Potent Inhibitor Of Fatty Acid Synthase Research Articles

Inhibitory Effects of the Polyphenols from the Root of Rhizophora apiculata Blume on Fatty Acid Synthase Activity and Human Colon Cancer Cells.

Marine mangrove vegetation has been traditionally employed in folk medicine to address various ailments. Notably, Rhizophora apiculata Blume has exhibited noteworthy properties, demonstrating efficacy against cancer, viruses, and bacteria. The enzyme fatty acid synthase (FAS) plays a pivotal role in de novo fatty acid synthesis, making it a promising target for combating colon cancer. Our study focused on evaluating the FAS inhibitory effects of both the crude extract and three isolated compounds from R. apiculata. The n-butanol fraction of R. apiculata extract (BFR) demonstrated a significant inhibition of FAS, with an IC50 value of 93.0 µg/mL. For inhibition via lyoniresinol-3α-O-β-rhamnopyranoside (LR), the corresponding IC50 value was 20.1 µg/mL (35.5 µM). LR competitively inhibited the FAS reaction with acetyl-CoA, noncompetitively with malonyl-CoA, and in a mixed manner with NADPH. Our results also suggest that both BFR and LR reversibly bind to the KR domain of FAS, hindering the reduction of saturated acyl groups in fatty acid synthesis. Furthermore, BFR and LR displayed time-dependent inhibition for FAS, with kobs values of 0.0045 min-1 and 0.026 min-1, respectively. LR also exhibited time-dependent inhibition on the KR domain, with a kobs value of 0.019 min-1. In human colon cancer cells, LR demonstrated the ability to reduce viability and inhibit intracellular FAS activity. Notably, the effects of LR on human colon cancer cells could be reversed with the end product of FAS-catalyzed chemical reactions, affirming the specificity of LR on FAS. These findings underscore the potential of BFR and LR as potent FAS inhibitors, presenting novel avenues for the treatment of human colon cancer.

The Effect of Nano-Encapsulated Orlistat on Prostate Cancer: An In vitro Study

Background: The targeted inhibition of fatty acid synthase (FASN) by Orlistat, a potent FASN inhibitor, has been shown to block tumor proliferation and induce apoptosis in cultured tumor cells. Since Orlistat is insoluble, its solubility in blood circulation is limited. Cancer nanotherapeutics are rapidly progressing and are being implemented to solve several limitations of conventional drug delivery systems. Aim: In this in vitro study, we aimed to overcome the limitation of orlistat changes by designing a nanoparticle and investigating its selective cytotoxic effects on cancer cells. Method: To investigate the effects of encapsulated Orlistat on prostate cancer, we measured apoptosis and the distribution of cells via flow cytometry, confocal microscope, and Western blot. Results: There is an improvement in the solubility of Orlistat with the effect of nanoparticles; it was observed that Orlistat decreased the expression of FASN and caused dose-dependent cytotoxicity with Caspase-3, Bax, and PARP activation in PC-3 cells, while it did not have a significant effect on normal prostate epithelial cells. Conclusion: The present results indicate that the limitation of Orlistat can be overcome by assembling a nano-micellar formulation that can induce apoptotic death in the PC-3 cell line but normal prostate epithelial cells.

Fasnall Induces Atypically Transient Stress Granules Independently of FASN Inhibition.

SummaryStress Granule formation has been linked to the resistance of some cancer cells to chemotherapeutic intervention. A number of studies have proposed that certain anti-tumor compounds promote cancer cell survival by inducing Stress Granule formation, leading to increased cellular fitness and apoptosis avoidance. Here we show that a potent fatty acid synthase inhibitor, fasnall, known for its anti-tumor capabilities, triggers the formation of atypical Stress Granules, independently of fatty acid synthase inhibition, characterized by high internal mobility and rapid turnover.

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

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

Mechanistic MALDI-TOF Cell-Based Assay for the Discovery of Potent and Specific Fatty Acid Synthase Inhibitors

Human cancers require fatty acid synthase (FASN)-dependent de novo long-chain fatty acid synthesis for proliferation. FASN is therefore an attractive drug target, but fast technologies for reliable label-free cellular compound profiling are lacking. Recently, MALDI-mass spectrometry (MALDI-MS) has emerged as an effective technology for discovery of recombinant protein target inhibitors. Here we present an automated, mechanistic MALDI-MS cell assay, which monitors accumulation of the FASN substrate, malonyl-coenzyme A (CoA), in whole cells with limited sample preparation. Profiling of inhibitors, including unpublished compounds, identified compound 1 as the most potent FASN inhibitor (1nM in A549 cells) discovered to date. Moreover, cellular MALDI-MS assays enable parallel profiling of additional pathway metabolites. Surprisingly, several compounds triggered cytidine 5'-diphosphocholine (CDP-choline) but not malonyl-CoA accumulation indicating that they inhibit diacylglycerol generation but not FASN activity. Taken together, our study suggests that MALDI-MS cell assays may become important tools in drug profiling that provide additional mechanistic insights concerning compound action on metabolic pathways.

Pyridine derivatives as anticancer lead compounds with Fatty Acid Synthase as the target: An in silico-guided in vitro study.

For the past few decades, structure-based drug discovery (SBDD) has become an inevitable technique in the drug development process for screening hit compounds against therapeutic targets. Here, we have successfully used the SBDD approach viz. virtual high-throughput screening to identify potential inhibitors against the Ketoacyl synthase (KS) domain of Fatty acid synthase (FASN). Overexpression of FASN, and subsequent enhancement of de novo lipogenesis is a key survival strategy of cancer cells. Hence, targeting lipid metabolism using FASN inhibitors has been considered as a promising method to induce metabolic stress, thereby posing a survival disadvantage to cancer cells. In the present study, we have successfully identified eight FASN inhibitors from Asinex Elite database by implementing in silico tools. Five of the hit compounds share a common ring structure, which enables characteristic binding interactions with FASN-KS. Among them, in vitro validation showed that SFA 22637550 possesses significant FASN inhibitory activity and antiproliferative effect in human cancer cells of various origins. The maximum sensitivity was exhibited towards HepG2 hepatocellular carcinoma cells (IC50 = 28 µM). The mode of cell death was found to be apoptosis with a significant increase in SubG0 population without affecting any other phases of the cell cycle. The current study puts forward an excellent core structure for the development of potent FASN inhibitors for successfully targeting cancer cell metabolism, thereby causing selective cell death.

Preclinical evaluation of novel fatty acid synthase inhibitors in primary colorectal cancer cells and a patient-derived xenograft model of colorectal cancer.

Fatty Acid Synthase (FASN), a key enzyme of de novo lipogenesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN expression is associated with poor prognosis. Potent FASN inhibitors (TVBs) developed by 3-V Biosciences demonstrate anti-tumor activity in vitro and in vivo and a favorable tolerability profile in a Phase I clinical trial.However, CRC characteristics associated with responsiveness to FASN inhibition are not fully understood. We evaluated the effect of TVB-3664 on tumor growth in nine CRC patient-derived xenografts (PDXs) and investigated molecular and metabolic changes associated with CRC responsiveness to FASN inhibition.CRC cells and PDXs showed a wide range of sensitivity to FASN inhibition. TVB-3664 treatment showed significant response (reduced tumor volume) in 30% of cases. Anti-tumor effect of TVB-3664 was associated with a significant decrease in a pool of adenine nucleotides and alterations in lipid composition including a significant reduction in fatty acids and phospholipids and an increase in lactosylceramide and sphingomyelin in PDXs sensitive to FASN inhibition. Moreover, Akt, Erk1/2 and AMPK were major oncogenic pathways altered by TVBs.In summary, we demonstrated that novel TVB inhibitors show anti-tumor activity in CRC and this activity is associated with a decrease in activation of Akt and Erk1/2 oncogenic pathways and significant alteration of lipid composition of tumors. Further understanding of genetic and metabolic characteristics of tumors susceptible to FASN inhibition may enable patient selection and personalized medicine approaches in CRC.

Abstract 2994: Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy

Abstract Objectives: Fatty acid synthase (FAS) is a key enzyme involved in fatty acid synthesis. It also plays an important role in tumorigenesis and is highly expressed in many cancers, including endometrial cancer. Orlistat is a weight loss medication that has been shown to be a potent inhibitor of FAS. The goal of this study was to evaluate the anti-tumorigenic potential of orlistat in endometrial cancer cell lines. Methods: The endometrial cancer cell lines ECC-1 and KLE were treated with varying doses of orlistat (0.01-500 μM). Cell proliferation was assessed using the MTT assay. Cell cycle progression was evaluated by Cellometer and apoptosis was assessed using the Annexin V assay. Reactive oxygen species (ROS) was measured using the DCFH-DA assay. Western immunoblotting was performed to determine changes in FAS, cellular stress, cell cycle progression and the AMPK/mTOR pathways. Results: Orlistat inhibited cell proliferation in a dose-dependent manner in both cell lines (IC50 = 81.7 μM in ECC-1; IC50 = 145.5 μM in KLE). Treatment with orlistat resulted in G1 arrest but did not affect apoptosis. Orlistat increased ROS at high doses (500 uM, p = 0.008 for ECC-1 cells and p = 0.012 for KLE cells) and induced expression of BIP and PERK. Western immunoblot analysis demonstrated that orlistat decreased expression of FAS, acetyl-CoA carboxylase (ACC), and carnitine palmitoyltransferase 1A (CPT1A), important proteins in fatty acid metabolism. Treatment with orlistat also increased expression of phosphorylated AMPK and decreased expression of the downstream S6 protein. Conclusions: Orlistat suppressed cell growth in endometrial cancer cell lines through inhibition of fatty acid metabolism, induction of cell cycle G1 arrest, and modulation of the AMPK/mTOR pathway. Given that patients with endometrial cancer have high rates of obesity, orlistat should be further investigated as a novel strategy for endometrial cancer treatment and prevention. Citation Format: Weiya Z. Wysham, Dario R. Roque, Jianjun Han, Hui Guo, Lu Zhang, Paola A. Gehrig, Chunxiao Zhou, Victoria L. Bae-Jump. Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2994.

Effects of Fatty Acid Synthase Inhibition by Orlistat on Proliferation of Endometrial Cancer Cell Lines.

Fatty acid synthase (FAS) is a key lipogenic enzyme that is highly expressed in endometrial cancer. Orlistat is a weight loss medication that has been shown to be a potent inhibitor of FAS. The goal of this study was to evaluate the anti-tumorigenic potential of orlistat in endometrial cancer cell lines. The endometrial cancer cell lines ECC-1 and KLE were used. Cell proliferation was assessed by MTT assay after treatment with orlistat. Cell cycle progression was evaluated by Cellometer and apoptosis was assessed using the Annexin V assay. Reactive oxygen species (ROS) was measured using the DCFH-DA assay. Western immunoblotting was performed to determine changes in FAS, cellular stress, cell cycle progression, and the AMPK/mTOR pathways. Orlistat inhibited cell proliferation by 61% in ECC-1 cells and 57% in KLE cells at a dose of 500μM. Treatment with orlistat at this concentration resulted in G1 arrest (p < 0.05) but did not affect apoptosis. Orlistat increased ROS and induced the expression of BIP (1.28-fold in ECC-1 compared to control, p < 0.05; 1.92-fold in KLE, p < 0.05) and PERK (2.25-fold in ECC-1, 1.4-fold in KLE, p < 0.05). Western immunoblot analysis demonstrated that orlistat decreased expression of important proteins in fatty acid metabolism including FAS (67% in ECC-1, 15% in KLE), acetyl-CoA carboxylase (40% in ECC-1, 35% in KLE), and carnitine palmitoyltransferase 1A (CPT1A) (65% in ECC-1, 25% in KLE) in a dose-dependent manner. In addition, orlistat at a dose of 500μM increased expression of phosphorylated-AMPK (1.9-fold in ECC-1, p < 0.01; 1.5-fold in KLE, p < 0.05) and decreased expression of phosphorylated-Akt (25% in ECC-1, p < 0.05; 37% in KLE, p < 0.05) and phosphorylated-S6 (68% in ECC-1, 56% in KLE). Orlistat inhibits cell growth in endometrial cancer cell lines through inhibition of fatty acid metabolism, induction of cell cycle G1 arrest, activation of AMPK and inhibition of the mTOR pathway. Given that patients with endometrial cancer have high rates of obesity, orlistat should be further investigated as a novel strategy for endometrial cancer treatment.

De Novo Palmitate Synthesis Supports Oncogenic Signalling and Tumor Growth Through Diverse Mechanisms: Implications for FASN-Targeted Therapeutics

Palmitate, the enzymatic product of fatty acid synthase (FASN), provides a substrate for the synthesis of longand short-chain fatty acids. Many recent studies have expanded our knowledge about the roles palmitate and lipid synthesis play in tumor cell biology beyond supporting energy metabolism and membrane building. The recent article described cell biology and pharmacology studies using a novel, selective small molecule FASN inhibitor, TVB-3166. They demonstrated that FASN inhibition disrupts oncogenic signalling and tumor growth in xenograft models through inhibition of pathways that include Wnt/beta-catenin and expression of c-Myc: potent oncogenes historically recalcitrant to direct pharmacological inhibition. Discussed here are how these findings advance our mechanistic understanding of the diverse biological roles of palmitate and its integration into various signalling pathways driving tumor cell proliferation and survival. These insights highlight the promising potential of selective, potent FASN inhibitors as a novel therapeutic strategy for cancer and other illnesses.

Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses.

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.

Fatty acid synthesis and NLRP3-inflammasome

Fatty acid synthesis is an important process of the lipogenesis [1]. Fatty acid synthesis is tightly regulated by key enzymes including fatty acid synthase (FASN) [1]. FASN and lipogenesis related enzymes are upregulated in various cancer cells and associated with oncogenic progression [1]. FASN is being an indicator of poor prognosis and a chemotherapeutic target in breast cancers [1]. Fatty acid synthesis has been implicated in immune response such as the differentiation of B lymphocytes and human monocytes [2, 3]. Recently, we demonstrated that upregulation of FASN-dependent fatty acid synthesis regulates inflammation through NLRP3 inflammasome activation in macrophages [4]. We have described a novel molecular mechanism that fatty acid synthesis under the regulation of FASN is critical for NLRP3-mediated caspase-1 activation. Genetic and pharmacologic inhibition of FASN suppressed NLRP3-mediated caspase-1 activation in macrophages, as well as IL-1β and IL-18 production in vivo [4]. Our study revealed that FASN-dependent lipid synthesis acts as an upstream signal for NLRP3 gene expression [4]. Inhibition of fatty acid synthesis by FASN knockdown or C75, a potent FASN inhibitors, suppressed the NLRP3 inflammasome activation in macrophages [4]. FASN is a multifunctional metabolic enzyme that catalyzes the terminal steps in the synthesis of long-chain saturated fatty acids [1]. The production of fatty acids supports membrane synthesis in proliferating cells [1]. Furthermore, FASN expression and activity is critical for AKT activation which promotes cell survival and enhances tumor cell growth and invasiveness [1]. We demonstrated that FASN regulates NLRP3 gene expression via AKT activation in macrophages [4]. The activation of AKT in response to lipopolysaccharide (LPS) stimulation is suppressed by FASN inhibition [4]. Consistently, inhibition of AKT by BX795, a selective AKT inhibitor, reduced NLRP3 gene expression [4]. Our results suggest that FASN regulates the activation of AKT signaling pathway in macrophages. Activation of FASN can promote glucose-dependent fatty acid synthesis, illustrating that glucose utilization is regulated by lipogenesis [1]. Because the metabolites from glycolysis are the main carbon sources for lipid synthesis, glycolysis can be regulated along with fatty acid synthesis. It has been shown that glycolysis is linked to changes in lipogenesis required for cell proliferation in androgen-dependent prostate cancer cells [5]. Similarly, we identified that increased glycolytic phenotype is linked to activation of FASN-dependent fatty acid synthesis during inflammation in vitro and in vivo [4]. Since FASN has a critical role in inflammation and cancer progression [1–3], several studies have attempted to identify an upstream regulator of FASN as a molecular target for anti-inflammatory or cancer treatment. Previous study has shown that the expression of FASN is increased by human epithelial growth factor receptor-2 (HER2) overexpression, and that the activation of the mammalian target of rapamycin (mTOR) by HER2 plays an important role in their overexpression through the selective translational activation in breast cancer cells [6]. Similarly, we identified that UCP2 regulates fatty acid synthesis during inflammation in macrophages. Deficiency of UCP2 reduced the expression of FASN and triglyceride synthesis in response to LPS in macrophages [4]. Our results demonstrated that UCP2 plays an upstream regulator of FASN-dependent lipid synthesis in macrophages. Several types of inflammation by different causes and mechanisms can promote cancer development [1]. Recent study has reported that 5-FU and Gemcitabine mediated NLRP3 inflammasome activation represent a new mechanism of resistance to chemotherapy treatment in cancer [7]. Chronic inflammation and fatty acid metabolism could be linked as critical events in carcinogenesis and tumor progression. We identified that FASN-dependent fatty acid synthesis regulates the NLRP3 inflammasome activation in macrophages [4]. Our results suggest that FASN-dependent fatty acid synthesis might be a target for regulating of by chemotherapy treatment in cancer. Collectively, we suggest that FASN could be a critical regulator of the NLRP3 inflammsome activation in cancer.

Abstract 2535: Structure-activity relationships of novel N-benzoyl arylpiperidine and arylazetidine FASN inhibitors

Abstract De novo synthesis of fatty acids in mammalian cells is catalyzed by Fatty Acid Synthase (FASN), a complex dimeric protein with seven catalytic domains that processes acetyl-CoA and malonyl-CoA into palmitic acid. Palmitate and other fatty acids are essential for normal physiological processes like energy storage and production and are key intermediates in the biosynthesis of hormones and other essential biomolecules. The role of FASN in cancer has been well documented over the past decade. While FASN is expressed at low levels in most normal tissue (except liver and adipose tissue), it is highly expressed in many tumors including prostate, breast, and colon. FASN overexpression and activity is correlated with poor prognosis and disease progression. Numerous publications describing anti-cancer effects of small molecule FASN inhibitors have appeared in the scientific and patent literature. Recently, several groups reported anticancer and antiviral activities of N-benzoyl arylpiperidine FASN inhibitors. In this work we describe the discovery of novel N-benzoyl arylpiperidine and arylazetidine compounds that potently inhibit FASN enzymatic activity in vitro and antiproliferative activity in FASN-sensitive cell lines. We conducted focused structure-activity (SAR) studies exploring the benzoyl group and the aryl substituent on the piperidine or azetidine heterocycle to optimize potency, improve properties and oral bioavailability, and achieve in vivo FASN-driven pharmacodynamic (PD) activity. X-ray crystal structures of these molecules confirm a unique binding mode within the KR subdomain of FASN. Using structural information and molecular modeling, we were able to rationalize SAR trends and design FASN inhibitors with excellent in vitro potency and cellular activity. These efforts resulted in the discovery of compounds exemplified by JNJ-54380482, a potent FASN inhibitor (IC50 = 26 nM) that inhibits proliferation of A2780 ovarian cells in lipid-reduced medium (LRM, IC50 = 8.9 nM). Engagement of FASN in cellular assays is confirmed by rescue of proliferation upon addition of palmitate. JNJ-54380482 is orally bioavailable in mice (F% = 51) and exhibits excellent plasma exposure when dosed at 10 mg/kg (Cmax = 2.6 µM, T1/2 = 5 h). In a PD study using H460 lung xenograft-bearing mice, oral treatment with 30 mg/kg of JNJ-54380482 results in 36-fold higher tumor levels of malonyl-CoA vs vehicle. In summary, we synthesized several hundred compounds having the N-benzoyl arylpiperidine and arylazetidine chemotypes. Utilizing structural information from X-ray co-crystals to understand SAR and guide the design of more potent molecules, we identified &amp;gt;100 analogs with highly potent FASN enzymatic (IC50 ≤ 50 nM) and antiproliferative activities in A2780 cells in LRM (IC50 ≤ 100 nM). Key examples from these series have good in vitro properties, are orally bioavailable, and demonstrate FASN target engagement in a mouse PD model. Citation Format: Peter J. Connolly, Gilles Bignan, James Bischoff, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Tianbao Lu, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Structure-activity relationships of novel N-benzoyl arylpiperidine and arylazetidine FASN inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2535. doi:10.1158/1538-7445.AM2014-2535

Inhibitory effects of grape skin extract and resveratrol on fatty acid synthase

BackgroundGrape skin, a rich source of phytochemicals, has been reported to possess remarkable anti-obesity activity. Fatty acid synthase (FAS) is a key enzyme catalyzing the synthesis of fatty acid de novo, and has been considered as an anti-obesity target. To elucidate the anti-obesity mechanism of grape skin, we investigated the effects of grape skin extract (GSE) and resveratrol, one of the phytochemicals in GSE, on FAS and FAS over-expressed 3 T3-L1 preadipocyte.MethodsPurified FAS was obtained from chicken liver. Dried grape skin was extracted by 50% ethanol and partitioned by ethyl acetate. Inhibitory effects of GSE and resveratrol on FAS including fast-binding inhibition, time-dependent inhibition, and enzyme kinetics were determined. Inhibitory effects of GSE and resveratrol on 3T3-L1 preadipocyte were also measured.ResultsGSE inhibited the overall reaction and β-ketoacyl reductase (KR) reaction of FAS with IC50 values of 4.61 μg/ml and 20.3 μg/ml. For inhibition by resveratrol, the relevant IC50 values were 11.1 μg/ml and 21.9 μg/ml, respectively. And both GSE and resveratrol showed time-dependent inhibition for FAS, with the kobs values of 0.028 min-1, and 0.040 min-1 respectively. They inhibited the overall reaction of FAS competitively with acetyl-CoA, noncompetitively with malonyl-CoA and in a mixed manner with NADPH. Moreover, the inhibition on KR domain by resveratrol was time-dependent with kobs value of 0.106 min-1. In 3 T3-L1 preadipocytes, resveratrol reduced lipid accumulation remarkably.ConclusionsGSE and resveratrol are potent FAS inhibitors and they bound reversibly to the KR domain of FAS to inhibit the reduction of the saturated acyl groups in fatty acid synthesis. Based on the valid data and deliberate analysis, we proposed that GSE and resveratrol have great medical potential and officinal value in treating obesity and related diseases.

Inhibition of fatty acid synthase by luteolin post-transcriptionally down-regulates c-Met expression independent of proteosomal/lysosomal degradation

The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in the progression of several cancers and associated with increased tumor invasion and metastatic potential. We determined previously that the polyphenol epigallocatechin-3-gallate inhibited HGF-induced c-Met phosphorylation in a variety of tumor cell lines in part by disrupting lipid rafts. Fatty acid synthase (FASN) is implicated in cancer progression and may regulate lipid raft function. We therefore examined the effects of luteolin, a potent FASN inhibitor, on c-Met signaling. Luteolin blocked HGF-induced c-Met phosphorylation and scattering of DU145 prostate cancer cells, but inhibition required at least a 4 h preincubation time. Western blot analysis indicated that inhibition of HGF-induced scattering by luteolin occurred coincident with reduction of total c-Met protein in DU145 cells. In addition, luteolin-induced c-Met down-regulation was mimicked by a pharmacologic inhibitor of FASN, C75, or short hairpin RNA knockdown of FASN. Consistent with a role for FASN, loss of c-Met in cells treated with C75 or luteolin was prevented by exogenous addition of palmitate. Luteolin-induced loss of c-Met primarily occurred at a post-transcriptional level and involved cell surface internalization but did not involve translation inhibition, nor was it dependent on the activity of the 26S proteosome or acidic lysosomes. Taken together, our study shows a novel connection between FASN activity and c-Met protein expression and suggests that luteolin could act as a novel HGF/c-Met inhibitor by reducing expression of this receptor.

Novel and potent inhibitors of fatty acid synthase derived from catechins and their inhibition on MCF-7 cells

Fatty acid synthase (FAS) is a potential target for cancer, but potent inhibitors against FAS are scarce. In this study, we found that activities of catechins on inhibiting FAS increased greatly by heating them in acid. The enhancement was positively correlated to H+ concentration. The inhibitory activities of the final products from different catechins were similar, all of which were less than 1 μg/mL. The product from ( − )-epigallocatechin gallate (EGCG) was stable at room temperature, and its inhibitory kinetics and reacting sites on FAS were obviously different from the known FAS inhibitors. It also affected the viability of MCF-7 cells more obviously than EGCG. A putative route of the reaction progress was proposed and the effective inhibitors were deduced to be oligomers of 2-hydroxy-3-(3′, 4′, 5′-trihydroxyphenyl) propenoic acid by analysis of their spectra. The work affords new and potent FAS inhibitors that would be promising candidates for the treatment of cancer.

C75, a Fatty Acid Synthase Inhibitor, Inhibits Feeding Activity and Pheromone Production in a Moth, Helicoverpa zea

Fatty acid synthesis produces long-chain fatty acids that are principal forms of stored energy and essential constituents of cellular membrane lipids. In animals fatty acid synthesis is catalyzed by fatty acid synthase (FAS) from acetyl-coenyzyme A (CoA) and malonyl-CoA. Cerulenin and C75, potent FAS inhibitors, can inhibit feeding in mammals. Using these inhibitors we examined the effect of feeding inhibition during H. zea larval stage. Growth of larvae injected (30 μg/g body weight) with C75 or cerulenin was significantly delayed during the first 8 hrs after injection, but recovered to normal levels within 20 hrs. During the first 8 hr period, the amount of consumed diet in the inhibitor treated larvae was significantly less than the control group. The retardation of larval development could be caused from the reduction of food intake after injection of the inhibitor. The result indicates that C75 or cerulenin inhibits fatty acid synthesis, resulting in feeding suppression in the larval moth as demonstrated in vertebrates. Pheromone production was significantly decreased in the isolated pheromone gland of H. zea females treated with FAS inhibitors. Pheromone production was inhibited by blocking fatty acid synthesis, even though PBAN stimulated pheromone biosynthesis. After topical application of D3-16: Acid to pheromone glands the relative labeled pheromone amount was increased when the gland was incubated with C75. This result indicates that a part of the pheromone amount could be synthesized from 16: Acid directly when fatty acid synthesis was blocked. These results indicate that the inhibitors have a potential possibility to control insect feeding activity and inhibit pheromone biosynthesis in moths.

Novel inhibitors of fatty‐acid synthase from green tea (Camellia sinensis Xihu Longjing) with high activity and a new reacting site

Recent studies have shown that FAS (fatty acid synthase) is a potential therapeutic target of obesity. In the present paper we report that extract of green tea (Camellia sinensis Xihu Longjing) inhibits FAS effectively with an IC50 value of 12.2 microg dry weight/ml. The ability of GTE (green tea extract) to inhibit FAS is more potent than that of two known inhibitors in green tea leaves, EGCG (epigallocatechin gallate) and ECG (epicatechin gallate). We find that (-)-CG (catechin gallate) is a very potent inhibitor of FAS, with an IC50 of 1.5 microg/ml, and may contribute to the high inhibitory effect of GTE on FAS. The inhibitory mechanism of (-)-CG is not mainly involved in its binding to the beta-oxoacyl reductase domain to which both (-)-EGCG and (-)-ECG mainly bind. By analyses of the inhibitory kinetics and the structure of the gallated catechins, we found that the acyl transferase domain may be the main site reacting with (-)-CG, the structure consisting of a B ring, a C ring and a gallate ring, which is possibly essential for its inhibitory efficacy. The polyphenols rather than the alkaloids are the main fractions contributing to the inhibitory effect of GTE on FAS. During separation we also found that the total ability of this portion to inhibit FAS increases by 15-fold, and this may be due to some novel potent inhibitor of FAS other than (-)-CG being formed.

Keemun black tea extract contains potent fatty acid synthase inhibitors and reduces food intake and body weight of rats via oral administration

The inhibitory effects of a black tea extract on fatty acid synthase were measured through inhibition kinetics. The Keemun black tea extract showed more potent inhibitory activity on fatty acid synthase than green tea extract. Additionally, the inhibitory ability of the black tea extract depended on the extracting solvent and the conditions used. Only 10–23% of the inhibitory activity from the black tea was extracted by the general method of boiling with water. The results suggested that the main fatty acid synthase inhibitors in black tea might be theaflavins. Which were more potent than epigallocatechin gallate or C75. The reaction site on the fatty acid synthase and the inhibition kinetics for the extract were different from those of epigallocatechin gallate or C75. In addition, Keemun black tea extract significantly reduced food intake, body weight and blood triglyceride of diet-induced obesity SD rats via oral administration.

The galloyl moiety of green tea catechins is the critical structural feature to inhibit fatty-acid synthase

It has been reported that inhibition of fatty-acid synthase (FAS) is selectively cytotoxic to human cancer cells. Considerable interest has developed in identifying novel inhibitors of this enzyme complex. Our previous work showed that green tea (−)-epigallocatechin gallate can inhibit FAS in vitro. To elucidate the structure–activity relationship of the inhibitory effects of tea polyphenols, we investigated the inhibition kinetics of the major catechins and analogues. Ungallated catechins from green tea do not show obvious inhibition compared with gallated catechins. Another gallated catechin, (−)-epicatechin gallate, was also found as a potent inhibitor of FAS and its inhibition characteristics are similar to (−)-epigallocatechin gallate. Furthermore, the analogues of galloyl moiety without the catechin skeleton such as propyl gallate also showed obvious slow-binding inhibition, whereas the green tea ungallated catechin not. Atomic orbital energy analyses suggest that the positive charge is more distinctly distributed on the carbon atom of ester bond of galloyl moiety of gallate catechins, and that gallated forms are more susceptible for a nucleophilic attack than other catechins. Here we identify the galloyl moiety of green tea catechins as critical in the inactivation of the ketoacyl reductase activity of FAS for the first time.