An inhibitor of fatty acid synthase thioesterase domain with improved cytotoxicity against breast cancer cells and stability in plasma

Article abstract of DOI:10.1124/jpet.119.258947

It is well recognized that many cancers are addicted to a constant supply of fatty acids (FAs) and exhibit brisk de novo FA synthesis. Upregulation of a key lipogenic enzyme, fatty acid synthase (FASN), is a near-universal feature of human cancers and their precursor lesions, and has been associated with chemoresistance, tumor metastasis, and diminished patient survival. FASN inhibition has been shown to be effective in killing cancer cells, but progress in the field has been hindered by off-target effects and poor pharmaceutical properties of candidate compounds. Our initial hit (compound 1) was identified from a high-throughput screening effort by the Sanford-Burnham Center for Chemical Genomics using purified FASN thioesterase (FASN-TE) domain. Despite being a potent inhibitor of purified FASN-TE, compound 1 proved highly unstable in mouse plasma and only weakly cytotoxic to breast cancer (BC) cells in vitro. An iterative process of synthesis, cytotoxicity testing, and plasma stability assessment was used to identify a new lead (compound 41). This lead is more cytotoxic against multiple BC cell lines than tetrahydro-4-methylene-2S-octyl-5-oxo-3R-furancarboxylic acid (the literature standard for inhibiting FASN), is stable in mouse plasma, and shows negligible cytotoxic effects against nontumorigenic mammary epithelial cells. Compound 41 also has drug-like physical properties based on Lipinski’s rules and is, therefore, a valuable new lead for targeting fatty acid synthesis to exploit the requirement of tumor cells for fatty acids.

Full text of DOI:10.1124/jpet.119.258947

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2019/07/12/jpet.119.258947.DC1
1521-0103/371/1/171–185$35.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
https://doi.org/10.1124/jpet.119.258947
J Pharmacol Exp Ther 371:171–185, October 2019
Copyright ª 2019 by The American Society for Pharmacology and Experimental Therapeutics
An Inhibitor of Fatty Acid Synthase Thioesterase Domain with
Improved Cytotoxicity against Breast Cancer Cells and Stability
in Plasmas
Leslie E. Lupien, Evan M. Dunkley, Margaret J. Maloy, Ian B. Lehner, Maxwell G. Foisey,
Maddison E. Ouellette, Lionel D. Lewis, Darcy Bates Pooler, William B. Kinlaw,
and Paul W. Baures
Division of Endocrinology and Metabolism, Department of Medicine, Norris Cotton Cancer Center (W.B.K.) and Section of
Clinical Pharmacology & The Clinical Pharmacology Shared Resource (L.D.L., D.B.P.), The Geisel School of Medicine (L.E.L.,
W.B.K.), and Program in Experimental and Molecular Medicine, Dartmouth-Hitchcock Medical Center (L.E.L.), Dartmouth
College, Lebanon, New Hampshire; and Department of Chemistry, Keene State College, Keene, New Hampshire (E.M.D.,
M.J.M., I.B.L., M.G.F., M.E.O., P.W.B.)
Received April 11, 2019; accepted July 1, 2019
ABSTRACT
It is well recognized that many cancers are addicted to a constant (the literature standard for inhibiting FASN), is stable in mouse
supply of fatty acids (FAs) and exhibit brisk de novo FA plasma, and shows negligible cytotoxic effects against non-
synthesis. Upregulation of a key lipogenic enzyme, fatty acid tumorigenic mammary epithelial cells. Compound 41 also has
synthase (FASN), is a near-universal feature of human cancers drug-like physical properties based on Lipinski’s rules and is,
and their precursor lesions, and has been associated with therefore, a valuable new lead for targeting fatty acid synthesis to
chemoresistance, tumor metastasis, and diminished patient exploit the requirement of tumor cells for fatty acids.
survival. FASN inhibition has been shown to be effective in
SIGNIFICANCE STATEMENT
killing cancer cells, but progress in the field has been hindered by
off-target effects and poor pharmaceutical properties of candi- An iterative process of synthesis and biological testing was
date compounds. Our initial hit (compound 1) was identified from used to identify a novel thioesterase domain FASN inhibitor
a high-throughput screening effort by the Sanford-Burnham that has drug-like properties, is more cytotoxic to breast
Center for Chemical Genomics using purified FASN thioesterase cancer cells than the widely used tetrahydro-4-methylene-
(FASN-TE) domain. Despite being a potent inhibitor of purified 2S-octyl-5-oxo-3R-furancarboxylic acid, and has negligible
FASN-TE, compound 1 proved highly unstable in mouse plasma effects on the growth and proliferation of noncancerous
and only weakly cytotoxic to breast cancer (BC) cells in vitro. An mammary epithelial cells. Our studies have confirmed the value
iterative process of synthesis, cytotoxicity testing, and plasma of using potent and selective FASN inhibitors in the treatment
stability assessment was used to identify a new lead (compound of BC cells and have shown that the availability of exogenous
41). This lead is more cytotoxic against multiple BC cell lines than lipoproteins may impact both cancer cell FA metabolism and
tetrahydro-4-methylene-2S-octyl-5-oxo-3R-furancarboxylic acid survival.
Introduction
Cancer cells exhibit fundamental metabolic alterations that
drive and maintain the malignant phenotype. Since the 1920s
it has been known that, in contrast to most normal tissues,
cancer cells avidly take up glucose and convert it to lactate
through the glycolytic pathway, irrespective of whether
oxygen is present (aerobic glycolysis, the Warburg effect)
(Warburg, 1956). Aerobic glycolysis not only provides cancer
cells with energy, but also diverts carbon to the synthesis of
cellular building blocks, including lipids (Zaidi et al., 2013).
Heightened de novo lipid synthesis, mediated through
This research was made possible by the Institutional Development Award
Program of the National Institutes of Health National Institute of General Medical
Sciences [Grant P20GM103506] (to P.W.B. and W.B.K.) as well as funding from the
National Institutes of Health National Cancer Institute [Grant R01CA58961] (to
W.B.K.). This research was also supported by the National Institutes of Health
National Cancer Institute [Grant P30CA023108] (to L.D.L. and D.B.P.) Support for
the Keene State College NMR Facility [National Science Foundation 1337206] (to
P.W.B.) is appreciated, as is support from The Friends of Norris Cotton Cancer
Center (to P.W.B. and W.B.K.).
https://doi.org/10.1124/jpet.119.258947.
s
This article has supplemental material available at jpet.aspetjournals.org. upregulation of the requisite enzymes, including fatty acid
ABBREVIATIONS: BC, breast cancer; C75, 4-methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid; FA, fatty acid; FASN, fatty acid synthase;
FASNi, fatty acid synthase inhibitor; FASN-TE, fatty acid synthase thioesterase; GSK264220A, N-[2-methyl-5-(1-piperidinylsulfonyl)-3-furanyl]-N9-
phenyl-urea; HPLC, high-performance liquid chromatography; LC, liquid chromatography; LC-MS/MS, liquid chromatography–tandem mass
spectrometry; LPL, lipoprotein lipase; PI, propidium iodide.
171

172
Lupien et al.
synthase (FASN), is considered a near-universal hallmark (https://www.cancer.gov/about-cancer/treatment/clinical-trials/
of most human tumor cells and their precursor lesions search/v?id5NCI-2016-01710&r51) (Dean et al., 2016). How-
(Menendez and Lupu, 2007; Hanahan and Weinberg, 2011; ever, progress in the field continues to be slowed by off-target
Ward and Thompson, 2012; Benjamin et al., 2015; Gonzalez- effects and poor pharmaceutical properties of candidate FASN
Guerrico et al., 2016). Increased FASN expression is associ- inhibitors, as reviewed in Menendez and Lupu (2007, 2017),
ated with cancer progression, higher risk of recurrence and Flavin et al. (2010), Liu et al. (2010), Kinlaw et al. (2016), and
metastasis, reduced response to classic chemotherapeutic Röhrig and Schulze (2016). The generation of more potent and
agents, multidrug resistance, and diminished patient survival selective FASN inhibitors has potential application to a wide
(Rysman et al., 2010; Liu et al., 2013; Wu et al., 2014; Jung range of cancer types, and is therefore an important goal.
et al., 2015; Heuer et al., 2017; Menendez and Lupu, 2017).
Imidazole-4,5-dicarboxylic acid is a useful scaffold for drug
FASN is a multifunctional, homodimeric enzyme. Each discovery (Baures, 2006) and has been used to create inhib-
monomer is a large polypeptide comprised of seven catalytic itors of human immunodeficiency virus protease (Baures,
domains working in concert to synthesize the 16-carbon 1999) to target HL-60 promyelocytic leukemia cells (Perchellet
saturated fatty acid (FA) palmitate from acetyl-CoA and et al., 2005) and in the design of inhibitors of protein-protein
malonyl-CoA (Buckley et al., 2017). Palmitate and palmitate- interactions (VanCompernolle et al., 2003). The ability to
derived lipids have integral roles in cell metabolism, mem- readily derivatize the scaffold and the conformational be-
brane architecture, protein localization, and intracellular havior resulting from a strong intramolecular hydrogen bond
signaling (Menendez and Lupu, 2007; Flavin et al., 2010; (Baures et al., 2002; Rush et al., 2005) were features that
Daniëls et al., 2014; Gonzalez-Guerrico et al., 2016). Studies enabled the inclusion of these compounds in the Molecular
of the mechanistic role of FASN have revealed that it fuels Library Small Molecule Repository for screening within the
cancer cell proliferation and malignant progression by: 1) National Institutes of Health Molecular Libraries Initiative
generating FA precursors required for membrane synthesis (Austin et al., 2004). A high-throughput bioassay was used to
and ATP production (Buckley et al., 2017; Luo et al., 2017; identify inhibitors of the FASN thioesterase (FASN-TE)
Jafari et al., 2019); 2) altering membrane composition and domain. This initial screening effort and a series of confirma-
fluidity to confer resistance to endogenous and exogenous tory screens identified compound 1 as the most potent in-
insults, including chemotherapy and membrane peroxidative hibitor against this domain from the library of 362,050
damage (Liu et al., 2008; Rysman et al., 2010); 3) modulating compounds. Compound 1 and several closely related deriva-
the composition of lipid raft membrane microdomains, which tives (Fig. 1) were found to be highly unstable in mouse plasma
house the membrane receptor tyrosine kinases that modulate and demonstrated limited cytotoxicity against breast cancer
key cellular processes including signal transduction, intracel- (BC) cells in vitro. Multiple rounds of rational modification
lular trafficking, cell polarization, and migration (Swinnen were used to overcome the stability issues of compound 1 and
et al., 2003; Menendez et al., 2005); 4) providing palmitate generate stable, potent, and selective FASNis. The new lead
for the covalent post-translational modification (S-acylation compound reported herein is a structurally unique FASN-TE
and palmitoylation) of tumor-promoting signaling proteins inhibitor that is more cytotoxic across a panel of BC cell lines
(e.g., Wnt, tubulin, and H/N/K-RAS) (Levental et al., 2010; than literature standards, such as (2)-4-methylene-2-octyl-5-
Anderson and Ragan, 2016; Heuer et al., 2017); 5) regulating oxotetrahydrofuran-3-carboxylic acid (C75), and has dis-
the formation of structures that drive metastasis and invasion played selectivity for BC cells. These studies have additionally
(Zaytseva et al., 2012; Benjamin et al., 2015; Singh et al., 2015; yielded insights into the different pathways that cancer cells
Ventura et al., 2015; Gonzalez-Guerrico et al., 2016; Wang use to acquire the FAs that they require.
et al., 2016; Jafari et al., 2019); and 6) generating oncogenic
signaling lipids, such as diacylglycerol and phosphatidylinositol
(Benjamin et al., 2015; Heuer et al., 2017; Wagner et al., 2017).
In these various ways, FASN modulates cancer cell prolifera-
tion, survival, extracellular matrix organization, migration and
invasion, and the expression and activity of signaling networks
required for maintaining the malignant phenotype (Menendez
and Lupu, 2017).
Materials and Methods
General. Reagents were obtained from commercial suppliers and
used without additional purification. Solvents were purchased in the
highest grade available from Fisher Scientific and passed through
a MBRAUN MB-SPS-800 purification system prior to use. Thin layer
chromatography was performed on 250 mm silica gel plates and
Participation of FASN in this plethora of cancer-promoting compounds were visualized using UV light. Column chromatogra-
phy was performed on silica gel (grade 60, 230–400 mesh; Fisher
pathways positions the enzyme as an attractive therapeutic
target. Pharmacologic or RNA interference–mediated inhibi-
tion of FASN has been shown to attenuate cancer cell growth,
induce apoptosis, and diminish metastatic potential of cancer
cells in many different models, while exerting no direct effects
on the growth and survival of normal cells (Kuhajda et al.,
1994; Pizer et al., 1996; De Schrijver et al., 2003; Kridel et al.,
2004; Menendez and Lupu, 2007, 2017; Zaytseva et al., 2012,
2014; Li and Cheng, 2014; Ventura et al., 2015; Röhrig and
Schulze, 2016; Buckley et al., 2017). Clinical efficacy of FASN
inhibitors (FASNis) alone or in combination therapy has
begun to be realized with 4-(1-(4-cyclobutyl-2-methyl-5-(5-
methyl-4H-1,2,4-triazol-3-yl)benzoyl)piperidin-4-yl)benzonitrile
Fig. 1. Structure-activity relationship summary of N,N9-disubstituted
imidazole-4,5-dicarboxamides active in confirmatory screens against
(TVB-2640), a compound developed by 3-V Biosciences FASN-TE.

Improved Cytotoxicity and Stability of a FASN Inhibitor
173
Scientific). Columns were prepared in plastic syringe bodies (130 Â (HPLC) column with a guard column (10 Â 2.1 mm) attached at
25 mm) filled to two-thirds capacity with silica gel. Fractions
containing only the desired product were pooled and concentrated
40°C. Compounds were eluted over 6 minutes with a gradient of
30%–90% CH3CN and 70%–10% H2O, each containing 0.1% CH3CO2H
under vacuum. 1H NMR spectra were recorded at 400 MHz in CDCl3 at a 0.75 ml/min flow rate. The column was washed with 90% CH3CN
with CHCl3 as the internal reference (d 7.24) or DMSO-d6 with DMSO for 1.5 minutes at the end of each injection, and then equilibrated with
as the internal reference (d 2.49). The 13C NMR spectrum of compound 30% CH3CN for 1.5 minutes before each new injection. The mass
41 was recorded at 100.5 MHz in CDCl3 with CHCl3 as the internal spectrum was recorded using electrospray ionization detection from
reference (d 77.00).
Synthesis. Synthesis of compound 1.4 from compound 1.1 was analyzed for purity on a Polaris 5 mm C18-A (50 Â 2.0 mm) HPLC
performed as shown in Scheme 1, as previously described (Solinas column and eluted with gradient of CH3CN/H2O containing
50 to 800 (mass-to-charge ratio). In addition, the samples were
a
et al., 2008). Briefly, freshly prepared compound 1.2 was used in the 0.1% CH3CO2H at a 0.2 ml/min flow rate. Compounds were detected
reaction with lysine derivative 1.3 to give compound 1.4 as a white or at 218 nm. The mobile phase gradient was as follows: 0 minutes, 4:6
off-white solid following extraction and precipitation. This intermedi- CH3CN/H2O → 1 minute, 4:6 CH3CN/H2O → 12 minutes, 10:0 CH3CN/
ate can be stored at room temperature indefinitely. Two equivalents H2O → 13 minutes, 10:0 CH3CN/H2O → 14 minutes, 4:6 CH3CN/H2O
of glycine benzyl ester hydrochloride and compound 1.5 were added → 20 minutes, 4:6 CH3CN/H2O.
to compound 1.4 in dichloromethane along with two equivalents of
Cell Lines and Tissue Culture. MCF-7, MDA-MB-231, BT-474,
diisopropylethylamine, and the reaction was stirred at room temper- DU4475, SKBR3, and T47-D BC cells, and HeLa cervical cancer cells
ature for 16–48 hours. The progress of each reaction was followed with
thin layer chromatography. Purification by column chromatography
using a gradient of hexanes and ethyl acetate afforded compound 1 as
were obtained from American Type Culture Collection and routinely
grown in phenol red–containing HyClone RPMI 1640 media with
10% (v/v) heat-inactivated FBS (GE Healthcare Life Sciences) and
a film with 1H NMR and liquid chromatography (LC)/mass spectrom- 1% penicillin-streptomycin. Cells were maintained at 37°C in a hu-
etry data consistent with that previously reported for this compound midified atmosphere containing 5% CO2. MCF10A mammary epithe-
(Solinas et al., 2009). Compounds 11–43 were prepared from com- lial cells were cultured in Dulbecco’s modified Eagle’s medium/F12
pound 1.4 using the same approach and the corresponding amino acid
growth media (Invitrogen) supplemented with 5% horse serum
or amine to replace compound 1.5. Compounds 15–24 required the (Invitrogen), 20 ng/ml epidermal growth factor (Peprotech), 0.5 mg/ml
synthesis of derivatives of compound 1.5 that were subsequently used hydrocortisone, 100 ng/ml cholera toxin, 10 mg/ml insulin (Sigma),
in the reaction. Compounds 11–43 were purified by column chroma- and 1% penicillin-streptomycin. Cell line characteristics are listed in
tography using a gradient of hexanes/ethyl acetate or dichlorome- Table 1. Lipoprotein-depleted and matched control FBS were attained
thane with 3% methanol (v/v). The purity and identity of the final
from Kalen Biomedical.
products were determined by LC, liquid chromatography–tandem
Cell Viability Assays. FASN and lipase inhibitors were purchased
mass spectrometry (LC-MS/MS), and 1H NMR spectroscopy. Addi- from Cayman Chemical (Ann Arbor, MI), diluted in DMSO, and stored in
tional details regarding the synthesis and characterization of these the dark at 220°C until use. These included: (2)-tetrahydro-4-methylene-
derivatives are provided in the Supplemental Material.
LC and LC-MS/MS Supporting Compound Purity and (20 mM), N-[2-methyl-5-(1-piperidinylsulfonyl)-3-furanyl]-N9-phe-
Identity. Compounds were analyzed for purity and identity by nyl-urea (GSK264220A) (50 mM), and orlistat (20 mM). Inhibitors
LC-MS/MS using a Dionex Ultimate 3000 component LC system and were diluted in tissue culture medium immediately before use;
2S-octyl-5-oxo-3R-furancarboxylic acid (20 mM stock), cerulenin
Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer
(LC-MS/MS) with HESI-II probe operating in positive ion mode.
Solutions of the compounds were injected onto a 1.9 mm Hypersil
control cells were cultured in medium containing the same concen-
tration of DMSO as the highest treatment condition.
The seeding densities for each cell line were determined for the
GOLD (50 Â 2.1 mm) high-performance liquid chromatography treatment durations to ensure that cells were both capable of logarithmic
Scheme 1. Synthesis of compound
from compound 1.1.
1

174
Lupien et al.
TABLE 1
Immortalized human cell lines used in this study and their respective biomarker status and subtype, where applicable
Cell Line
Source
Tumor Type
Subtype
Luminal B
TNBC, IM subtype
N/A
Luminal A
Basal
Claudin-low or basal-like
N/A
Luminal
Luminal A
ER
PR
HER2
BT-474
DU4475
HeLa
Primary breast
Mammary gland
Cervix
Pleural effusion
Primary breast
Pleural effusion
Pleomorphic liposarcoma
Pleural effusion
Pleural effusion
Ductal carcinoma
Epithelial cell
Adenocarcinoma
Invasive ductal carcinoma
Epithelial cell
Adenocarcinoma
Liposarcoma
Adenocarcinoma
Invasive ductal carcinoma
1
2
1
2
1
2
MCF-7
1
2
2
1
2
2
1
2
1
2
2
2
MCF10A
MDA-MB-231
LiSa-2
SKBR3
T47-D
2
1
1
2
ER, estrogen receptor; HER2, ErbB2; IM, immunomodulatory; N/A, not applicable; PR, progesterone receptor; TNBC, triple-negative breast cancer.
growth and remained subconfluent at the experimental endpoint. Cells
were seeded into 96-well plates, allowed to adhere overnight, treated ery of $70% of compound from mouse plasma after 1-hour incuba-
with freshly diluted drugs on day 2, and cultured for 72 hours prior to tion at 37°C. Recovery was considered with respect to drug in solvent
assessment. Initial cytotoxicity screens were conducted with MCF-7 BC or plasma on ice (presumed to be the most stable conditions) versus
Analysis of Compound Stability. We defined stability as recov-
cells, with cells being treated for 72 hours in standard media at
concentrations ranging from 5 to 100 mM of inhibitor. Cell viability
was assessed using alamarBlue Cell Viability Reagent (Invitrogen),
according to the manufacturer’s guidelines. Extended viability screens
plasma at 37°C. Some compounds may have been subjected to enzymatic
degradation in the plasma, while others may have bound covalently to
plasma proteins.
Malonyl-CoA LC-MS/MS Assay. BT-474 BC cells were treated
were carried out using the ATP-based CellTiter-Glo 2.0 Assay (Promega). with FASNi (50 mM) for 4 hours in standard RPMI 1640 media,
Luminescence was read using a LMAX II luminometer (Molecular washed with PBS, harvested with trypsin, and frozen as cell
Devices). Background luminescence was calculated from blank/Cell- pellets at 220°C. Cell pellets were resuspended at 10,000 cells/ml
Titer-Glo–alone wells. Luminescence values (relative light units) were
in cold acetonitrile:methanol:water (40:40:20) containing 1 mg/ml
normalized to vehicle and compiled as percentage of DMSO control. Half- 13C-malonyl-CoA internal standard. Calibrator and quality con-
maximal inhibitory concentration (IC50) determination was performed trol samples were made in acetonitrile:methanol:water (40:40:20)
using a nonlinear regression curve-fitting algorithm: log(inhibitor) containing internal standard. All samples were vortexed and
versus response-variable slope (four parameter) with Graph Pad centrifuged 4300g for 20 minutes at 4°C. Supernatants were
Prism 6.0.
collected and 25 ml was injected onto a Dionex Ultimate 3000
Cell Death and Apoptosis Assay. Apoptosis was assessed using HPLC system in tandem with a TSQ Vantage mass spectrometer. HPLC
a Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and separation was achieved with a gradient of 5% → 95% methanol
propidium iodide (PI) (ThermoFisher Scientific), according to the
manufacturer’s instructions. Flow cytometry data were read with the
and 95% → 5% 5 mM ammonium acetate over 3 minutes at a flow rate
of 0.3 ml/min on a 40°C Phenomenex Luna C18 2.1 Â 50 mm, 1.6 mm
8-Color MACSQuant-10 (Miltenyi Biotec) and analyzed using FlowJo column with 2.1 Â 10 mm C18 guard cartridge. The mass spectrometer
software. The percentage of apoptosis is shown by the percentage of
was operated in positive ion mode using a HESI-II probe, with spray
annexin V1 cells in the population, where total cell death may be voltage 3750 V, vaporizer and capillary temperatures at 380°C and 241°C,
calculated as the sum of the annexin V1/PI1, annexin V1/PI2, and respectively, and sheath and auxiliary gases at 20 and 5, respectively.
annexin V2/PI1 populations.
Single reaction monitoring mode was used for the following ion transitions:
Statistics. Statistical significance was evaluated using two-tailed malonyl-CoA 854.055 → 303.040 mass-to-charge ratio with S-Lens 191 and
unpaired student’s t tests with correction for multiple comparisons collision energy 35 and 13-C-malonyl-CoA 857.061 → 350.090 mass-to-
(the Holm-Sidak method), where applicable, on the mean values of at
charge ratio with S-Lens 187 and collision energy 29. The calibration curve
least three independent in vitro experiments. Values of P , 0.05 was linear over the concentration range 0.01–5 mg/ml, with all quality
were deemed significant: *P , 0.05, **P , 0.01, ***P , 0.001. Error controls having a CV , 15%.
is presented as mean 6 S.D.
His6-Tagged FASN-TE Protein Expression and Purification.
Plasma Stability Assay. Each FASN inhibitor (200 nM in a total A pET15b plasmid coding an N-terminal His6-tagged FASN-TE, with
volume of 50 ml) was tested for stability both in HPLC solvent a thrombin recognition sequence separating the His6 tag from the
(70% acetonitrile) and in mouse plasma following 1-hour incubation FASN-TE domain, was generously provided by W. Todd Lowther
under several conditions (on ice, at 25°C, and at 37°C). Immediate (Wake Forest University, Winston-Salem, NC). This was used for the
recovery (no incubation) of compound from plasma at 25°C was also
tested. After incubation, 200 ml of acetonitrile containing 80 nM of
protein expression and purification of FASN-TE with slight modifica-
tion from a previously described procedure (Pemble et al., 2007). We
a stable internal standard (compound ILP-III-15) (Supplemental Fig. used a solution of Bacterial Protein Extraction Reagent (Thermo
1) was added to each sample. Samples were immediately vortexed and Scientific) with lysozyme and DNase I at a ratio of 5 ml/g of cells to lyse
centrifuged at 12,000 rpm for 5 minutes. Supernatants were dried
the cells. Following the addition of streptavidin-agarose (Thermo
under nitrogen at 45°C, resuspended in 50 ml 70% acetonitrile, and Scientific) to remove the biotinylated-thrombin, the protein solution
transferred to the autosampler where 5 ml was injected for LC-MS/MS was passed through Ni21-sepharose and the cleaved protein was
analysis.
collected in the flow-through. This step on Ni21-sepharose ensured
Stability Analysis by LC-MS/MS. Samples were injected onto that no residual His6-tagged FASN-TE remained in the protein
a 50 Â 2.1 mm, 1.9 mm Hypersil GOLD column fitted with a 10 Â solution. The FASN-TE domain was dialyzed into 20 mM HEPES,
2.1 mm C18 guard at 40°C. Isocratic elutions were achieved with
60% acetonitrile, 40% water, and 0.1% acetic acid at a rate of 0.75 ml/
min on the Dionex/Thermo LC-MS/MS system. Mass spectrometry
pH 7.5, 100 mM NaCl, and 1 mM DTT. For the enzyme assays, the
protein was diluted in H2O and dialyzed against 25 mM HEPES, pH
7.5, 12.5 mM NaCl, 0.001% Brij-35, and 125 mM sarcosine, resulting
parameters were: spray voltage 3750 V, vaporizer temperature 474°C, in a stock protein concentration of 2.6 mg/ml (75 mM). A 0.71 M
capillary temperature 202°C, sheath gas pressure 30, and auxiliary tris-(2-carboxyethyl)phosphine stock solution was used to yield
gas pressure 25. Selected reaction monitoring, collision energy, and a 1 mM tris-(2-carboxyethyl)phosphine concentration to protect
S-Lens parameters were tuned selectively for each compound.
cysteines from oxidation.

Improved Cytotoxicity and Stability of a FASN Inhibitor
175
FASN-TE Inhibition Experiments. Prior to each experiment,
stock protein was diluted to 7.5 mM with 200 mM Tris, pH 7.5, 100 mM
NaCl, 0.01% Brij-35, and 1.0 M sarcosine. The FASN-TE domain
was used to conduct IC50 determinations using 4-methylumbelliferyl
heptanoate (Sigma Aldrich) as the substrate with enzyme alone
versus solutions of varying concentrations of compounds 1 and 41. A
stock solution of compound 41 was prepared in 25 mM in DMSO, while
a stock solution of compound 1 was prepared at 6.25 mM in DMSO.
Serial dilutions of the stocks were prepared, and 1.0 ml of each was
added to 155 ml of the pooled enzyme solution. A DMSO control was
prepared analogously. These solutions were incubated for 30 minutes
at room temperature before adding 50 ml from each into three wells of
a 96-well plate (Costar black polystyrene, round bottom). A substrate
stock concentration was prepared at 100 mM in DMSO and diluted to
60 mM in water containing 10% DMSO prior to the assay. To each well
containing enzyme with or without an inhibitor 50 ml of the substrate
solution was then added, giving a final substrate concentration of
30 mM. The final enzyme concentration in the assay was 3.75 mM. The
final concentrations of compound 1 in the enzyme assay were 20, 10, 5,
2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 mM, respectively. The
final concentrations of compound 41 in the enzyme assay were 80, 40,
20, 10, 5, 2.5, 1.25, 0.625, 0.313, and 0.156 mM. The plate was shaken
for 10 seconds and fluorescence was recorded (360/40 ex, 460/40 em),
in which the shaking was repeated and read every minute for 4 hours.
In the absence of inhibitors, the fluorescence reached a maximum in
approximately 60 minutes. The background signal resulting from the
nonenzymatic hydrolysis of the substrate was subtracted from the
enzyme plus the substrate alone control solution and all of the wells
that contained compound 1 or 41. Initial velocities were determined at
or prior to the point that 10% substrate hydrolysis was evident as
determined by the percentage of maximum fluorescence intensity.
Data for each concentration were collected in triplicate, and three
experiments were used to determine an average IC50 curve for
Rational Design of Structurally Related Compounds to
Improve the Stability and In Vitro Cytotoxicity of
Compound 1
Modifications of the Benzyl Ester Moiety. Compound 1
was weakly cytotoxic to MCF-7 BC cells (IC50 5 117 mM)
(Supplemental Fig. 120) and was unstable in mouse plasma
(0% recovery, ,5 minutes). Heat inactivation of mouse
plasma abrogated the instability of compound 1, suggesting
the presence of esterase or protease activity. We hypothe-
sized that the lability in mouse plasma was due to the benzyl
ester bond based on this evidence and the fact that amino
acid derivatives like compound 2, containing tert-butyl
esters, were recovered at higher percentages (30%–59%)
from mouse plasma after 1 hour at 37°C (data not shown).
Replacing the glycine benzyl ester moiety in compound 1 for
a leucine tert-butyl ester resulted in complete recovery of the
compound (ILP-III-15) from mouse plasma. This leucine
derivative was thus used as the internal standard for our
compound stability assay (Supplemental Fig. 1).
Our design, synthesis, and testing efforts started with the
most active hit, compound 1, and focused on improving the
stability of the benzyl ester bond. This led to derivatives 11–14
that replaced part or all of the benzyl ester (Fig. 2A). Each of
these derivatives was substantially more cytotoxic against
MCF-7 BC cells in vitro than compound 1 (with IC50 values
ranging from 8 to 25 mM), but only compound 13 showed
marked improvement in plasma stability (40% recovery after
1 hour at 37°C) (Fig. 2, B and C). The less than complete
recovery of compound 13 suggested that another activity such
as hydrolysis of the glycine amide bond in compound 1 was
possible. A second approach to blocking the action of the
protease/esterase activity against these compounds was thus
employed. The benzyl ester was modified with an amide bond
(compound 15) and coupled to steric bulk at the amide
nitrogen (compound 16), the benzyl carbon (compounds 17
and 18), or the a-carbon (compounds 19 and 20) of the amino
acid (Fig. 3). Compound 16 was recovered at only 29% from
37°C plasma after 1 hour, and the addition of steric bulk did
not improve the cytotoxicity of the compounds (data not
shown). Combining a methyl group at the amide nitrogen
with one at the a-carbon of the amino acid (compounds 21 and
22) or the benzyl carbon (compounds 23 and 24) likewise
showed no improvement in the cytotoxicity (data not shown),
leading us to abandon this series.
compounds 1 and 41. Half-maximal inhibitory concentration (IC50
)
determination was performed using a nonlinear regression curve-
fitting algorithm: log(inhibitor) versus response-variable slope (four
parameter) with Graph Pad Prism 6.0.
Results
Synthesis
The methodology for synthesizing compounds 1–10 has
been previously reported (Perchellet et al., 2005; Solinas
et al., 2008, 2009); these methods were also used in the
synthesis of compounds 11–43 (Scheme 1). The millimolar
scale for the synthesis of compounds 11–43, the thin layer
chromatography eluant, and Rf value, as well as the yield of
purified compounds are shown in Supplemental Table 1. The
synthetic procedures and details for reaction intermediates, the
reaction scheme, structures, 1H NMR spectra, and LC/mass
spectrometry analysis are also provided (Supplemental Figs.
2–18; Supplemental Scheme 1; Supplemental Table 2). Com-
The Benzylamine Series. Benzylamine derivative 25
was flagged as active in the initial high-throughput screen
with purified FASN-TE, and also formed the base structure of
compound 13, which demonstrated improved cytotoxicity in
our assays. (The original bioassay can be viewed in PubChem
by searching AID 602261 under PubChem Bioassay. The SID
number for compound 15 is 24833677. A direct link to the
bioassay data for compound 15 is as follows: https://pubchem.
ncbi.nlm.nih.gov/bioassay/602261#sid524833677&section5Top.)
Compounds based on a substituted benzylamine (compounds
26–37) were thus prepared and tested. Substituents on the
aromatic ring impacted both the cytotoxicity and the plasma
stability; however, even the more potent of these compounds
was not sufficiently stable to justify further investigation
pounds 11–43 were characterized by 1H NMR spectroscopy, 13
C
NMR spectroscopy (compound 41 only), and LC/mass spec-
trometry using the total ion count (Supplemental Figs. 19–85).
Additional HPLC traces with UV detection at 218 nm to
assess for purity are shown in Supplemental Figs. 86–118.
(The confirmatory bioassays can be viewed in PubChem by
searching AID 624326 or AID 624327 under PubChem
Bioassay. The SID number for compound 1 is 49733438.
Direct links to the confirmatory bioassay data for com-
pound 1 are as follows: https://pubchem.ncbi.nlm.nih.gov/ (Fig. 4).
bioassay/624326#sid549733438 and https://pubchem.ncbi.
Cyclohexylamine Derivatives. Compound
9 demon-
nlm.nih.gov/bioassay/624327#sid549733438.)
strated activity against purified FASN-TE in initial confirmatory

176
Lupien et al.
Fig. 2. Structure, cytotoxicity, and mouse plasma stability data for compounds 11–14. (A) Structure of derivatives that modify the ester bond of
compound 1. (B and C) MCF-7 BC cells were treated with FASNi for 72 hours in standard media and assessed using the alamarBlue cell viability assay.
(B) Concentration-response curves for compounds 11–14. Data are presented as mean 6 S.D. (N 5 3–5 experiments). Here and in all ensuing figures, the
IC50 values were generated using a nonlinear regression curve-fitting algorithm: log(inhibitor) vs. response-variable slope (four parameters) with Graph
Pad Prism 6.0. (C) Compiled IC50 and mouse plasma stability results, represented by percentage of compound remaining after 1 hour incubation in
mouse plasma at 37°C (N.D., not determined).
assays (FASN-TE IC50 5 1.56 mM). While the potency of
Extended Characterization of a New Lead, Com-
compound 9 was approximately 10-fold less than compound pound 41. The ability of compound 41 to directly bind and
1 (FASN-TE IC50 , 0.16 mM), this compound demon- inhibit FASN was assessed using a malonyl-CoA LC-MS/MS
strated greater plasma stability than other initial hits and assay. Treatment of BT-474 BC cells with compound 41
was, therefore, used as the inspiration for a series of cyclo- (4 hours, 50 mM) resulted in significant accumulation of the
hexylamine derivatives (Fig. 5). A boost in the cytotoxicity committed substrate of de novo lipogenesis, malonyl-CoA
(IC50 ∼16–24 mM) and plasma stability (90% recovery after (Fig. 7A). The identity of compound 41 as a FASNi targeting
1 hour at 37°C) was observed for compound 38 when the the thioesterase domain was next confirmed using a FASN-TE
substituent was just a cyclohexylamine. The improvement enzyme activity assay. Here, both compounds 1 and 41
in cytotoxicity observed for the benzylamine series when an displayed concentration-dependent FASN-TE enzyme inhibi-
alkyl group was added at the 4-position (compounds 34–36) tion compared with hydrolysis observed with substrate alone
led us to synthesize the racemic cis- and trans-4-tert- (with data corrected for nonenzymatic hydrolysis of the sub-
butylcyclohexylamine derivative, compound 39. This modi- strate) (Fig. 7B). Together, these data show that compound 41
fication further improved cytotoxicity (IC50 ∼7–16 mM), with binds and inhibits FASN-TE, resulting both in reduced
plasma stability of 80%. The diastereomeric cis- and trans- thioesterase enzyme activity and accumulation of the cyto-
isomers, compounds 40 and 41, were prepared for compar- toxic substrate, malonyl-CoA.
ison. Both diastereomers were equally cytotoxic to BC cells
Cytotoxicity of compound 41 was compared with the
(∼14–18 mM); however, the cis-isomer (compound 40) was FASNi (2)-C75 following 72-hour continuous treatment in
recovered at 62%, while the trans-isomer (compound 41) was standard media (Fig. 8). The cytotoxic effects of compound
98% recovered from mouse plasma. Diastereomers 42 and 43 41 exceed those observed with treatment of (2)-C75 across
were comparable in cytotoxicity to compound 41 but dem- BC cell lines (Fig. 8, A and B). The FASNi-mediated changes
onstrated far less stability in mouse plasma (Fig. 5).
in cell viability could be attributable to a mix of growth
The four most promising compounds from this series inhibition or cytostasis and cell death. Cells were thus
(compounds 38, 39, 41, and 42) were directly compared in stained with Annexin V Alexa Fluor 488 and PI and assessed
terms of cytotoxicity and plasma stability (Fig. 6). It was on the by flow cytometry following 72-hour continuous treatment to
bases of these cytotoxicity and stability results, and structural quantify FASNi-induced apoptosis, more specifically. FASN
considerations, that compound 41 was chosen as the new lead inhibition resulted in significant concentration-dependent
compound for extended studies.
apoptosis in BC cells, with the impact of compound 41

Improved Cytotoxicity and Stability of a FASN Inhibitor
177
a urea-based inhibitor of lipoprotein lipase (LPL) and
endothelial lipase (Keller et al., 2008; Nomura and Casida,
2016). LPL is the major enzyme for extracellular hydrolysis
of triglyceride transported in lipoprotein particles, and thus
renders this source of FAs accessible to cells (Kuemmerle
et al., 2011; Kinlaw et al., 2016). Concentration-dependent
cytotoxicity was observed when BC cells were treated with
GSK264220A continuously for 72 hours, with most effects
on viability observed at concentrations greater than 25 mM
(Fig. 10A). Combined treatment with GSK264220A and
compound 41 caused increased cytotoxicity over either drug
alone (Fig. 10B). We hypothesized that the cytotoxic effects
of lipogenesis and lipolysis inhibition might additionally be
augmented by removing lipoprotein substrates from the
media, and found that this was, in fact, the case. MDA-MB-
231 cells cultured in 10% lipoprotein-depleted FBS media
and treated with a combination of compound 41 and
GSK264220A (100 mM) showed increased cytotoxicity over
compound 41 alone, or the combination in media containing
lipoproteins (Fig. 10C).
Discussion
Here, we have summarized the stepwise process through
which compound 41 was rationally designed, established its
efficacy against cancer cell lines in vitro, and highlighted the
therapeutic potential of targeting fatty acid synthesis in BC
cells through the FASN-TE domain. Initial screening data
that yielded hits 1–10 was useful, both in providing basic
structure-activity relationship data for inhibition of purified
FASN-TE and in offering clues for how to modify compound 1
to improve cytotoxicity and plasma stability. The fact that
heat-denatured plasma led to improved stability of compound 1
pointed to the action of an esterase or protease. We hypothesized
that the lability in mouse plasma was predominantly due to the
benzyl ester bond and showed that replacing the glycine
Fig. 3. Structure of ester-to-amide derivatives of 1 along with methyl
groups that add steric bulk.
exceeding that of (2)-C75 at equimolar concentration (Fig. 8, benzyl ester moiety in compound 1 for a leucine tert-butyl
C and D). ester (compound ILP-III-15) resulted in complete recovery
Compound 41 exhibited minimal cytotoxic effects against of the compound from mouse plasma.
nontumorigenic MCF10A mammary epithelial cells, creating
An important goal at the start of this project was to obtain
the potential for a large therapeutic window (Fig. 8, E and F). a compound in this structural class worthy of investigating in
This is in contrast to other FASNis, including (2)-C75 and a mouse model of BC, and thereby gauge the suitability of
cerulenin, where substantial cytotoxicity was observed against FASN-TE inhibitors for further development. Therefore, the
MCF10A cells at high concentrations. Effects of compound 41 and plasma stability of compound 1 was an important issue to
other FASNis were validated using several different measures of solve and led us to prepare derivatives 11–14, which replaced
cell viability including the CellTiter-Glo ATP-based assessment all or part of the benzyl ester. Each of these derivatives was
(reported here), the DNA-based Hoechst assay, the protein- substantially more cytotoxic against MCF-7 BC cells than
based sulforhodamine B assay, and the alamarBlue redox compound 1 (IC50 5 117 mM) (Supplemental Fig. 120);
indicator (used for initial profiling studies, data not shown) however, only compound 13 showed improvement in plasma
(Figs. 2–5).
stability. The stability of compound 13 was similar to that
The cytotoxic effects of FASNi treatment are concentration of compound 2, further supporting the hypothesis that lack
and time dependent, (Supplemental Fig. 121) and are im- of metabolic stability results in part from hydrolysis of the
pacted by the availability of exogenous lipoproteins in the glycine amide bond of compound 1. The biphenyl substructure
media (Fig. 9). This latter observation conforms with previous in compounds 13 and 14 is a privileged scaffold found both in
publications (Kuemmerle et al., 2011; Zaidi et al., 2012, 2013; compounds that bind multiple proteins (Hajduk et al., 2000)
Daniëls et al., 2014; Schug et al., 2015; Svensson et al., 2016; and in compounds known to be FA mimetics (Proschak et al.,
Cao et al., 2017) and underscores the importance of both 2017). Thus, while compound 13 was a valuable intermediary
endogenous and exogenous FA sources for cancer cell survival in testing the significance of the benzyl ester bond to plasma
and proliferation.
stability, we did not think it represented an optimal structure
With this in mind, we next investigated the potential of to move forward.
targeting FA synthesis and exogenous FA uptake simulta-
We simultaneously prepared derivatives 15–24 with the
neously using our FASNi compound 41 and GSK264220A, dual goal of blocking the action of the protease/esterase

178
Lupien et al.
Fig. 4. Structure, cytotoxicity, and mouse plasma stability data for compounds 25–37. (A) Benzylamine derivatives 25–37: structure, compiled IC50
values, and mouse plasma stability results, represented by percentage of compound remaining after 1-hour incubation in mouse plasma at 37°C. (B and
C) Cytotoxicity concentration-response curves for compounds 25–37 against MCF-7 BC cells following 72-hour continuous treatment in standard media,
assessed using the alamarBlue cell viability assay, N 5 2–5 experiments. Data shown as mean 6 S.D.
activity by adding methyl groups as steric bulk and poten- of peptides (Biron et al., 2008). No improvements in cytotox-
tially improving the stability and drug-like properties of the icity were observed with derivatives 15–24, leading us to
compounds—effects that have been tied to the N-methylation discontinue this series.
Fig. 5. Structure, cytotoxicity, and mouse
plasma stability data for compounds 9
and 38–43. (A) Structural comparison of
cyclohexylamine derivatives based on com-
pound 9. (B) Cytotoxicity of compounds
38–43 against MCF-7 BC cells following
72-hour continuous treatment in standard
media, assessed using the alamarBlue cell
viability assay, N 5 4–6 experiments. (C)
Compiled IC50 and mouse plasma stability
results, represented by percentage of com-
pound remaining after 1-hour incubation in
mouse plasma at 37°C.

Improved Cytotoxicity and Stability of a FASN Inhibitor
179
Fig. 6. Cytotoxicity and stability of promising cyclohexylamine derivatives (compounds 38, 39, 41, and 42). (A) Cell viability was assessed using the
ATP-based CellTiter-Glo assay following 72-hour continuous treatment with FASNi in standard media. A comparison of the concentration-response
trends generated for each FASNi in three different BC cell lines (MCF-7, MDA-MB-231, and BT-474), N 5 2–5 experiments per compound. (B) Compiled
IC50 values for each of the four FASNi. (C) Viability of noncancerous MCF10A mammary epithelial cells following 72-hour treatment with FASNi in
standard media. Data not fitted to a nonlinear curve. (D) Compound stability represented as percentage of recovery compared with control compound
(ILP-III-15).
Benzylamine derivative 25 was flagged as active in the initial
Compound 9 was active in the confirmatory assays, albeit
high-throughput screen with the purified thioesterase domain, approximately 10 times less potent (IC50 5 1.56 mM) at the
and since it also formed the base structure of compound 13, purified thioesterase domain compared with compound 1
a series of benzylamine derivatives (25–37) were prepared. The (IC50 , 0.16 mM). However, compound 9 showed improved
plasma stability of derivative 25 was comparable to com- plasma stability compared with other initial hits; therefore, it
pound 13 at room temperature or on ice, but lower at 37°C was used as the inspiration for a series of cyclohexylamine
(Supplemental Table 2). Addition of a methyl group at the derivatives (Fig. 5). (The SID number for compound 9 is
amide nitrogen (derivative 26) improved recovery from plasma 26724152. Direct links to the confirmatory bioassay data for
to 65% at 37°C, but derivative 26 was still not as cytotoxic as compound 9 are as follows: https://pubchem.ncbi.nlm.nih.gov/
compound 13. Either the cytotoxicity or the plasma stability, bioassay/624326#sid526724152 and https://pubchem.ncbi.
or both, were reasons enough for derivatives 27–37 to be nlm.nih.gov/bioassay/624327#sid526724152.) A boost in the
uninteresting, and this series was subsequently abandoned.
cytotoxicity (IC50 ∼16–24 mM) and plasma stability (90% recovery
Fig. 7. On-target inhibition of FASN by compound 41. (A)
Treatment of BT-474 lipogenic, HER21 BC cells with
compound 41 (4 hours, 50 mM) directly inhibited FASN activity,
as shown by significant accumulation of the committed
substrate for de novo lipogenesis, malonyl-CoA (*P , 0.05,
one-way ANOVA; biologic duplicate and technical triplicate
samples). (B) The ability of compounds 1 and 41 to specifically
target the thioesterase domain of FASN was assessed using
a FASN-TE substrate hydrolysis assay (N 5 3 experiments).
Under these conditions, the FASN-TE IC50 value of compound
1 was 1.47 6 0.17 mM with a Hill slope of 0.773, whereas that
of compound 41 was 14.58 6 1.16 mM with a Hill slope of
0.824.

180
Lupien et al.
Fig. 8. Cytotoxicity of compound 41 exceeds that of (2)-C75 across a range of cancer cell lines. Cells were treated with FASNi continuously for 72 hours
in standard 10% FBS RPMI 1640 and assessed using the ATP-based CellTiter-Glo viability assay (A, B, E, and F) or annexin V/PI cell death/apoptosis
assay (C and D). (A) Concentration-response curves for each BC cell line. (B) IC50 values for compound 41 vs. (2)-C75. (C and D) FASN inhibition causes
concentration-dependent apoptosis in MDA-MB-231 and BT-474 BC cells, as detected by annexin V/PI flow cytometry. The apoptosis observed with
compound 41 treatment significantly exceeds that of (2)-C75 at several concentrations, as indicated. Two-tailed, unpaired t tests were performed to
compare the effects of (2)-C75 at equimolar concentrations, *P # 0.05; **P # 0.01; ***P # 0.001. (E) Compound 41 displays minimal cytotoxicity against
MCF10A mammary epithelial cells. (F) Similar cytotoxicity trends observed across cancer cell lines; negligible impact on MCF10A mammary epithelial
cell viability. N $ 5 experiments for all treatments.

Improved Cytotoxicity and Stability of a FASN Inhibitor
181
Fig. 9. Availability of media lipoproteins impacts cytotoxicity and cell death from FASNi treatment. Increased cytotoxicity was observed with 72-hour
treatment of compound 41 in RPMI 1640 media containing 10% lipoprotein-depleted serum (LPDS) vs. the matched serum control. This trend was
observed in all cell lines tested and reflected in both assessments of (A) cell viability (CellTiter-Glo) and (B) apoptosis (Annexin/PI flow cytometry). No
significant differences in cell viability were observed in cells grown for 72 hours in matched or LPDS media alone (data not shown). N 5 3–7 experiments
per assay. Error expressed as S.D. from the mean, *P # 0.05; **P # 0.01; ***P # 0.001.
after 1 hour at 37°C) was observed for compound 38, when the probe for any increase in cytotoxicity; however, both diaster-
substituent was a cyclohexylamine alone. The racemic cis- and eomers 42 and 43 were comparable in cytotoxicity to compound
trans-4-tert-butylcyclohexylamines were both commercially 41 and were far less stable in mouse plasma.
available and relatively inexpensive; therefore, they were used
Compound 41 was selected for further investigation on the
to synthesize compound 39 to assess whether cytotoxicity gains basis of its promising stability and cytotoxicity parameters
could be made, as observed in the benzylamine series between (Figs. 6 and 8), the fact that substituted cyclohexylamines
compounds 25 and 35. This was indeed the case, and the mixture are not readily available or prepared, and that there was
of diastereomers only demonstrated a small decrease in plasma no obvious structure-activity information from another se-
stability compared with compound 39. We reasoned the cytotox- ries to suggest what modifications could be made to pursue
icity and plasma stability should be different for the two additional gains in cytotoxicity. Compound 41 was verified as
diastereomers in compound 39; therefore, we next synthesized having on-target inhibitory effects against the FASN-TE do-
individual diastereomers 40 and 41 to make the comparison. The main using a malonyl-CoA accumulation assay and a FASN-TE
trans-stereoisomer (compound 41) had superior plasma stability enzyme activity assay (Fig. 7). To further evaluate com-
and cytotoxicity against BC cells than that of the cis-stereoisomer pound 41, we used a range of BC cell lines that varied
(compound 40). A second pair of diastereomeric cyclohexylamine by tumor type and biomarker status (estrogen receptor,
derivatives, diastereomers 42 and 43, were prepared to further progesterone receptor, and HER2) (Table 1). Importantly,

182
Lupien et al.
Fig. 10. Simultaneous inhibition of FA synthesis and uptake (via lipolysis) results in increased cytotoxicity of BC cells in vitro. (A) Continuous
treatment with lipase inhibitor, GSK264220A (GSK), for 72 hours resulted in concentration-dependent cytotoxicity in MDA-MB-231 and BT-474 BC
cells, and is impacted by the presence of lipoproteins in the media at high concentrations (˃50 mM). (B) Simultaneously targeting FA synthesis and
uptake using compound 41 and GSK264220A causes increased cytotoxicity in MDA-MB-231 and BT-474 BC cells. The effects of continuous combined
treatments are compared with that of compound 41 alone (supplemented with DMSO to match the highest vehicle exposure). (C) The impact of media
lipoproteins on the cytotoxic effects of compound 41 and combined treatments of compound 41 and GSK at 100 mM. Viability was measured using the
ATP-based CellTiter-Glo assay. (B and C) Data points at a concentration of 0 mM of compound 41 represent the relative viability of the vehicle control
condition or the effect of GSK alone (without FASNi present). N 5 3–5 experiments for all treatments.
compound 41 demonstrated concentration-dependent cy- has no major issues related to these filters (Fig. 11). Only the
totoxicity across this panel of BC cell lines, was more potent rotatable bonds are outside of the parameters favored for small-
than (2)-C75, and also more selective than classic FASNi, molecule drug discovery, while the intramolecular hydrogen
as evidenced by negligible cytotoxicity against the non- bond formed in this scaffold is recognized as a beneficial feature
tumorigenic mammary epithelial MCF10A cell line.
This new lead compound represents an improved FASNi to
in the design of orally available drugs (Doak et al., 2014).
In addition to the insights into the inhibition of the FASN-
investigate the importance of this target in BC. We have TE domain provided by this work, important pathophysiolog-
shown that compound 41 is stable in mouse plasma and more ical information regarding the role of lipogenesis in breast
cytotoxic across a wide array of BC cell lines than other cancer biology has also emerged. The addiction of several
widely used FASNis. Compound 41 has drug-like proper- types of cancer cells to fatty acid synthesis is well recognized
ties and structure, making it a promising lead for continued (Menendez and Lupu, 2007; Baenke et al., 2013; Röhrig and
development toward a clinical therapeutic. Valuable guide- Schulze, 2016). We, and others, have previously reported that
lines for drug discovery include the rule-of-five set of in addition to de novo FA synthesis, the uptake of exogenous
properties (Lipinski et al., 1997; Lipinski, 2000, 2004), lipids may provide an alternative source of FA to cancer cells
the number of rotatable bonds (Veber et al., 2002), and cell (Ruby et al., 2010; Kuemmerle et al., 2011; Zaidi et al., 2013;
permeability (Egan et al., 2000). Lagorce et al., 2008 used Rozovski et al., 2015, 2016, 2018; Cao et al., 2017), and that
these rules and others to create an online screening tool for this process is facilitated by the expression of LPL and the cell
filtering lead compounds against the adsorption, distribution, surface FA uptake channel, CD36. This prompts the hypoth-
metabolism, excretion, and toxicity properties, thereby facil- esis that cancer cells may occupy various positions along
itating decision making at early stages of drug discovery (http:// a spectrum of dependence upon FASN-mediated lipogenesis
fafdrugs4.mti.univ-paris-diderot.fr). Importantly, compound 41 and LPL-driven lipolysis to satisfy their requirement for FA.

Improved Cytotoxicity and Stability of a FASN Inhibitor
183
potent and specific FASN inhibitors, such as compound 41
and its derivatives, become available.
Acknowledgments
The authors thank W. Todd Lowther (Wake Forest University,
Winston-Salem, NC) for the plasmid coding of the N-terminal His6-tagged
thioesterase domain of FASN.
Authorship Contributions
Participated in research design: Lupien, Lewis, Pooler, Kinlaw,
Baures.
Conducted experiments: Lupien, Dunkley, Maloy, Lehner, Foisey,
Ouellette, Pooler, Baures.
Contributed new reagents or analytic tools: Lewis.
Performed data analysis: Lupien, Lewis, Pooler, Baures.
Wrote or contributed to the writing of the manuscript: Lupien,
Lewis, Pooler, Kinlaw, Baures.
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between the two modes of lipid acquisition may be fluid, with
the removal of exogenous lipoproteins eliciting increased
expression of genes involved in FA synthesis, including FASN.
Our current finding that sensitivity to FASN-TE inhibition is
enhanced by either inhibition of lipase activity and/or removal
of lipoproteins from tissue culture media strongly supports the
concept that FA synthesis and uptake both support BC cell
survival. This notion has begun to be realized across a variety
of different cancer types, including not only BC (Kuemmerle
et al., 2011; Song et al., 2012; Slebe et al., 2016) but also
colorectal cancer (Notarnicola et al., 2012; Tang et al., 2012),
prostate cancer (Wang et al., 1995; Cerhan et al., 1997;
Connolly et al., 1997; Fair et al., 1997; Tokuda et al., 2003;
Brown et al., 2006; Gazi et al., 2007; Narita et al., 2008),
hepatocellular carcinoma (Li et al., 2016; Cao et al., 2017),
lung cancer (Sundaram et al., 2018), and pancreatic cancer
(Vasseur and Guillaumond, 2016).
Several groups have linked obesity or high-fat diets to
cancer prognosis, as well as implicated tumor-adjacent adipo-
cytes in altering the metabolism, growth, and progression
of solid tumors (Tokuda et al., 2003; Gazi et al., 2007;
Notarnicola et al., 2012; Nieman et al., 2013; Abel et al.,
2014; Massa et al., 2016; Cozzo et al., 2017; Gonzalez-Reyes
et al., 2018; Luo et al., 2018). It thus appears that, at least for
some tumors, simultaneous targeting of both lipogenesis and
uptake/lipolysis will provide the largest anticancer effect.
The importance of interactions between these complemen-
tary pathways will come into sharper focus as increasingly

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Address correspondence to: Paul W. Baures, Department of Chemistry,
Keene State College, 229 Main Street, Keene, NH 03435. E-mail: pbaures@
keene.edu