Discovery of GSK837149A, an inhibitor of human fatty acid synthase targeting the b-ketoacyl reductase reaction ´ ´ ´ ´ Marıa Jesus Vazquez1, William Leavens2, Ronggang Liu3, Beatriz Rodrıguez1, Martin Read2, ´ Stephen Richards2, Deborah Winegar4 and Juan Manuel Domınguez1 GlaxoSmithKline GlaxoSmithKline GlaxoSmithKline GlaxoSmithKline R&D, R&D, R&D, R&D, ´ ´ Biological Reagents and Assay Development Department, Centro de Investigacion Basica, Tres Cantos, Spain Analytical Chemistry Department, Medicines Research Center, Stevenage, UK Cardiovascular and Urogenital Centre of Excellence for Drug Discovery, King of Prussia, PA, USA Metabolic Centre of Excellence for Drug Discovery, Research Triangle Park, Durham, NC, USA Keywords breast cancer; fatty acid synthase; GSK837149A; ketoacyl reductase; obesity Correspondence ´ J M Domınguez, GlaxoSmithKline R&D, ´ ´ Centro de Investigacion Basica, C ⁄ Santiago ´ Grisolıa 4, 28760-Tres Cantos, Spain Fax: +34 91 807 4062 Tel: +34 91 807 4000 E-mail: juan.m.dominguez@gsk.com (Received 28 November 2007, revised 21 January 2008, accepted 30 January 2008) doi:10.1111/j.1742-4658.2008.06314.x GSK837149A has been identified as a selective inhibitor of human fatty acid synthase (FAS) The compound was first isolated as a minor impurity in a sample found to be active against the enzyme in a high-throughput screening campaign The structure of this compound was confirmed by NMR and MS studies, and evaluation of the newly synthesized molecule confirmed its activity against FAS The compound and other analogs synthesized, all being symmetrical structures containing a bisulfonamide urea, act by inhibiting the b-ketoacyl reductase activity of the enzyme GSK837149A inhibits FAS in a reversible mode, with a Ki value of $ 30 nm, and it possibly binds to the enzyme–ketoacyl-ACP complex Although initial results suggest that cell penetration for these compounds is impaired, they still can be regarded as useful tools with which to probe and explore the b-ketoacyl reductase active site in FAS, helping in the design of new inhibitors Fatty acids are essential to all living cells and have a wide diversity of biological functions As components of phospholipids, triglycerides and other complex lipids, fatty acids provide structural integrity to cellular membranes and cell walls and serve as a source of energy In the free state, fatty acids can act in the transmission of cellular signals or can be used to modify proteins during post-translational processing The de novo synthesis of fatty acids is accomplished by the complex, multicomponent enzyme fatty acid synthase (FAS; EC 3.2.1.85) FAS catalyzes the formation of long-chain fatty acids from acetyl-CoA and malonylCoA in a cyclic sequence of reactions, adding two carbon units per cycle, each reaction being catalyzed by a different enzymatic activity The elongating chain is covalently linked to an acyl carrier protein (ACP), which transports it through the active sites where each reaction is catalyzed, namely malonyl-CoA ⁄ acetylCoA-ACP-transacylase (MAT), b-ketoacyl synthase (KS), b-ketoacyl reductase (KR), dehydratase (DH), and b-enoyl reductase (ER) Once the synthesized fatty acid has reached the desired length, it is released from the ACP by means of a thioesterase (TE) activity FAS exists in two basic forms in nature Type I FAS is a multienzyme that integrates all the active sites catalyzing the individual reactions into one single polypeptide chain Type I FAS can be further classified as animal type I FAS, present as a homodimeric protein, and microbial type I FAS, which is present as oligomers of higher order (hexamers or dodecamers), including two types of polypeptide [1] In contrast, type II FAS, present in bacteria, plants, and eukaryotic mitochondria, is Abbreviations ACP, acyl carrier protein; DH, dehydratase; ER, b-enoyl reductase; FabG, b-ketoacyl reductase enzyme of type II FAS; FAS, fatty acid synthase; HTS, high-throughput screening; KR, b-ketoacyl reductase; KS, b-ketoacyl synthase; MAT, malonyl-CoA ⁄ acetyl-CoA-ACPtransacylase; Q-TOF, quadrupole TOF; TE, thioesterase 1556 FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS ´ M J Vazquez et al composed of individual monofunctional proteins, each of which catalyzes one individual reaction Whereas type I FAS produces only palmitate or stearate, type II FAS is able to synthesize fatty acids of different lengths, degrees of saturation, branching, and hydroxylation [2] The homodimeric animal type I FAS was initially believed to be arranged in an antiparallel, head-to-tail mode with a large interdomain region that served as an area of contact between the two monomers This region was thought to be critical for the correct association of the two monomers required for functional activity [3,4] Such an arrangement would give rise to two identical ‘catalytic chambers’, each formed by the active sites of the two polypeptide chains The detailed ˚ structure of mammalian type I FAS at 4.5 A resolution has recently become available [5] and has revealed a very different organization The two monomers are arranged in a parallel conformation, forming an intertwined dimer with two catalytic chambers, each chamber being composed of active sites from the same monomer except for the KS site The shape of this dimer is rather asymmetric, and hence the sizes of the catalytic chambers are significantly different from one another This fact, together with the considerable distances among the active sites within each chamber, suggests that the enzyme structure must be flexible and that domain motions must occur during each catalytic cycle in order to ensure the accessibility of the elongating fatty acid to the individual active sites Clinical studies conducted over the last decade have shown that a biologically aggressive subset of carcinomas constitutively expresses high levels of type I FAS and that upregulation of FAS gene expression is an early event in the development of certain types of cancer [6] Moreover, two inhibitors of FAS, cerulenin and C-75, have demonstrated significant antitumor activity Cerulenin has been shown to inhibit the growth of neoplasic cells in vitro, with the inhibition being reversed only with high, supraphysiological concentrations of palmitate [7] C-75, on the other hand, has demonstrated efficacy in vivo against xenografts of breast cancer cells in nude mice [8] Although the mechanisms behind the antitumor effect of FAS inhibition are still under discussion, it is thought that the intracellular accumulation of toxic levels of malonylCoA and ⁄ or impairments in cellular membrane structure may contribute to the inhibition of cell growth Recent data obtained with breast cancer and endometrial adenocarcinoma cells showing that FAS differentially modulates the sensitivity of the cells to estrogen have led to the proposal that FAS inhibition may provide an alternative breast cancer therapy that may FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS GSK837149A, inhibitor of human fatty acid synthase avoid the onset of endometrial hyperplasia associated with current tamoxifen-based therapies [9] In addition to this role in oncogenicity, FAS has also been considered as a possible target for the treatment of obesity Both cerulenin and C-75 have been shown to reduce food intake and cause profound weight loss in mice [10] These effects correlate with reciprocal changes in the expression of orexigenic and anorexigenic neuropeptides in the hypothalamus The mechanisms responsible for these effects are thought to be related to increased malonyl-CoA levels in the hypothalamus [11,12] Despite the increased interest in FAS as a therapeutic target for cancer and obesity, there remain few selective human FAS inhibitors The two best characterized FAS inhibitors, cerulenin and C-75, are affected by several drawbacks that limit their use, the main one being their irreversible behavior Cerulenin is a natural antibiotic produced by the fungus Cephalosporium ceruleans, and is known to inhibit the KS reaction by covalently binding to the Cys residue in the corresponding active site [13] However, this compound cannot be considered to be a selective inhibitor of human FAS, as it inhibits both type and type FAS [14], and possesses high chemical reactivity, which is responsible for its low specificity Indeed, interference of cerulenin with other cellular processes besides fatty acid synthesis (e.g protein acylation, cholesterol synthesis, and proteolysis) has been described [15] Moreover, this reactivity is also responsible for the low chemical stability of the compound In addition, the observed effects of cerulenin in in vivo experiments have been poorly reproducible [16], suggesting that strain differences between mice may influence the response to this compound C-75 is a weakly potent (Ki 16 mm) synthetic inhibitor designed through molecular modeling based on the mechanism of ketoacyl synthesis at the KS active site [17] Despite this targeted design, it has been shown to interact at several sites in FAS; that is, it is not a selective KS inhibitor [18] Certain activities ascribed to C-75 appear to be paradoxical (increase in malonyl-CoA and stimulation of carnitine palmitoyltransferase-1) [19], hence complicating the interpretation of the effects observed with this compound The antiobesity drug Orlistat has been reported to inhibit FAS by interacting with its TE domain Crystal structures of Orlistat covalently bound to FAS at the TE domain have recently become available [20] Like the other molecules described above, Orlistat is not a selective inhibitor of FAS, and indeed the antiobesity effects of Orlistat are thought be primarily related to the irreversible inhibition of gastric and pancreatic lipases [21] Some flavonoids have been 1557 ´zquez et al M J Va GSK837149A, inhibitor of human fatty acid synthase High-throughput screening (HTS) to identify human FAS inhibitors Aproximately 550 000 compounds belonging to the GlaxoSmithKline collection were screened in the search for inhibitors of human FAS As the enzyme consumes 14 NADPH molecules during the synthesis of one molecule of palmitate, an assay monitoring NADPH consumption, based on substrate-induced quenching technologies [24], was used in the HTS campaign The compounds were tested at a single concentration of 10 lm in the assay mixture, using a final assay volume of lL In total, 12 735 compounds yielding significant inhibition of FAS (detected by the impaired ability of the enzyme to consume NADPH in the assay) were selected These compounds were grouped in different chemical clusters, prioritized according to their chemical features, and evaluated in a dose–response manner for their potency against the enzyme Among the compounds selected, SKF-100601 was identified as the most promising one because of its biological and chemical properties (structure shown in Fig 1) This compound appeared to inhibit FAS F N N F O N N N F O S N N O N N O O S S O O N N GSK837149A Fig Chemical structure of SKF-100601 and GSK837149A 1558 The impure SKF-100601 sample (103 mg) was subjected to preparative reverse-phase chromatography in a Supelco ABZ Plus column As shown in Fig 2, four 20 100 15 10 50 0 10 12 14 Time (min) O SKF-100601 N Identification of the active component 1/IC50 (mL·µg–1) Results reversibly with a pIC50 of 6.0, and acted exclusively on the KR activity, as demonstrated by experiments such as those described later in this article In addition, its chemical structure looked suitable for the introduction of a diversity of modifications that could contribute to the modulation of its pharmacological properties A modest chemical effort was therefore initiated, consisting in searching for analogs in corporate databases as well as in affording the synthesis of the molecule All of the 222 analogs selected failed to inhibit FAS when they were tested at 100 lm in the assay Furthermore, the newly synthesized molecule also failed to inhibit the enzyme It was concluded that the activity observed in the original sample used in HTS was probably due to an impurity that was absent in the newly synthesized molecule If this hypothesis was correct, the potency of the active component should be at least one order of magnitude higher than that found for the HTS sample, as it was determined that the HTS sample was 85% pure With this in mind, and considering the interesting biological properties described above, it was decided to undertake the purification and identification of the FAS inhibitor present in the SKF-100601 sample used in the HTS campaign Sum of Abs 190–600 nm reported to inhibit FAS, but it is unclear whether this family of compounds act in the KS or in the KR domain [22] The discovery of hydroxyquinolinones [23] as FAS inhibitors has also been reported recently, but details of the mechanisms of action of these molecules at the enzyme level have not been described In this article, we describe the discovery of GSK837149A, the first selective human FAS inhibitor known to act specifically and selectively on the KR activity of the enzyme N Fig Preparative chromatography of SKF-100601 The chromatogram corresponds to the injection of a 0.5 mL sample containing 30 mg of the impure SKF-100601 material that had been identified as inhibiting FAS in the HTS Samples were collected, dried, dissolved in dimethylsulfoxide, and tested for their inhibition in the FAS assay from 50 lgỈmL)1 to 0.8 ngỈmL)1 The line corresponds to the elution profile as the summed response of all wavelengths from 190 to 600 nm in arbitrary units, whereas the bars show the inhibitory activity of the fractions collected: their ability to inhibit FAS is expressed as the reverse of the IC50 in lgỈmL)1 for graphical purposes No value is reported when no inhibition was found even at the highest concentration tested FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS ´ M J Vazquez et al GSK837149A, inhibitor of human fatty acid synthase major peaks were identified Samples corresponding to these peaks, as well as to the rest of the elution profile, were pooled, dried, and tested for their ability to inhibit FAS Only the fractions corresponding to the peak eluting at 7.56 were able to inhibit the enzyme Notably, the major component of the mixture, which eluted at 10 min, did not show any inhibition of FAS The active component was subjected to liquid chromatography high-resolution MS, confirming the presence of a single entity, which yielded the mass spectrum shown in Fig 3A The parent ion with an m ⁄ z of 555.1238 Da was observed, as well as masses of 446.0598 Da and 398.0923 Da corresponding to fragments of the parent ion Elemental composition analysis was performed on the parent ion, giving a molecular formula of C23H22N8O5S2 with a 0.2 p.p.m error The purified active sample was finally subjected to NMR analysis The spectrum obtained is shown in Fig 3B Comparison of this spectrum with that of purified SKF-100601 (obtained from the preparative chromatography described above) suggested that the trifluorophenyl moiety present in SKF-100601 was absent in this active component Simple cleavage of the urea to yield the corresponding aniline was ruled out Indeed, the relatively sharp peak for the amino group of urea pointed to a dimeric structure with the urea moiety bridging between the two halves of the A molecule This, combined with the molecular formula and mass values deduced above, led us to propose the structure shown in the lower part of Fig as the one corresponding to the compound inhibiting FAS Confirmation of the activity On the basis of these analytical studies, 70 mg of the compound corresponding to the structure shown in Fig 1, which was registered as GSK837149A, was prepared The compound was tested in the FAS assay in parallel with the SKF-100601 sample used in the HTS campaign As shown in Fig 4, the new compound was able to inhibit FAS and showed a significantly higher potency than the SKF-100601 sample Fitting of these concentration–response curves to Eqn (1) (see Experimental procedures) yielded pIC50 values of 7.8 and 6.0 for GSK837149A and for the sample of SKF-100601 respectively These differences in potency suggest that GSK837149A represents 1–2% of the impure SKF100601 sample The new compound was also able to inhibit the FAS activity of crude cytosolic extracts from rat liver homogenates (data not shown), demonstrating that the ability to inhibit FAS is genuine and is not an artefact generated by the use of recombinant enzyme, and also demonstrating that such inhibition is not restricted to the human enzyme but extends to FAS enzymes from other mammals 555.1238 100 100 80 446.0598 80 40 398.0923 20 0 100 300 200 400 500 600 700 800 900 1000 Inhibition (%) % 60 60 40 m/z 20 B 0.14 0.12 0.1 –11 0.08 –10 –9 –8 –7 –6 –5 –4 log [compound] (M) 0.06 0.04 0.02 10 Chemical shift (p.p.m.) Fig Analytical data on the active component identified (A) Mass spectrum (B) Proton NMR spectrum FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS Fig Concentration–response curves of the newly synthesized GSK837149A and the impure SKF-100601 sample Evaluation of FAS activity was performed as described in Experimental procedures at different concentrations of compounds Data were fitted to Eqn (1) to calculate the pIC50 value for GSK837149A and the apparent pIC50 for SKF-100601 as if it was pure and responsible for the inhibition caused The data correspond to the average of triplicate determinations ± SEM d, GSK837149A; s, SKF-100601 1559 ´zquez et al M J Va GSK837149A, inhibitor of human fatty acid synthase Analysis of the individual activities catalyzed by FAS There are several methods reported in the literature for analysis of the individual FAS reactions [18,25], but not all of them are adequate for the rapid analysis of a large number of samples such as those derived from an HTS campaign We decided to investigate the possibility of using suitable substrates for some of the individual reactions and monitoring them spectrophotometrically through the consumption of NADPH as described in Dodds et al [26] for the bovine enzyme Therefore, acetoacetyl-CoA was used to monitor KR, b-hydroxybutyryl-CoA was used to monitor the activity of DH, and crotonoyl-CoA was used to monitor the activity of ER NADPH consumption was observed in all the cases (data not shown), confirming that human FAS is able to utilize these CoA derivatives as substrates The fact that NADPH is also consumed when b-hydroxybutyryl-CoA is used also confirms that the enzyme is able to catalyze all the reactions following the one corresponding to the substrate used The reactions were also monitored by HPLC, using an appropriate method to isolate and quantify the intermediate substrates and products Figure 5A shows that when acetoacetyl-CoA and NADPH were used as substrates, butyryl-CoA was formed in parallel to the consumption of acetoacetylCoA, whereas traces of b-hydroxybutyryl-CoA and of crotonoyl-CoA (the latter was present in very small A B amounts and was difficult to quantify properly) were also observed No traces of free CoA were detected On the other hand, when acetyl-CoA and malonylCoA were used as substrates, their disappearance ran in parallel with the release of free CoA, whereas no other intermediate of the FAS reaction was detected (Fig 5D) When b-hydroxybutyryl-CoA was used (Fig 5B), the formation of butyryl-CoA occurred simultaneously with the consumption of b-hydroxybutyryl-CoA, and a steady-state concentration of the intermediate crotonoyl-CoA was also detected Finally, when crotonoyl-CoA was used, slow formation of butyryl-CoA was seen as well as significant levels of b-hydroxybutyryl-CoA (Fig 5C), suggesting that DH is able to promote the reverse reaction quite efficiently, as already reported [27] Therefore, the use of these substrates can be exploited to analyze the effect of FAS inhibitors on the KR, DH and ER reactions in a highly efficient manner simply by observing the effect on NADPH consumption Identification of the inhibited FAS activity and selectivity studies GSK837149A was used in the reactions described above to identify which of the enzymatic reactions was inhibited by the compound Concentration–response studies were conducted on these reactions: as shown in Fig 6, the compound is able to inhibit KR, as well as the global FAS reaction, but neither DH nor ER 2 1.5 nmol nmol 1.5 1 0.5 0.5 0 50 100 150 50 C D 0.8 nmol nmol 1.5 0.5 0.6 0.4 0.2 0 50 100 150 Time (min) 1560 100 Time (min) Time (min) 200 250 50 100 150 Time (min) 200 250 Fig Analysis of the reactions catalyzed by FAS in the presence of different substrates The reactions were performed in duplicate as described under Experimental procedures, except for the reaction volume, which was 100 lL, with 30 lM NADPH and 10 nM FAS and the substrates described below At the desired times, the reactions were stopped in an ice bath, and 50 lL was injected in the HPLC system to analyze the components of the reaction mixture following the method described in Experimental procedures The time courses correspond to the reactions run with 40 lM acetoacetylCoA (A), 40 lM b-hydroxybutyryl-CoA (B), 40 lM crotonoyl-CoA (C), and lM acetylCoA and 20 lM malonyl-CoA (D) The following products were monitored: acetyl-CoA (d), malonyl-CoA ( ), CoA (.), acetoacetylCoA (s), b-hydroxybutyryl-CoA (h), crotonoyl-CoA (D), and butyryl-CoA ( ) FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS ´ M J Vazquez et al GSK837149A, inhibitor of human fatty acid synthase Inhibition (%) 100 80 60 40 20 –10 –9 –8 –7 –6 –5 –4 log [GSK837149A] (M) Fig Concentration–response curves of GSK837149A on the individual activities catalyzed by FAS The reactions were performed as described in Experimental procedures with 30 lM NADPH, 10 nM FAS, and the following substrates: lM acetyl-CoA and 20 lM malonyl-CoA (d); 40 lM acetoacetyl-CoA (s); 40 lM b-hydroxybutyryl-CoA (h); and 40 lM crotonoyl-CoA (D) Data were fitted to Eqn (1) to calculate the pIC50 values The data presented here correspond to the average of triplicate determinations ± SEM Because the other activities involved in FAS (i.e MAT, KS, TE, and the binding to ACP) were not tested, it was not possible to rule out any effect on these at this point However, the similar potencies found for the compound in the global FAS reaction (i.e the reaction promoted in the presence of acetylCoA and malonyl-CoA besides NADPH) and in the KR reaction suggest that KR is the only activity being inhibited Moreover, several analogs of GSK837149A were prepared: as shown in Fig 7, a good linear corre9 lation (r2 = 0.97) was observed between the pIC50 values obtained when the global FAS and the KR activities were measured Therefore, the possibility that these compounds act on FAS by inhibiting another activity in addition to KR seems unlikely To check the selectivity of these inhibitors, their effect on the b-ketoacyl reductase enzyme of type II FAS (FabG), was tested, following the procedures described in Patel et al [28] Neither GSK837149A nor any of the analogs caused any inhibition of FabG when they were present at 100 lm, suggesting that the compounds are selective in inhibiting the human KR of type I FAS The reversible nature of the inhibition caused by GSK837149A on FAS was also demonstrated: as shown in Fig 8, FAS could be rescued from the inhibitory effect of GSK837149A by simply removing the unbound compound by filtration and diluting to reconstitute the initial volume The drop-off in the effect of the compound corresponded to the dilution made, and as the enzymatic assay was run immediately after diluting the filtered sample, the dissociation step seems to be rapid The same result was obtained when other analogs of GSK837149A were tested in the global FAS assay and in the KR activity assay (data not shown) 100 80 Inhibition (%) 120 60 40 20 pIC50 on KR 8.5 –20 –10 7.5 –9 –8 –7 –6 –5 log [GSK837149A] (M) 6.5 5.5 5.5 6.5 7.5 8.5 pIC50 on global FAS Fig Correlation among pIC50 values obtained in the assays monitoring the global FAS reaction and KR FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS Fig Reversibility of inhibition by GSK837149A The experiment was performed by concentration and subsequent dilution (25-fold) of the enzyme and inhibitor mixtures, using filters of a suitable pore size to allow the separation of the free compound from the enzyme, as described in Experimental procedures All samples were compared with their corresponding controls treated similarly in the absence of compound The values in the horizontal axis correspond to the concentration of compound in the sample before any concentration and dilution cycle The lines represent the results of fitting the data to Eqn (1) d, untreated sample (no dilution); h, sample concentrated and diluted once (25-fold dilution); D, sample concentrated and diluted twice (625-fold dilution) 1561 ´zquez et al M J Va GSK837149A, inhibitor of human fatty acid synthase Enzymatic mode of inhibition Table Summary of inhibition patterns caused by GSK837149A on the KR reaction catalyzed by FAS With the aim of finding the mode of inhibition of GSK837149A on the KR activity of FAS substrates, titrations were performed at different concentrations of the inhibitor, varying the concentration of one substrate while keeping the concentration of the other fixed at saturating (i.e 10 times the Km value) or nonsaturating (i.e at the Km value) levels The results are summarized in Fig and in Table The inhibition constants obtained in all cases suggest that the Ki value for GSK837149A is between 25 and 35 nm, in agreement with the observed pIC50 value This compound showed a clear competitive inhibition with respect to NADPH that was not altered by the use of saturating concentrations of acetoacetyl-CoA, suggesting that the compound binds to the same enzyme form that NADPH does On the other hand, the inhibition pattern with respect to acetoacetyl-CoA was clearly uncompeitive In all, these results suggest that the KR activity catalyzed by FAS follows a compulsory ordered kinetic mechanism, acetoacetyl-CoA being the first substrate to bind, and that the inhibitor binds to the enzyme–acetoacetyl-CoA complex to eventually form a ternary dead-end complex enzyme–acetoacetylCoA–GSK837149A Discussion In the search for novel inhibitors of human FAS, we have identified GSK837149A as a potent and selective inhibitor of the enzyme The high potency of this compound has allowed its detection in an HTS campaign, even though it was present as a minor impurity in the sample of a different compound, and such potency has also enabled us to track it during the isolation and Varied substrate Concentration of fixed substrate Inhibition pattern Ki (nM) NADPH NADPH Acetoacetyl-CoA · Km 10 · Km · Km Competitive Competitive Uncompetitive 35 ± 24 ± 34 ± purification process from the original sample NMR and high-resolution MS data were pooled, and drawing on experience from related compounds that had been fully assigned by means of NOE experiments, a putative dimeric structure was proposed Such a structure, shown in Fig 1, satisfied the NMR data in all respects and also gave the required mass of 555.1233 Da The synthesis of the compound corresponding to this structure, GSK837149A, led us to confirm that it was responsible for the inhibition of FAS The results obtained demonstrate that GSK837149A inhibits the b-ketoacyl reductase activity of the enzyme Our experimental data demonstrate that neither DH nor ER activities are inhibited by the compound The other activities involved in the FAScatalyzed cycle (MAT, KS, TE, and the binding to ACP) were not tested; however, the similarity between the inhibition potencies on KR and on the global FAS reaction suggests that KR is the only activity of the enzyme being inhibited Moreover, this similarity in potency extends to other analogs of GSK837149A covering three orders of magnitude in potency, and indeed an excellent correlation is observed among the pIC50 values for KR and global FAS If another enzymatic reaction was inhibited, such a correlation would be 1/v0 / v0 C 10 / v0 B A 1.5 0.5 0 0 0.02 0.04 0.06 / [AcAcCoA] 0.08 0.01 0.02 0.03 / [NADPH] 0.04 0.01 0.02 0.03 0.04 / [NADPH] Fig Double reciprocal plots of the inhibition caused by GSK837149A of the KR reaction of FAS with different varied substrates The experiment was run as described in Experimental procedures at a fixed nonsaturating concentration of NADPH (A) and at fixed nonsaturating (B) and saturating (C) concentrations of acetoacetyl-CoA Initial rates are expressed in lM NADPH consumed per and concentrations are in lM The lines correspond to the results of fitting by nonlinear regression all the experimental data to Eqn (2) (B,C) or Eqn (3) (A) The resulting inhibition constants are summarized in Table Concentrations of the inhibitor were nM (s), 16 nM (d), 31 nM (h), 62.5 nM ( ), 125 nM (D) and 250 nM ( ) 1562 FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS ´ M J Vazquez et al difficult to explain unless the structure–activity relationships sustaining these inhibitions were the same for KR as for the other sites affected, which seems very unlikely, especially in the light of the differences among the individual active sites in FAS [5] As stated previously, the human FAS inhibitors most extensively characterized so far are not selective for the human enzyme In fact, most of them act irreversibly through covalent bond formation due to the high chemical reactivity of the molecules rather than to their similarity with intermediates of the enzymatic reaction, which would lead to a more selective mechanism-based inhibition GSK837149A has shown to act reversibly, as its inhibition disappeared rapidly after dilution, suggesting that the kinetics of association and dissociation are fast The selectivity of the compound is endorsed by its inability to inhibit FabG, the KR counterpart in type II FAS The potency of GSK837149A is such that its Ki value (25–35 nm) is similar to the concentration of enzyme used in the experiments reported in this article (10 nm) Under these conditions, inhibitor depletion is significant, and therefore the treatment of enzyme inhibition data through classic steady-state methods is questionable The quadratic equation derived by Morrison [29] was used to recalculate the pIC50 values that had been obtained with Eqn (1) (see Experimental procedures), and essentially identical values were yielded Analogously, the considerations established by Williams & Morrison [30] were taken into account for Ki determination in the mode of inhibition studies However, the results shown in Fig are clear, and there are no reasons to suspect that these are erroneous, as might have been the case if a noncompetitive pattern had been obtained In any case, other simpler, graphical methods considering inhibitor depletion, such as those described in Henderson [31], have been used and have yielded identical Ki values and inhibition modes (data not shown) The fact that the compound behaves as a competitive inhibitor with respect to NADPH and an uncompetitive inhibitor with respect to acetoacetyl-CoA suggests a compulsory order in the binding of the substrates, with the reduced nucleotide being the second substrate to bind There are numerous precedents in the literature for compulsory ordered mechanisms in reductases and dehydrogenases depending on nicotinamide adenine dinucleotides, but these mechanisms usually start with the binding of the dinucleotide to the free enzyme [32] A random mechanism has recently been described for the b-ketoacyl reductase of Streptococcus pneumoniae [24] However, we are not aware of many compulsory ordered mechanisms with FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS GSK837149A, inhibitor of human fatty acid synthase the nucleotide binding last in sequence At this point, it is not possible to ignore the possibility that this result may be an artefact introduced by the use of a non-natural substrate such as acetoacetyl-CoA Nonetheless, the result may also have implications for understanding the mechanism that FAS utilizes to carry out each catalytic cycle, suggesting that the growing ketoacyl–ACP complex must reach the KR active site to allow NADPH to bind GSK837149A and several related analogs have been tested for cellular activity in a whole cell fatty acid synthesis assay in HepG2 cells, using [14C]acetate as a precursor, and in human primary adipocytes by monitoring triglyceride accumulation (data not shown) Unfortunately, all the compounds were inactive in these assays Subsequent evaluation in a cell permeability assay confirmed very low cell permeability for GSK837149A Several chemical modifications of the parent compound have been tried, including alkylation of urea and sulfonamide, removal of the sulfonamide groups, replacement of urea by other linkers, and preparation of asymmetrical analogous ureas In total, about 150 compounds have been synthesized, but permeability has not improved and no activity in whole cell has been detected Although this result prevents the immediate therapeutic application of this chemical family, the relevance of the inhibitors is still noticeable because of their high-affinity binding to the enzyme and the novelty of their mechanism of action It is conceivable that they could be utilized as probes to investigate the KR active pocket, to aid the rational design of new molecules inhibiting FAS by binding at such sites Experimental procedures Materials Human FAS was prepared by the Biological Reagents group at GlaxoSmithKline as described below St pneumoniae b-ketoacyl-ACP reductase (FabG) was provided by the same group, following the procedures described in Patel et al [28] Unless otherwise stated, all chemical reagents used were from Sigma-Aldrich (St Louis, MO, USA) Expression and purification of human FAS Human FAS was cloned from a human testis cDNA library created from testis RNA purchased from Clontech (Mountain View, CA, USA) The gene was cloned into the Sal1 ⁄ Not1 sites of pSPORT1 and subsequently engineered into a pFastBac-1 expression system (Invitrogen, Carlsbad, CA, 1563 ´zquez et al M J Va GSK837149A, inhibitor of human fatty acid synthase USA) with a C-terminal His6 tag The protein was expressed in baculovirus-infected Spodoptera frugiperda Sf9 cells under the polyhedron promoter For large-scale production, FAS was expressed at 27 °C in a 10 L or 100 L cell bag using a Wave bioreactor (GE Healthcare, Giles, Buckinghamshire, UK) Sf9 cells were infected using a multiplicity of infection of 1.0 The cells were harvested 48 h after infection by centrifugation at 10 000 g, washed twice with NaCl ⁄ Pi, and flash frozen in a dry ice ethanol bath They were subsequently thawed and mixed at a : (mLỈg)1) ratio with lysis buffer (100 mm NaCl in 25 mm Hepes at pH 7.5) containing 20 mm imidazole Homogenization was carried out by mechanical disruption using a Brinkmann polytron (Brinkmann Instruments Inc., Westbury, NY, USA), removing debris by centrifugation for h at 25 000 g The soluble fraction was filtered through a 1.2 lm filter (PALL, East Hills, NY, USA) and then loaded onto a nickel-chelating Sepharose column (GE Healthcare) equilibrated in lysis buffer with 20 mm imidazole All chromatography steps were performed at °C After loading, the column was washed with five column volumes of lysis buffer with 20 mm imidazole followed by five column volumes of 30 mm imidazole in lysis buffer FAS was eluted with a 30–300 mm imidazole gradient in lysis buffer over two column volumes followed by an eight column volume hold at 300 mm imidazole in lysis buffer Fractions containing FAS were pooled and concentrated using PALL JumboSep filters The sample was then applied to a Superdex 200 column (GE Healthcare) equilibrated with 100 mm NaCl, mm dithiothreitol, mm EDTA and 15% glycerol in 50 mm Tris ⁄ HCl at pH 8.0 Fractions were collected and checked for FAS activity, and active samples were pooled and concentrated using JumboSep filters, aliquoted, and frozen at )80 °C Measurement of FAS activity Fatty acid synthesis catalyzed by FAS was followed by monitoring NADPH consumption During the HTS campaign, this was done by measuring the release of the NADPH-induced quenching of resorufin as detailed in Vazquez et al [24], as this procedure is suited for miniaturization and thus enables the analysis of a large number of samples, minimizing costs and protein expenses In all other cases, the consumption of NADPH was monitored spectrophotometrically through the decrease in the absorbance at 340 nm at 25 °C in 384-well clear-bottomed polystyrene plates (Corning, Corning, NY, USA), using a 384-well plate spectrophotometer (SpectraMax Plus; Molecular Devices, Sunnyvale, CA, USA) The reactions were carried out in a final volume of 50 lL containing lm acetyl-CoA, 20 lm malonyl-CoA, 30 lm NADPH and 10 nm enzyme in 50 mm sodium phosphate buffer at pH 7.0 The extinction coefficient of NADPH for 50 lL 1564 in these plates has been determined to be 3120 m)1 Initial rates were calculated from the slope of the progress curves during the first When the inhibition caused by compounds was being determined, these were added in neat dimethylsulfoxide, ensuring that the final dimethylsulfoxide concentration was 1% in all cases When dealing with compounds of a known molecular mass, their potencies were determined by means of the negative logarithm of the IC50 (i.e the pIC50), as the experiments were carried out by performing serial dilutions of the compounds, and hence the range of concentrations was evenly distributed in a logarithmic rather than a linear scale pIC50s were calculated by fitting the data to Eqn (1), using the nonlinear regression function of grafit 5.0.8 (Erithacus Software Limited) Equation (1) is a modified version of the classic isotherm equation % Inhibition ẳ Min ỵ Max Min ỵ 10logẵI ỵ pIC50ị n 1ị where % inhibition was calculated from the ratio of the initial velocities in the presence and absence of compound, ‘Min’ and ‘Max’ are the lower and higher asymptotes of the sigmoid curve obtained, [I] is the concentration of inhibitor in molar units, and n is the Hill coefficient Measurement of KR, DH and ER activities The activities of the individual reactions catalyzed by FAS were monitored spectrophotometrically through the decrease in the absorbance caused by NADPH at 340 nm at 25 °C in 384-well clear-bottomed polystyrene plates (Corning), using a 384-well plate spectrophotometer (SpectraMax Plus; Molecular Devices) The reactions were carried out in a final volume of 50 lL containing 30 lm NADPH and 10 nm enzyme in 50 mm sodium phosphate buffer at pH 7.0, as well as either 40 lm acetoacetyl-CoA (for determining KR), 40 lm b-hydroxybutyryl-CoA (for DH), or 40 lm crotonoyl-CoA (for ER) The concentrations of these substrates have been determined previously to correspond to their apparent Km values Initial rates were calculated from the slope of the progress curves during the first When the inhibition caused by compounds was being determined, these were added in neat dimethylsulfoxide, ensuring that the final dimethylsulfoxide concentration was 1% in all cases For determining the inhibition pattern caused by GSK837149A, initial velocity data were obtained using the same buffer conditions with variable concentrations of acetoacetyl-CoA (8–500 lm) at a fixed nonsaturating concentration of NADPH (30 lm), as well as at variable concentrations of NADPH (8–250 lm) at fixed nonsaturating and saturating concentrations of acetoacetyl-CoA (20 lm and 200 lm, respectively) Data analysis was performed by fitting the experimental data to the appropriate equations FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS ´ M J Vazquez et al GSK837149A, inhibitor of human fatty acid synthase with the nonlinear regression function of grafit 5.0.8 (Erithacus Software Limited), using Eqns (2,3) for competitive and uncompetitive inhibition respectively: v0 ẳ VẵS   Km ỵ ẵKI ỵ ẵS ic 2ị v0 ẳ VẵS  Km ỵ ẵS ỵ 3ị ½IŠ Kiu  where Kic denotes the inhibition constant for competitive inhibition and Kiu the inhibition constant for uncompetitive inhibition, [I] and [S] are the concentrations of the inhibitor and the substrate being varied respectively, Km is the Michaelis constant for such substrate, and V is the maximum velocity Reversibility experiments The reversibility of the inhibition caused by GSK837149A was tested by filtration followed by dilution of the concentrated retentate using Ultrafree MC filters (Millipore, Billerica, MA, USA) with a cut-off of 10 kDa Samples of 10 nm FAS in 50 mm sodium phosphate buffer at pH 7.0 were incubated with a given concentration of compound for 30 min, and then filtered by centrifugation according to the manufacturer’s instructions The retentate sample, whose volume was ⁄ 25th of that of the original sample, was diluted 25-fold to recover the original volume Immediately afterwards, it was tested for FAS activity or subjected to a new filtration and dilution cycle (625-fold dilution) and finally tested Chromatographic procedures Preparative chromatography was accomplished using a system from Waters (Milford, MA, USA) with a Supelco ABZ Plus column (100 · 21.2 mm, lm particle size) Samples were injected in 0.5 mL containing 30 mg of material A 15 linear gradient was used, starting with water with 0.1% (v ⁄ v) formic acid and ending with 95% acetonitrile with 0.05% (v ⁄ v) formic acid at a flow rate of 20 mLỈmin)1 Analytical chromatography for liquid chromatography high-resolution MS experiments was carried out in an Agilent 1100 instrument (Agilent Inc, Santa Clara, CA, USA) with a Phenomenex Luna C18(2) reverse-phase column (150 · 2.1 mm, lm particle size (Phenomenex, Torrance, CA, USA) Samples of lL were injected, and a linear gradient elution was carried out at a flow rate of 0.4 mLỈ min)1, starting from water containing 0.1% (v ⁄ v) formic acid and ending with acetonitrile containing 0.1% (v ⁄ v) formic acid in 21 The system was left in these final conditions for min, and then decreased linearly to the initial FEBS Journal 275 (2008) 1556–1567 ª 2008 FEBS conditions over min; this was followed by an equilibration period of prior to the next injection Chromatographic analysis of the individual reactions catalyzed by FAS was also performed in an Agilent 1100 system, using a Kromasil C18 ODS-2 column (250 · 4.6 mm, lm particle size) (Eka Chemicals, Bohus, Sweden) Samples of 50 lL were injected and subjected to a gradient of methanol ⁄ ammonium acetate (50 mm in water at pH 7.4), progressing linearly from 10 : 90 to 45 : 55 in 20 at a constant flow rate of mLỈmin)1 MS Positive ion mass spectra were acquired as accurate mass centroided data using a Micromass two hybrid quadrupole TOF (Q-TOF) mass spectrometer (Waters), equipped with a Z-spray interface, over a mass range of 80–1100 Da, with a scan time of 0.95 s and an interscan delay of 0.07 s Reserpine was used as the external mass calibrant ([M + H]+ = 609.2812 Da) The mass spectrometer was operated in W reflectron mode to give a resolution (full width at half maximum) of 16 000–20 000 Ionization was achieved with a spray voltage of kV, a cone voltage of 30 V, and cone and desolvation gas flows of 5–10 and 500 LỈmin)1 respectively The source block and desolvation temperatures were maintained at 120 °C and 250 °C respectively The elemental composition was calculated using masslynx v3.5 (Waters) for the [M + H]+, and the mass error was quoted as p.p.m NMR spectroscopy NMR spectra were acquired in dimethylsulfoxide solution on a Varian UNITY 400 MHz spectrometer (Varian Inc., Palo Alto, CA, USA) using a mm inverse geometry probe Two or three milligrams of solid were dissolved in 0.6 mL of solvent Standard acquisition parameters were 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