. Ory D.S. Schaffer J.E. Fatty acid transport protein 1 and long chain acyl CoA synthetase 1 interact in adipocytes.J. Lipid Res. 2005; 47: 665-672Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). A number of studies have shown that fatty acid transport, particularly for protonated fatty acids, may also occur by diffusion or flip-flop between the two faces of the membrane (31Hamilton J.A. Transport of fatty acids across membranes by the diffusion mechanism.Prostaglandins Leukot. Essent. Fatty Acids. 1999; 60: 291-297Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 32Hamilton J.A. Brunaldi K. A model for fatty acid transport into the brain.J. Mol. Neurosci. 2007; 33: 12-17Crossref PubMed Scopus (126) Google Scholar, 33Kamp F. Guo W. Souto R. Pilch P.F. Corkey B.E. Hamilton J.A. Rapid flip-flop of oleic acid across the plasma membrane of adipocytes.J. Biol. Chem. 2003; 278: 7988-7995Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 34Kamp F. Hamilton J.A. pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids.Proc. Natl. Acad. Sci. USA. 1992; 89: 11367-11370Crossref PubMed Scopus (260) Google Scholar, 35Kamp F. 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A recent review of the diffusional model of fatty acid transport suggests that flip-flop is tied to the metabolic conversion to acyl-CoA, consistent with our model of vectorial acylation (32Hamilton J.A. Brunaldi K. A model for fatty acid transport into the brain.J. Mol. Neurosci. 2007; 33: 12-17Crossref PubMed Scopus (126) Google Scholar). Together, these reports provide compelling evidence for targeting fatty acid transport to limit the cellular uptake of fatty acids and thus lipotoxicity. Previously, we developed a live cell high-throughput screening (HTS) assay using a fat1Δfaa1Δ yeast strain to identify chemical compounds that will inhibit fatty acid uptake (P. N. Black and C. C. DiRusso, United States Patent 7,070,944) (38Li H. Black P.N. DiRusso C.C. A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors.Anal. Biochem. 2005; 336: 11-19Crossref PubMed Scopus (41) Google Scholar). In this system, transport is a function of mouse FATP2 expressed from a yeast promoter and long-chain fatty acid activation is provided by the endogenous ACSL Faa4p. We demonstrated that this HTS assay is rapid to execute, inexpensive to implement, and has appropriate sensitivity for HTS. In the present work, we conducted a pilot study using a 2,080 compound library from MicroSource Discovery Systems, Inc. (Gaylordsville, CT), called SpectrumPlus® to improve and validate this live cell HTS method. We also devised secondary screens to eliminate false-positives in the yeast system and further evaluated the potential hit compounds using the human Caco-2 cell line. The members of the largest family of structurally related compounds identified in this screen are derivatives of a tricyclic phenothiazine core from which atypical antipsychotic drugs have been developed. Among these are chlorpromazine and the related compound clozapine. Many of the drugs in this class cause adverse side effects, including weight gain, hypertriglyceridemia, hyperglycemia, and ketoacidosis, which in severe cases have led to patient death (reviewed in Ref. 39Newcomer J.W. Second-generation (atypical) antipsychotics and metabolic effects: a comprehensive literature review.CNS Drugs. 2005; 19: 1-93PubMed Google Scholar). Consequently, several of these drugs have received black box warning labels. The mechanism by which these drugs cause these clinical symptoms is unknown. Our identification of these compounds as inhibitors of fatty acid uptake leads us to suggest that the metabolic dysregulation associated with the administration of these drugs is caused in whole or in part by reduced fatty acid uptake, resulting in the disruption of normal cellular lipid trafficking. The SpectrumPlus compound library (2,080 compounds) was obtained from MicroSource Discovery Systems, Inc. The library includes five subsets of compounds: 1) Genesis Plus, composed of 960 compounds that represent new and classical therapeutic agents as well as established experimental inhibitors and receptor agonists; 2) Pure Natural Products Collection, a unique collection of 720 diversified pure natural products and their derivatives, including simple and complex oxygen heterocycles, alkaloids, sequiterpenes, diterpenes, pentercyclic triterpenes, sterols, and many other diverse representatives; 3) Agro Plate, containing 80 compounds representing classical and experimental pesticides, herbicides, and purported endocrine disruptors; 4) Cancer Plate, including 80 cytotoxic agents, antiproliferative agents, immune suppressants, and other experimental and therapeutic agents; and 5) Spectrum Plus Plate, containing 240 biologically active and structurally diverse compounds. The compounds are supplied as 10 mM solutions in DMSO and were diluted in PBS to a final concentration of 80 μM for screening in yeast using a Caliper RapidPlate 96/384 Dispenser (Caliper Life Sciences, Hopkinton, MA). Yeast extract, yeast peptone, and yeast nitrogen base without amino acids and dextrose were obtained from Difco (Detroit, MI). Complete amino acid supplement and single amino acids were from Sigma (St. Louis, MO). Fatty acid-free bovine serum albumin (FAF BSA) and other chemical reagents were also from Sigma, unless stated otherwise. Corning® white with clear flat-bottom 96-well and 384-well microplates were used for screening of yeast. For Caco-2 cells, tissue culture-treated 96-well transparent with clear flat-bottom polystyrene microplates were from Fisher Scientific (Pittsburgh, PA). The fluorescent long-chain fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was obtained from the Molecular Probes Division of Invitrogen. S. cerevisiae strain LS2086 containing deletions within the FAT1 and FAA1 genes (fat1Δfaa1Δ; MATa ura3-52 his3Δ200 ade2-101 lys2-801 leu2-3,112 faa1::HIS3 fat1Δ::G418) (26Zou Z. Tong F. Faergeman N.J. Borsting C. Black P.N. DiRusso C.C. Vectorial acylation in Saccharomyces cerevisiae. Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex.J. Biol. Chem. 2003; 278: 16414-16422Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) was used for the primary screen. For most experiments, yeast minimal medium with dextrose (YNBD) contained 0.67% yeast nitrogen base (YNB), 2% dextrose, adenine (20 mg/l), uracil (20 mg/l), and amino acids as required [arginine, tryptophan, methionine, histidine, and tyrosine (20 mg/l); lysine (30 mg/l); and leucine (100 mg/l)]. When a rich medium was required (e.g., toxicity studies in yeast), yeast complete media with adenine (YPDA) was used. Growth in liquid culture and on plates was at 30°C. Caco-2 cells were maintained in Earl's minimal essential medium with 20% FBS in a 95% air/5% CO2 atmosphere at 37°C, as described previously (40Levy E. Mehran M. Seidman E. Caco-2 cells as a model for intestinal lipoprotein synthesis and secretion.FASEB J. 1995; 9: 626-635Crossref PubMed Scopus (178) Google Scholar). For growth and differentiation, the BD Biosciences Intestinal Epithelium Differentiation Media Pack was used. Cells were plated in basal seeding medium at a density of 2.5 × 105 cells/cm2 on a collagen-coated black-clear 96-well plate (BD Biosciences). Entero-STIM medium was added to each well after removal of the basal seeding medium 24 h later. Both media contained mito-serum extender. After another 24 h, cells were washed once with complete balanced Hanks' solution containing Ca2+ and Mg2+ without phenol red before the C1-BODIPY-C12 uptake assay. For expression of the human FATP2 in yeast, the cDNA encoding the protein was amplified using PCR from a cDNA within NIH Image Clone I.D. 30348317 using forward primer 5′-gcatagccctcgagatgctttccgccatctacaca-3′ and reverse primer 5′-cgcagaccaagctttcagagtttcagggtttttagc-3′. The amplified DNA was cloned into the yeast expression vector pDB121 to give pDB126. In this construct, the expression of targeted human FATP2 is under the control of the GAL10 promoter (41DiRusso C.C. Connell E.J. Faergeman N.J. Knudsen J. Hansen J.K. Black P.N. Murine FATP alleviates growth and biochemical deficiencies of yeast fat1Δ strains.Eur. J. Biochem. 2000; 267: 4422-4433Crossref PubMed Scopus (41) Google Scholar). The human FATP2 plasmid and pRS416GAL4-ER-VP16 encoding a synthetic transcriptional activator, which is a protein fusion between the Gal4 DNA binding domain, a β-estradiol-responsive regulatory domain, and a VP16 RNA polymerase activation domain (42Stafford G.A. Morse R.H. Chromatin remodeling by transcriptional activation domains in a yeast episome.J. Biol. Chem. 1997; 272: 11526-11534Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), were cotransformed into strain LS2086 for HTS assays. Expression of human FATP was induced by the growth of yeast cells overnight in selective medium containing 10 nM β-estradiol. The level of human FATP2 protein was routinely measured using an antibody directed toward a T7 epitope fused to the C terminus of human FATP2 in Western blots of cell extracts. For screening of the 2,080 compound MicroSource library, a Biomek® FX laboratory automated workstation (Beckman Coulter, Inc., Fullerton, CA) was used. LS2086 yeast cells expressing human FATP2 (pDB126/fat1Δfaa1Δ) or transformed with the empty vector pDB121 (negative control cells) were pregrown in YNBD without leucine and uracil (YNBD −leu −ura); the cells were subsequently subcultured to absorbance at 600 nm = 0.02 in the same medium containing 10 nM β-estradiol to induce expression and incubated overnight at 30°C with shaking. Mid-log-phase yeast cells [0.8–1.2 optical density at 600 nm (OD600)] were harvested and resuspended in PBS at a cell density of 6 × 107/ml before dispensing to a 384-well assay plate (22.5 μl/well). Wells in the first two rows of each plate received the negative control cells, and all other wells received cells expressing human FATP2. Compounds (2.5 μl) were then added to a final concentration of 80 μM. After 2 h of incubation at 30°C, the C1-BODIPY-C12 uptake assay was performed. To each well, a mixture of C1-BODIPY-C12 (final concentration, 1.25 μM), FAF BSA (final concentration, 0.75 μM), and trypan blue (final concentration, 2.1 mM) was added to give a final volume of 100 μl. Trypan blue quenched the extracellular fluorescence, and only cell-associated fluorescence was measured after 30 min on a Bio-Tek Synergy HT multidetection microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) with filter sets of 485 ± 20 nm excitation and 528 ± 20 nm emission. On each plate, two rows each of negative control cells and positive control cells were incubated with 0.8% DMSO in PBS and assayed simultaneously to calculate a Z′ factor for each plate. The results presented are the values obtained for two screening experiments (see Tables 1, TABLE 3, TABLE 4, TABLE 5, TABLE 6, TABLE 7 below).TABLE 1Compounds that reduced cell-associated fluorescence in humanized yeastCompound IdentifierNamePercentage Transport InhibitionaPercentage inhibition calculated from the ratio of fluorescence in arbitrary fluorescence units (AFU) for cells with compound compared with positive control cells with 0.8% DMSO alone. Results are from individual samples in two high-throughput screening trials (trial 1, trial 2).Comment01500138Benzethonium chloride100, 10001500184Chlorpromazine100, 10001500315Gentian violet100, 100Quenching agent01503610Benzalkonium chloride100, 10001503253Methylbenzethonium chloride100, 10001503227Perhexiline maleate100, 10001503223Pararosaniline pamoate100, 100Quenching agent02300061Clomipramine HCl100, 10001500575Thioridazine HCl100, 10001505204Almotriptan100, 10000100325Digitonin100, 100Membrane disruptor01503934Perphenazine91, 10001503200Centrimonium bromide100, 9601504079Tomatine97, 98Antimicrobial agent01503936Perciazine72, 9601503637Methiothepin maleate59, 10001500127Anthralin81, 8200310035Sanguinarine sulfate53, 64Antibiotic/antifungal agent00201664Celastrol75, 97Quenching agent01503207Cyclobenzaprine HCl80, 8601505205Olmesartan medoxomil80, 9801502237Harmol hydrochloride89, 8301500473Phenazopyridine hydrochloride53, 5101500994Flufenazine HCl54, 9901504017Sapindoside A75, 92Antifungal agent01503239Hycanthione62, 6901500898Emodin74, 6001500685Clozapine66, 5000300038Juglone53, 47Antifungal agent01504119Rhodomyrtoxin B64, 400300556Chrysarobin34, 4200211118Diacetlydideisovaleryl-rhodomyrtoxin45, 6001500759Quinalizarin70, 3401500602Gossypol40, 44Quenching agent01500510Promethazine44, 6601500509Promazine42, 4601500505Prochlorperazine edisylate45, 4700200022Aklavin HCl44, 6901504074Embelin44, 58a Percentage inhibition calculated from the ratio of fluorescence in arbitrary fluorescence units (AFU) for cells with compound compared with positive control cells with 0.8% DMSO alone. Results are from individual samples in two high-throughput screening trials (trial 1, trial 2). Open table in a new tab TABLE 3Compounds related to fatty acids with aliphatic, hydrocarbon chainsCompound StructureCompound NamePercentage InhibitionaPercentage inhibition compared with positive controls without compound. Results are for two experiments.Benzalkonium chloride [ammonium, alkyldimethyl(phenylmethyl)-, chloride]100, 100Centrimonium bromide (hexadecyl-trimethyl-ammonium, bromide)100, 96Embelin (2,5-dihydroxy-3-undecyl-cyclohexa-2,5-diene-1,4-dione)44, 57Batyl alcohol [3-(octadecyloxy)-1,2-propanediol]0, 0Avocadyne [(2,4-dihydroxyheptadec-16-ynyl) acetate]0, 0a Percentage inhibition compared with positive controls without compound. Results are for two experiments. Open table in a new tab TABLE 4Compounds related to phenothiazineCompound StructureCompound NamePercentage InhibitionaSee Table 3 for details.Chlorpromazine [3-(2-chlorophenothiazin-10-yl)-N,N-dimethyl-propan-1-amine]100, 100Thioridazine HCl [10-(2-(1-methyl-2-piperidyl)ethyl)-2-(methylthio)phenothiazine]100, 100Perphenazine {2-[4-[3-(2-chlorophenothiazin-10-yl)propyl]piperazin-1-yl]ethanol}91, 100Flufenazine [2-(4-{3-[2-(trifluoromethyl)-10H-phenothiazin-10-yl] propyl}piperazin-1-yl)ethanol]54, 99Periciazine {10-[3-(4-hydroxy-1-piperidyl)propyl] phenothiazine-2-carbonitrile}72, 96Promethazine (N,N-dimethyl-1-phenothiazin-10-yl-propan-2-amine)45, 47Promazine (N,N-dimethyl-3-phenothiazin-10-yl-propan-1-amine)52, 46Prochlorperazine edisylate [2-chloro-10-(3-(1-methyl-4-piperazinyl)propyl)phenothiazine edisylate]45, 47Isothiopendyl HCl [10H-pyrido(3,2-b) (1,4)benzothiazine, 10-(2-(dimethylamino)propyl)-]0, 0Triflupromazine {N,N-dimethyl-3-[2-(trifluoromethyl)phenothiazin-10-yl]propan-1-amine}0, 0a See Table 3 for details. Open table in a new tab TABLE 5Compounds related to cyclobenzaprineCompound StructureCompound NamePercentage InhibitionaSee Table 3 for details.Clomipramine [3-chloro-5-(3-(dimethylamino)propyl)-10,11-dihydro-5H-dibenz(b,f)azepine]100, 100Methiothepin maleate [(+−)-1-(10,11-dihydro-8-(methylthio)dibenzo(b,f)thiepin-10-yl)-4-methylpiperazine]59, 100Cyclobenzaprine [1-propanamine, 3-(5H-dibenzo(a,d)cyclohepten-5-ylidene)-N,N-dimethyl- (9CI)]80, 86Clozapine [8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo(b,e) (1,4)diazepine]66, 50Imipramine [1-(3-dimethylaminopropyl)-4,5-dihydro-2,3,6,7-dibenzazepine]0, 26Amitriptyline HCl [10,11-dihydro-5-(γ-dimethylaminopropylidene)-5H-dibenzo(a,d)cycloheptene]0, 0Loratadine [ethyl 4-(8-chloro-5,6-dihydro-11H-benzo(5,6)cyclohepta(1,2-b)pyridin-11-ylidene)-1-piperidinecarboxylate]0, 0a See Table 3 for details. Open table in a new tab TABLE 6Compounds related to anthralinCompound StructureCompound NamePercentage InhibitionaSee Table 3 for details.Anthralin (1,8,9-trihydroxyanthracene)81, 82Emodin (1,3,8-trihydroxy-6-methyl-9,10-anthracenedione)74, 60Juglone (5-hydroxy-1,4-naphthoquinone)53, 47Chrysarobin (6-methylanthracene-1,2,8-triol)34, 42Quinazalin (1,2,5,8-tetrahydroxyanthracene-9,10-dione)70, 34Rutilantinone [1-naphthacenecarboxylic acid, 1,2,3,4,6,11-hexahydro-2-ethyl-2,4,5,7,10-pentahydroxy-6,11-dioxo-, methyl ester, (1R,2R,4S)-]3, 54Aklavin [methyl (1S,2S)-4-(4-dimethylamino-5-hydroxy-6-methyl-oxan-2-yl)oxy-2-ethyl-2,5,7-trihydroxy-6,11-dioxo-3,4-