Comparative docking of dual conformations in human fatty acid synthase thioesterase domain reveals potential binding cavity for virtual screening of ligands

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4’ phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (?.164 kcal/mol) and B (?.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: ?5.872 kcal/mol and B form: ?5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.     

Computational screening of fatty acid synthase inhibitors against thioesterase domain

Thioesterase (TE) domain of fatty acid synthase (FAS) is an attractive therapeutic target for design and development of anticancer drugs. In this present work, we search for the potential FAS inhibitors of TE domain from the ZINC database based on similarity search using three natural compounds as templates, including flavonoids, terpenoids, and phenylpropanoids. Molecular docking was used to predict the interaction energy of each screened ligand compared to the reference compound, which is methyl γ-linolenylfluorophosphonate (MGLFP). Based on this computational technique, rosmarinic acid and its eight analogs were observed as a new series of potential FAS inhibitors, which showed a stronger binding affinity than MGLFP. Afterward, nine docked complexes were studied by molecular dynamics simulations for investigating protein–ligand interactions and binding free energies using MM-PB(GB)SA, MM-3DRISM-KH, and QM/MM-GBSA methods. The binding free energy calculation indicated that the ZINC85948835 (R34) displayed the strongest binding efficiency against the TE domain of FAS. There are eight residues (S2308, I2250, E2251, Y2347, Y2351, F2370, L2427, and E2431) mainly contributed for the R34 binding. Moreover, R34 could directly form hydrogen bonds with S2308, which is one of the catalytic triad of TE domain. Therefore, our finding suggested that R34 could be a potential candidate as a novel FAS-TE inhibitor for further drug design.     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

Structural and functional analyses of a saturated acyl ACP thioesterase, type B from immature seed tissue of Jatropha curcas

The distinguishing structural and functional domains of plant acyl-acyl carrier protein (ACP) thioesterases and their complex interaction with the ACP-linked fatty acid substrate complex have remained elusive. E. coli based heterologous expression and characterisation of many plant thioesterases reported so far have not been extended and linked to in silico modelling studies to explain the diversity in plant thioesterase substrate specificities. In this study, a thioesterase cDNA isolated from immature seed tissues of Jatropha curcas was found to be type B and specific to stearoyl acyl ACP when expressed in E. coli K27fadD88, a lipid utilisation mutant. Homology modelling and molecular docking of a selected region of the isolated JcFatB protein predicted that it had high affinity towards both stearate (18:0) and palmitate (16:0). Structural analysis of the sequence confirmed the presence of a transit peptide that is processed in multiple steps. The enzyme is localised in the chloroplasts and has an N-terminal inner chloroplast transmembrane domain characteristic of type B plant thioesterases. Docking of ligands with JcFatB and its comparison with a modelled Jatropha thioesterase type A provided further evidence for native substrate preferences of Jatropha thioesterases. This study provides essential clues to develop future methods for large-scale bacterial production of free fatty acids and for design of strategies to modulate the seed oil composition in this important non-edible, seed oil plant.     

酰基辅酶A硫酯酶在脂肪酸代谢途径中的作用

酰基辅酶A硫酯酶(acyl-CoA thioeaterase,ACOT)属于广泛的硫酯水解酶的一类酶,分属于α/β水解酶超家族和“HotDog”超家族.ACOT作用范围很广,可以裂解支链脂肪酸、短链和长链饱和及不饱和酰基辅酶A等多种含辅酶A的底物,在脂肪酸代谢途径中发挥信号转导、基因调控等生理生化作用.因此研究ACOT...     

Demonstration of dimethylnonanoyl-CoA thioesterase activity in rat liver peroxisomes followed by purification and molecular cloning of the thioesterase involved.

Peroxisomes play an indispensable role in cellular fatty acid oxidation in higher eukaryotes by catalyzing the chain shortening of a distinct set of fatty acids and fatty acid derivatives including pristanic acid (2,6,10,14-tetramethylpentadecanoic acid). Earlier studies have shown that pristanic acid undergoes three cycles of beta-oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA (DMN-CoA) which is then transported to the mitochondria for full oxidation to CO(2) and H(2)O. In principle, this can be done via two different mechanisms in which DMN-CoA is either converted into the corresponding carnitine ester or hydrolyzed to 4,8-dimethylnonanoic acid plus CoASH. The latter pathway can only be operational if peroxisomes contain 4,8-dimethylnonanoyl-CoA thioesterase activity. In this paper we show that rat liver peroxisomes indeed contain 4,8-dimethylnonanoyl-CoA thioesterase activity. We have partially purified the enzyme involved from peroxisomes and identified the protein as the rat ortholog of a known human thioesterase using MALDI-TOF mass spectrometry in combination with the rat EST database. Heterologous expression studies in Escherichia coli established that the enzyme hydrolyzes not only DMN-CoA but also other branched-chain acyl-CoAs as well as straight-chain acyl-CoA-esters. Our data provide convincing evidence for the existence of the second pathway of acyl-CoA transport from peroxisomes to mitochondria by hydrolysis of the CoA-ester in peroxisomes followed by transport of the free acid to mitochondria, reactivation to its CoA-ester, and oxidation to CO(2) and H(2)O. (c)2002 Elsevier Science.