154
Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 154-167
ATP Citrate Lyase Inhibitors as Novel Cancer Therapeutic Agents
Xu-Yu Zu1,#, Qing-Hai Zhang1,#, Jiang-Hua Liu1, Ren-Xian Cao1, Jing Zhong1, Guang-Hui Yi2,
Zhi-Hua Quan1 and Giuseppe Pizzorno3,*
1
Clinical Research Institution, the First Affiliated Hospital, University of South China, Hengyang, Hunan 421001, P.R.
China; 2Institute of Cardiovascular Disease, School of Medicine, University of South China, Hengyang, Hunan 421001,
P.R. China; 3Director, Human Health and Environment Program, Desert Research Institute Summerlin, 10530 Discovery Drive, Las Vegas, NV 89135, USA
Received: November 1, 2011; Accepted: January 12, 2012; Revised: February 6, 2012
Abstract: ATP citrate lyase (ACL or ACLY) is an extra-mitochondrial enzyme widely distributed in various human and
animal tissues. ACL links glucose and lipid metabolism by catalyzing the formation of acetyl-CoA and oxaloacetate from
citrate produced by glycolysis in the presence of ATP and CoA. ACL is aberrantly expressed in many immortalized cells
and tumors, such as breast, liver, colon, lung and prostate cancers, and is correlated reversely with tumor stage and differentiation, serving as a negative prognostic marker. ACL is an upstream enzyme of the long chain fatty acid synthesis, providing acetyl-CoA as an essential component of the fatty acid synthesis. Therefore, ACL is a key enzyme of cellular lipogenesis and potent target for cancer therapy. As a hypolipidemic strategy of metabolic syndrome and cancer treatment,
many small chemicals targeting ACL have been designed and developed. This review article provides an update for the
research and development of ACL inhibitors with a focus on their patent status, offering a new insight into their potential
application.
Keywords: ACL inhibitors, ATP citrate lyase, cancer therapy, citrate, lipogenesis, small chemicals.
1. INTRODUCTION
Cancer treatment has been improved considerably during
the past 50 years. This malignant disease, however, still
holds its frightening impact and only about 50% of patients
could be cured [1]. Increasing evidence has highlighted the
importance of cellular metabolism in carcinogenesis [2-4].
Metabolic changes, such as increased lipogenesis, frequently
occurs in malignant cells and the impact of metabolic dysregulation on tumor development and progression has long
been recognized [3, 5]. ATP citrate lyase is a key enzyme
linking glucose and lipid metabolism, which converts citrate
produced by glycolysis to acetyl-CoA and oxaloacetate in
the presence of ATP and coenzyme A. The acetyl-CoA is in
turn used for cholesterol and long-chain fatty acid biosynthesis [2, 6-10]. Hereby, ACL acts as a mediator between increased lipogenesis and so-called Warburg effect in cancer
cells. Up to date, numerous reports have shown marked elevation of ACL expression and enzyme activity in immortalized cells and tumors, including urinary bladder, breast,
liver, stomach, colon, lung, brain and prostate cancers [1117]. Reduction of ACL activity with genetic or pharmacologic strategies significantly inhibits cancer cell proliferation in a dose-dependent manner and suppresses tumor
growth in animals. As increased understanding of ACL
*Address correspondence to this author at the Director, Human Health and
Environment Program, Desert Research Institute Summerlin, 10530 Discovery Drive, Las Vegas, NV 89135, USA; Tel: 702-822-5380;
Fax: 702-944-2355; E-mail: [email protected]
#
These authors contributed equally to this work.
2212-3970/12 $100.00+.00
enzymology, mechanism-based and active site-directed inhibitors have been developed. This review article updates the
current research and development of ACL small molecule
inhibitors, with a focus on their patent status.
2. ATP CITRATE LYASE
2.1. Structure of ATP Citrate Lyase
ATP citrate lyase [ATP citrate (pro-3S)-lyase, ACL or
ACLY; EC 4.1.3.8] is an extra-mitochondrial enzyme [18].
In prokaryotic and lower-rank eukaryotic (e.g. fungi) cells,
ACL is composed of two subunits, but in mammalian cells,
ACL is a 110kDa polypeptide forming a functional homomeric tetramer [19]. In humans, there are two ACL isoforms.
The ACL isoform-I consists of 1101 amino acids, and isoform-II is 10 residues shorter [20]. ACL protein contains five
functional domains, named from N-terminus domains 3, 4, 5,
1 and 2 Fig. (1). Domains 1 and 2 make up the -subunit
(residues 487-820) and domains 3-5 compose the -subunit
(residues 2-425). Domain 1 binds CoA and domain 2 contains a phosphorylation site His765, regulating enzyme activity [20]. Domains 3 and 4 adopt an ATP-grasp fold and bind
nucleotide [6, 21, 22]. Domain 5 interacts with domain 2,
producing one of the two "power helices" to whose aminoterminus the phosphohistidine residue bind to in the phosphorylated ACL [23-25]. Between domains 5 and 1 lies a
stretch of residues that can be phosphorylated on three serine
or threonine residues [26-28]. When citrate binds to ACL, a
loop formed by residues 343-348 interacts through hydrogen
bonds with hydroxyl and carboxyl groups on the prochiral
© 2012 Bentham Science Publishers
ACL as Novel Cancer Therapeutic Target
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
A
155
820
425
487
750
2
767
B
3
4
5
1
2
CS
a -subunit
b -subunit
Fig. (1). Human ATP-citrate lyase structure. A. Complex with citrate. The protein is shown as a ribbon diagram, while citrate is shown as
a stick model in magenta. Residues 2-31 and 108-243 form domain 4, which is colored green. Domain 3 includes residues 32-107 and is red.
Domain 5 includes residues 244-425 and is yellow. Domain 1 includes residues 487-624 and is cyan. Domain 2 includes residues 625-820
and is blue. The terminal residues seen in the electron density are labeled with their residue numbers. The ATP-grasp fold, formed by domains 3 and 4, is at the bottom of the diagram and ATP/ADP would be expected to bind to the back, as oriented here [32]. B. Arrangement of
five domains.
center of citrate, and Arg379 forms a salt bridge with the
pro-R carboxylate of citrate.
2.2. Function of ATP Citrate Lyase
ACL links glycolysis to fatty acid/lipid synthesis. Much
of the bioenergetic supply is produced by the glycolysis of
glucose, in cancer cells in particular, and glycolytically derived pyruvate enters the truncated tricarboxylic acid cycle
(TCA cycle, also known as citric acid cycle), where citrate is
preferentially exported to cytosol via the tricarboxylate
transporter [29-31]. In the cytosol, citrate is cleaved by ACL
to produce cytosolic acetyl-CoA and oxaloacetate. The enzyme reaction catalyzed by ACL includes four steps [32,
33]:
ATP +E E-P + ADP
(1)
E-P + citrate E•citryl-P
(2)
E•citryl-P + CoA E•citryl-CoA + Pi
(3)
E•citryl-CoA E + acetyl-CoA + oxaloacetate
(4)
Where E represents enzyme (i.e., ACL). ACL is phosphorylated by ATP on histidine residue at the active site to
form E-P in step 1. The phosphoryl group is transferred to
citrate in step 2. Citryl-phosphate binds with the enzyme,
symbolized by E•citryl-P. Phosphate is released in the attack
by CoA to form a citryl-CoA thioester bond in step 3. In the
last step, citryl-CoA is cleaved into acetyl-CoA and oxaloacetate.
Cytosolic acetyl-CoA serves three important biosynthesis: 1) lipogenesis in the liver, adipose tissue, and mammary
gland [7, 9]; 2) acetylcholine biosynthesis in the nervous
tissue [34]; and histone acetylation in nuclei to control DNA
accessibility and gene transcription. More recently, ACL is
found to play a critical role in beta-cell survival [10, 35],
platelet activation [36], and tumorigenesis [37]. Therefore,
ACL has been attractive and its protein chemistry, enzyme
kinetics and substrate specificity, and transcriptional and
post-translational regulations have been extensively studied
[18, 38-44].
2.3. Regulation of ATP Citrate Lyase
2.3.1. Transcriptional Regulation
At transcriptional level, ACL is regulated by sterol regulatory element-binding protein-1 (SREBP-1) [44], and insulin and glucose metabolites are important stimulatory factors
[45-47]. In vitro, insulin/glucose stimulates ACL expression
in primary hepatocytes through the SREBP-1 pathway, but
transcription factor Sp1 acts as a repressor [48]. Decreased
binding affinity of Sp1 to the G/C-rich region at -64 to -41bp
in ACL promoter is observed in response to insulin and glucose [48-50]. The PI3K/Akt signaling pathway is involved in
insulin-stimulated ACL expression, and tissue-specific
growth factors that activate the PI3K/Akt pathway appear to
play a similar role [51]. But nateglinide as an insulin secretagogue has recently been shown to suppress the expression of ATP citrate lyase [52]. Glucose plays a synergistic
156 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
role in ACL expression through the PI3K/Akt signaling, indicating its importance in regulating ACL activity [13, 53,
54].
2.3.2. Posttranslational Regulation
ACL activity is also regulated by phosphorylation and
dephosphorylation. ACL has a catalytic autophosphorylation
site, His760 that is phosphorylated by ATP as the first step in
citrate cleavage [55]. This His760 can also be phosphorylated by nucleoside diphosphate kinase (NDPK) from rat
liver and PC12 cells [56]. ACL is also phosphorylated at
other sites, including Ser3, Ser450 and Thr446. Evidence
shows that the phosphorylation of these sites in ACL is increased during cell differentiation [55] and in response to
biologically active agents, such as glucagon, insulin, adrenergic agonists, vasopressin, and transforming growth
factor 1 [57]. In response to insulin, ACL is phosphorylated
on Ser450 by cAMP-dependent protein kinase and insulinstimulated kinase [58, 59]. Ser450 can also be phosphorylated by Akt in primary adipocytes [28], indicating that the
PI3K/Akt pathway is involved not only in ACL transcription, but also in ACL activity regulation through phosphorylation. In addition, Ser450 and Thr446 as well are also phosphorylated by glycogen synthase kinase-3 [10, 55]; and Ser3
of ACL is phosphorylated by cAMP-dependent protein
kinase. The later abolishes homotropic allosteric regulation
of ACL by citrate and increases enzyme activity [41, 56].
3. ATP CITRATE LYASE AND CANCER
It is well known that bioenergetics of cancer cells is altered, including increased glucose uptake and glycolysis,
lactic acid production and lipogenesis [54]. The increased
lipogenesis is a hallmark of cancer and an early event in tumorigenesis, providing lipids essential for cell growth and
division [60]. It is understood that cancer cells are highly
dependent on the de novo fatty acid synthesis for cellular
lipids [61]. On the other hand, cancer cells typically depend
more on glycolysis, the anaerobic breakdown of glucose for
ATP, even in the presence of available oxygen. This phenomenon is known as Warburg effect [62]. The elevated
glucose catabolism produces excessive glycolytic endproduct, pyruvate that is then converted either to lactate or
acetyl-CoA for de novo fatty acid synthesis [61]. Glycolysis
may suppress tumor cell differentiation by increasing cytosolic acetyl-CoA and resultant lipid synthesis [13].
Cytosolic acetyl-CoA is an essential component of
de novo fatty acid synthesis. ACL integrates glucose glycolysis with lipid synthesis by converting citrate to acetyl-CoA
Fig. (2). Citrate is a metabolite inhibitor of glycolysis and a
precursor of acetyl-CoA for de novo fatty acid synthesis.
Therefore, ACL activity may affect not only lipid synthesis
but also glucose glycolysis for ATP production, and ACL
expression is pervasively up-regulated in immortalized cells
and various tumors, including in urinary bladder, breast,
liver, stomach, colon, lung, brain and prostate tumors [11-13,
16]. The phosphorylated ACL (active form) is correlated
with tumor stage, differentiation grade, and prognosis. In
cultured cancer cells, increased ACL expression promotes
cancer cell proliferation and survival [13, 59] and enhances
aggressive biological behaviors, such as clonogenic growth,
Zu et al.
migration and invasion [16, 60, 61]. Genetic and pharmacologic abrogation of ACL activity in cancer cells results in
dose-dependent inhibition of cell proliferation and tumor
growth, and the effectiveness of this treatment rely on the
glycolytic phenotype of tumor cells. Cancer cells with a high
rate of glucose aerobic glycolysis are much more sensitive to
ACL inhibition than those with a low glycolytic rate [63]. In
glucose-dependent cancer cells, selective ACL inhibition or
knockdown promotes cell differentiation, elevates mitochondrial membrane potential, decreases cell viability, and stimulates apoptosis [54, 64]. Hence, therapeutic strategies targeting ACL need taking the glycolytic status of cells into consideration. ACL inhibition may also down-regulate lactate
dehydrogenase, allowing pyruvate to be imported into mitochondria and enter Krebs cycle [63]. Furthermore, it is noteworthy that ACL inhibition may affect certain oncogenic
gene expression by suppressing histone acetylation due to
reduction of cytosolic acetyl-CoA [65].
In summary, although it is controversial whether increased ACL expression and activity in cancer cells is a
cause of tumorigenic process, it is clear that ACL as an enzyme linking glucose and lipid metabolism is a promising
target for cancer therapy. The research and development of
ACL inhibitors represent a novel strategy of cancer treatment.
4. ATP CITRATE LYASE INHIBITORS
In view of the importance of ACL in glucose and lipid
metabolism, investigators have done much work for designing and developing ACL inhibitors for hypolipidemic treatment of metabolic and malignant diseases [66]. In particular,
mechanism-based, active-site directed, or tight-binding small
chemicals have been developed, having an irreversible, specific inhibition on ACL. Followed is a summary of some
representative ACL inhibitors.
4.1. Hydroxycitrate
Hydroxycitric acid (HCA) [67] is derived from fruit rinds
of Garcinia species, including G. cambogia, G. indica, and
G. atroviridis that grow prolifically on the Indian subcontinent and in Western Sri Lanka [68]. Hydroxycitric acid is
also produced in calyxes of Hibiscus subdariffa and H. rosasinensis that are cultivated in several tropical and semitropical countries. Nowadays, the extracts of Garcinia fruits or
flowers are popularly used as an ingredient in diet supplements or drinks. Studies with Garcinia extracts have showed
that ingested HCA is absorbed in the gastrointestinal tract
and enters the systemic circulation [69]. However, bioavailability of HCA is low and its competitive competence with
endogenous citric acid is weak. Administration of HCA at
250mg/kg twice a day for three weeks is innocuous with no
effects on weight, behavior, and survival of animals [63].
The safety dose to humans is up to 13.5g of hydroxycitrate
per day [16].
HCA has two diastereomers due to the existence of two
chiral centers in the molecule. Therefore, there are four
stereoisomers of HCA [70], comprising two pairs of enantiomers which are (2S,3S)-2-hydroxy citrate and (2R,3R)-2hydroxycitrate, (2S,3R)-2-hydroxycitrate and (2R,3S)-2- hy-
ACL as Novel Cancer Therapeutic Target
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
157
Glucose
Glucose transporter
Lactate
Cellular membrane
Glucose
HK
Lactate
LDH
NAD+
Pentose phosphate pathway
2NADP +
NAD+
2NADPH
Glucose-6P
NADH
CO 2
Glycolytic pathway
Pyruvate
Pyruvate
NADP+
ATP
NADPH
3
CO 2
Acetyl-CoA
CO 2
FA
S
Malonyl-CoA
Acetyl-CoA
ACC
Oxaloacetate
Oxaloacetate
Citric acid cycle
2
CO 2
NAD+
1
ACL
Citrate
Citrate
NADH
Fatty acids
Pentose sugars
NADH
Malate
Malate
4
NADP +
Mitochondria
CO 2
Pyruvate
Malic enzyme
Cholesterol
NADPH
Oncogene transcriotion
histone acetylation
Nucleus
Fig. (2). De novo synthesis of fatty acids from carbohydrate precursors and possible anticancer mechanisms of ACL inhibition. Upon
cellular uptake by glucose transporters, glucose is phosphorylated by hexokinases (HKs) to glucose-6-phosphate, most of which enters the
glycolytic pathway generating pyruvate and ATP. In the mitochondria, pyruvate is converted to acetyl-CoA, entering the citric acid cycle.
Depending on the oxygen tension, citrate can be oxidized to carbon dioxide and oxaloacetate, generating ATP via oxidative phosphorylation,
or can be transported into cytosol, converted by ATP citrate lyase (ACL) to acetyl-CoA and oxaloacetate. Acetyl-CoA is the requisite building block for fatty acid and cholesterol synthesis. NADPH required for fatty acid synthesis is produced by malic enzyme or via the pentose
phosphate pathway. Under anaerobic conditions, pyruvate may also be used as electron acceptor, producing lactate by lactate dehydrogenase
(LDH), which is excreted from the cell [54]. When genetic and pharmacologic downregulation of ACL is applied, there are four possible
approaches against cancer, labeled with blue-colored numbers. They are inhibition of fatty acid (1) and cholesterol (2) synthesis, glycolytic
pathway (3) by cytosolic citrate, and histone acetylation, affecting oncogenic gene expression (4).
droxycitrate, respectively Fig. (3). Each of the stereoisomers
can form a -lactone ring and, in general, HCA is a mixture
of nonlactone and lactone forms. Nonlactone can be converted to lactone in 1N HCl with a high yield [71].
It was found forty years ago that Garcinia’s constituent
(2S,3S)-2-hydroxycitrate, also named 4S-hydroxycitrate or ()-hydroxycitrate, is a strong competitive ACL inhibitor [72].
Later studies showed that among the four hydroxycitrate
stereoisomers, only (2S,3S)-2-hydroxycitrate possesses inhibitory effect on rat and human ACLs [72-74], and its lactone form is less effective [73]. The other three HCA stereoisomers are not potent inhibitors of ACL with low affinity (30
- 132M) [75]. Therefore, inhibitory potency of (-)-hydroxycitrate depends on its stereochemistry. It is noteworthy that
ACL inhibitory (2S,3S)-2-hydroxycitrate does not have substrate activity for this enzyme, but the other three do, in spite
of lack of inhibitory activity.
Inhibitory activity of (-)-hydroxycitrate on ACL has been
extensively investigated. It was reported that ACL activity in
colonocyte was decreased by 86.6% at 7mM of HCA [76].
Under glycolytic condition, hydroxycitrate suppressed migration of human glioblastoma cells U87 and Glioblastoma
cells LN229 with IC50 at 16.7 and 12.1mM, respectively
[16]. Hydroxycitrate at 10-500M could inhibit growth of
human bladder cancer cells T-24 or colon tumor cells HT-29
by 5-60%. HCA also inhibits cell invasion and increases 3hydroxy-3-methylglutaryl-CoA reductase and low-density
lipoprotein (LDL) receptor activity in HepG2 cells [77].Used
together with lipoic acid, HCA induces significant tumor
growth retardation and enhances survival in MBT-2 bladder
transitional cell carcinoma, B16-F10 melanoma and LL/2
Lewis lung carcinoma mouse syngenic cancer models [63].
A combination of lipoic acid,hydroxycitrate and cisplatin/
methotrexate shows more tumor suppression efficacy than
158 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
COOH
COOH
HO
HO
S
S
H
H
COOH
HOOC
H
H
O
O
S
H
R
COOH
OH
HO
OH
HOOC
H
HOOC
OH
O
H
(2S,3S)-HCA
O
H
R
H
R
COOH
H
H
HO
OH
H
O
COOH
OH
H
H
(2R,3R)-HCA
O
COOH
COOH
R
H
COOH
S
H
COOH
S
OH
R
COOH
H
R
COOH
S
H
COOH
COOH
S
R
H
COOH
H
Zu et al.
HOOC
O
OH
H
(2S,3R)-HCA
O
H
R
OH
COOH
S
H
H
(2R,3S)-HCA
Fig. (3). Structures of HCA stereoisomers. Upper panels show the nonlactone forms, and lower panels demonstrate the lactone forms [71].
cisplatin or methotrexate alone [78, 79]. In rat, (-)-hydroxycitrate reduces the synthesis of both cholesterol and fatty
acids and thus decrease plasma triglyceride level by inhibiting ACL [69, 80, 81]. Nevertheless, HCA inhibits lipogenesis in brown adipose tissue in response to glucose feeding,
but not in starved rat [82]. Therefore, (-)-hydroxycitrate is a
potential agent used for hypolipidaemic treatment of metabolic or malignant diseases [81].
4.2. Non-hydroxycitrate Citric Acid Analogues
Methionine sulfoximine [83] is a well-known irreversible
inhibitor of glutamine synthetase [6, 83, 84]. Since ACL
shares the similar reaction mechanism with glutamine synthetase, Dolle et al. [84] designed and synthesized a few citric acid analogs derived from this glutamine synthetase inhibitor.
Diastereomers (+)-12a, b are citric acid analogs by replacing the primary carboxylate groups with a sulfoximinoyl
Fig. (4A), leading to occurrence of enzyme-mediated phosphorylation at 14a, b rather than 12a, b. These compounds
mimic the enzyme-bound intermediate (citrate phosphate
anhydride) and thus are potential tight-binding inhibitors of
ACL. Unfortunately, testing results showed that only the (+)12a had a weak, reversible inhibition on rat ACL activity
with a Ki at 250M, 10-fold greater than the Km for citrate,
but the relative Vmax was only 25% of the normal substrate.
This data suggest that the sulfoximinoyl functional group can
mimic, to some extent, the carboxylate group in the active
site of the enzyme, probably due to its hydrogen-bonding
ability [84].
Via covalently modifying the active-site nucleophiles, a
novel class of citrate analogues has been developed [85]. The
(-)-5 and (+)-6 are the representative compounds in which
the epoxides are potentially active-site-directed. Other compounds include chlorocitrates (+)-10, (+)-11 and thiocitrates
12-16 Fig. (4B) [86]. Among these agents, epoxyaconitic
acid (-)-5 [87] showed modest reversible inhibition on rat
ACL with Ki at 18M, 22 fold higher than (+) -6 (Ki =
400M).
Earlier studies reported enantiomers of 2-monofluorocitrate Fig. (4C) as substrates and competitive inhibitors of
ACL, but their inhibitory activity was relatively weak [73μM
for the (2S,3S) and 192μM for the (2R,3R) forms] [88]. A
similar enzymatic kinetics emerges from 2-vinylcitrates
which, despites having very high Ki values, are cleaved by
ACL [89]. Therefore, enantiomers (+) and (-)-2,2-difluorocitrate Fig. (4C) were designed [90], and these difluorocitrate isomers have significant stronger competitive inhibition to ACL than any compounds published thus far. These
compounds have competitive inhibition against citrate at Ki
of 0.7μM for (+)-2,2-difluorocitrate and 3.2μM for (-)-2,2difluorocitrate. Their inhibitory patterns with either ATP or
CoA as a substrate are uncompetitive or mixed and the inhibition constants are much weaker. Neither isomer undergoes
carbon-carbon bond cleavage as a substrate, nor is there evidence of irreversible time-dependent inactivation. When
ACL is incubated with CoA and difluorocitrate, the maximal
intrinsic ATPase rate is 10% of the citrate-induced for (+)enantiomers and 2% for (-)-enantiomers.
4.3. Radicicol
Radicicol [91] Fig. (5), a 14-membered macrolide, is a
potent tranquilizer, inducing morphological changes of various transformed cells and cell cycle arrest in G1 and G2
phases, as well as angiogenesis inhibition [92]. Radicicol
targets Hsp90, showing strong binding in a manner competitive with ATP [93, 94]. Recent studies have shown that radicicol is also a noncompetitive inhibitor of ACL. Kinetic
analysis demonstrated that radicicol and its derivative BR-1
Fig. (5) inhibited the activity of rat liver ACL with no apparent effect on Km but decreased Vmax. As an inhibitor of ACL,
radicicol has a Ki at 13M for citrate and 7M for ATP.
Guay et al. [95] reproted that 50M radicicol inhibited ACL
activity along with decrease in insulin secretion by about
50% in the presence of 5 and 10mM glucose or 0.3mM
palmitate in vitro.
ACL as Novel Cancer Therapeutic Target
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
159
A
O
O
NR
NR
S
HOOC
HOOC
(
(
S
HOOC
OH
HOOC
)-12a:R=H
)-14a:R=P(O)(O - ) 2
(
(
OH
)-12b:R=H
)-14b:R=P(O)(O - ) 2
B
O
O
HOOC
HOOC
COOH
COOH
HOOC
HOOC
(+)-6
(-)-5
Cl
Cl
COOH
HOOC
HO
(
O
)-10
(
SR
COOH
)-11
SH
HOOC
COOH
HO
COOH
O
COOH
R
HOOC
COOH
COOH
HO
(-)-12:R=H
(-)-13:R=SMe
COOH
HOOC
COOH
COOH
15: R=SH
16: R=SSMe
(-)-14
C
F
F
COOH
COOH
HO
COOH
COOH
COOH
HO
(2R,3R)-2-fluorocitrate
(2S,3S)-2-fluorocitrate
F F
COOH
COOH
F F
COOH
HO COOH
(3S )2, 2 -d i f luorocitrate
COOH
COOH
HO COOH
(3R)2, 2 -d i f luorocitrate
Fig. (4). Representatives of non-hydroxycitrate citric acid analogues. A. Sulfoximine-containing citric acids. B. Epoxide-containing citric
acids, chloro-containing citric acids and thiol-containing citric acid. C. Fluoro-containing citric acid.
4.4. Antimycins
Antimycins [96] Fig. (6) were antibiotics isolated from
Streptomyces sp [97]. More recently, novel derivatives were
identified and named antimycins A2 to A6 and each of them
is a mixture of two closely related isomers [97]. Antimycins
are characterized with a carboxy phenol amido unit, a ninememberedcyclic bis-lactone, and two alkyl side chains with
variations of carbon length. Although, well identified as antibiotics and fungicides, more antimycins have been extensively investigated in energy metabolism in eukaryotic or-
ganisms due to their inhibition on electron flow in the mitochondrial respiratory chain between cytochromes b and c1
[97]. Lately, Barrow et al. reported that antimycins are inhibitors of ACL against the substrate citrate with promising
Ki values: 29.5M for antimycin Al, 4.2M for antimycin
A2, 60.1M for antimycin A3, 64.8M for antimycin A4,
55.0M for antimycin A7, and 4.0M for antimycin A8 [97].
4.5. Butanedioic Acid Derivatives
A series of 2-substituted butanedioic acids have been
developed as inhibitors of ACL based on the concept of link-
160 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
CH3
O
O
Zu et al.
H
O
H
HO
Radicicol
O
Cl
OH
CH3
O
O
H
O
O
H
O
NH
NH
O
2
S
O
O
Cl
OH
NH
BR-1
Fig. (5). Radicicol and representative of radicicol derivatives.
O
NH
H
OH
O
O
O
O
NH
R1
O
R2
O
Antimycin A1a R1 = CH(CH3)CH2CH3, R2 = (CH2)5CH3
Antimycin A1b R1 = CH2CH(CH3)2, R2 = (CH2)5CH3
Antimycin A2a R1 = CH(CH3)2, R2 = (CH2)5CH3
Antimycin A2b R1 = CH2CH2CH3, R2 = (CH2)5CH3
Antimycin A3a R1 = CH(CH3)CH2CH3, R3 =(CH2)3CH3
Antimycin A3b R1 = CH2CH(CH3)2, R2 =(CH2)3CH3
Antimycin A4a R1 = CH(CH3)2, R2 =(CH2)3CH3
Antimycin A4b R1 = CH2CH2CH3, R2 =(CH2)3CH3
Antimycin A7a R1 = CH(CH3)2, R2 =(CH2)2CH(CH3)2
Antimycin A7b R1 = CH2CH2CH3, R2 =(CH2)2CH(CH3)2
Antimycin A8a R1 = CH(CH3)CH2CH3, R2 =(CH2)2CH(CH3)2
Antimycin A8b R1 = CH2CH(CH3)2, R2 =(CH2)2CH(CH3)2
Fig. (6). Basic structure of antimycins.
ing a lipophilic group to a “citrate-like” head. The most
promising compounds are butanedioic acid derivatives with a
substitute in the second position with an appropriate length
(8 atom) spacer for a 2, 4-dichlorophenyl group. The compounds 1a-c Fig. (7) have reversible Ki’s at 1-3M against
rat ACL and show a competitive inhibitory mechanism
against citrate and noncompetitive with respect to CoA. Further studies suggest that these compounds interact with ACL
by binding to butanedioic acid moiety to the citrate site, supporting the competitive inhibition mechanism against citrate.
Unfortunately, butanedioic acid derivatives could not suppress cholesterol or fatty acid synthesis in cultured cells
(HepG2) due to the poor cell-permeability of these polar
chemicals [6].
Novel 2-substituted butanedioic acid derivatives have
demonstrated better promise as ACL inhibitors [2, 98]. SB-
ACL as Novel Cancer Therapeutic Target
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
Cl
(CH2)6
X
HO
Cl
COOH
COOH
161
1a: X=S
1b: X=CO
1c: X=CH2
1
Cl
Cl
(CH2)6
Cl
COOH
HO HO COOH
OH
(CH2)6
COOH
O
Cl
O
SB-204990
SB-201076
Fig. (7). Representatives of butanedioic acid derivatives.
201076 [66] Fig. (7) was found to be equally potent in inhibiting rat (Ki = 1±0.05M) or human (Ki = 1±0.1M) ACL.
SB-204990 [66] Fig. (7), the cell-penetrant -lactone prodrug
of SB-201076, was found to be inactive to ACL in the assay
without cellular context, because SB-201076 is formed inside the cells by hydrolysis of lactone. Pre-incubation of SB204990 with either diabetic or control platelet suspension for
30 minutes at 37°C did not affect ACL activity, but a longer
incubation led to 61% inhibition for diabetic and 37% for
control platelets [36]. Oral administration of SB-201076
dose-dependently reduces plasma cholesterol (up to 46%)
and triglyceride levels (up to 80%) in rats and plasma cholesterol (up to 23%) and triglyceride levels (up to 38%) in dogs
[2].
competitive to citrate at Ki of 16M [101] and to CoA at Ki
of 3 M [8].
Other biological effects of SB-204990 are observed.
In vitro studies showed SB-204490 at 0.1mmol/L decreases
acetyl-CoA content in platelet cytoplasm along with suppression of MDA synthesis and platelet aggregation [36]. Interestingly, SB-204990 can reduce D-[6-14C] glucose-dependent lipid synthesis in a dose-dependent manner, resulting in
proliferation inhibition and death of tumor cells with active
aerobic glycolysis [13]. Recently, SB-204990 has been
shown to be effective to induce apoptosis in greater than
50% of cancer cells in an in vitro apoptosis assay at a concentration of less than 50M [66].
4.7. 2-Hydroxy-N-arylbenzenesulfonamide
4.6. Dicarboxylic Acid Derivatives
MEDICA compounds are a class of ,'-methylsubstituted , -dicarboxylic acids, defined as MEDICA 616 upon the number of carbon atoms. MEDICA 16 [99] Fig.
(8) is a ,'-dimethyl hexadecanedioic acid with hypolipidemic effect, leading to 60 and 45% reduction of cholesterol
and high density lipoprotein in rats, respectively [100]. Recently, MEDICA 16 was reported to be an ACL inhibitor
HOOC
(CH2)10
COOH
MEDICA 16
Fig. (8). Representatives of dicarboxylic acid derivatives.
Sulfur-substituted fatty acid analogue 3-thiadicarboxylic
acid Fig. (8) is a novel dicarboxylic acid derivative that can
inhibit ACL and fatty acid synthase activity [102]. Treatment
with 3-thiadicarboxylic acid reduced plasma levels of triglycerides from 5.8 to 2.7mmol/L and cholesterol from 11.0 to
7.7mmol/L in rats and suppressed the activity of the ratelimiting enzyme in cholesterol biosynthesis, 3-hydroxy-3methylglutaryl-CoA reductase by 58%. It is believed that
hyperlipidem in experimental nephrosis could be ameliorated
by 3-thiadicarboxylic acid via decreasing the overproduction
of very-low-density lipoprotein [102].
With attempts to identify a cell-permeable ACL inhibitors, Li et al. [103] identified 2-hydroxy-N-arylbenzenesulfonamide [104] as a modest inhibitor of ACL with 50%
inhibition of ACL activity (IC50) at 1.1M) [103]. Among
approximately 50 analogs synthesized by Li et al. [103], 11
showed greater than 50% inhibition at 10M Fig. (9) and the
compound 9 showed an IC50 of 0.13μM and had no cytotoxicity up to 50M. Long-term oral dosing of compound 9 in
high-fat-fed mice lowered down plasma cholesterol, triglyceride, and glucose and blocked weight gain. Those data suggest that 2-hydroxy-N-arylbenzenesulfo-namide 9 is the most
potent ACL inhibitor with appreciable cell permeability.
4.8. Purpurone
Purpurone is a purple, non-crystalline glassy solid.
HRFAB mass spectrum ([M + H]+, m/z = 698.1673) established its molecular formula of C40H27NO11 Fig. (10).
HOOC
S
(CH2)10
S
COOH
3-thiadicarboxylic acid
162 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
Zu et al.
OH
OH
Cl
Cl
OH
HO
OH
O S
NHR
O
Compound
IC50a (mM)
R
N
O
4
t-Bu
1.1
t-Bu
CH2
HO
2.3
5
Cl
O
CH2
OH
OH
OH
Cl
6
7.6
F
F
OH
t-Bu
7
5.6
Purpurone
t-Bu
Ph
Ph
0.19
8
Ph
OMe
9
0.13
Ph
Fig. (10). Structure of purpurone.
viability and cellular ATP level of HepG2 cells at 100μg/ml.
Recently, a new rapid synthesis of purpurone has been developed, which will promote investigation and development
of new purpurone derivatives as inhibitors of ACL [106].
4.9. Other Agents
10
6.9
n-Hex
10.9
11
Me
N
COMe
8.1
12
OPh
Me
O
13
0.37
O
i-Pr
O
14
0.34
O
Ph
36
Fig. (9). In vitro SAR summary. In vitro data are at least two separate measurements using recombinant hACL [103].
Purpurone was isolated from the sponge Iotrochota sp. and
has antioxidant activity. Purpurone exhibits its inhibitory
activity on ACL in a dose-dependent manner, and has an
IC50 of 7μ [105]. Purpurone reduced fatty acid synthesis,
but not cholesterol [105]. Purpurone had no effect on the
Apart from ACL inhibitors discussed above, a few other
chemicals have partial or indirect inhibitory activity to ACL
and are discussed below.
4.9.1. 2, 3-Butanedione and Phenylglyoxal
As arginine-targeted agents, both 2, 3-butanedione and
phenylglyoxal could inactivate ACL in a concentrationdependent manner. Phenylglyoxal induces rapid inactivation
of ACL by 85%, but butanedione does to a lesser degree
(35%). Inhibition to ACL by these two compounds is at least
in part due to their modifications of arginine residues at the
CoA binding site. Phenylglyoxal-modified ACL shows a
decrease in Vmax, but its Km for substrates does not alter significantly. However, ACL substrates, CoA or CoA plus citrate, could protect the enzyme against inactivation by 2, 3butanedione and phenylglyoxal in rats [38].
4.9.2. L-Glutamate
Glutamate, as the most abundant excitatory neurotransmitter in the vertebrate nervous system, is found to be a specific inhibitor of ACL from adult rat brain and liver [34,
107]. Glutamate, especially L-glutamate, has a time-dependent inhibitory effect on ACL activity in the presence of both
ATP and MgCI2 and a high concentration of ATP could reverse this process [107]. Szutowicz et al. found that Lglutamate is a competitive inhibitor of ACL (Ki = 0.3mM)
with no effect on Km and Vmax at a low concentration of
MgCL2, suggesting excessive Mg2+ ions is indispensable for
glutamate inhibition [107]. The inhibitory activity of glutamate may arise from the production by glutamine synthetase
of ADP, a known product inhibitor of ACL [34].
ACL as Novel Cancer Therapeutic Target
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
phosphorylation at histidine by 67%, inactivating ACL
[111]. Vanadate has also inhibitory activity to succinyl-COA
synthease (SCS) [111].
4.9.3. Deoxycholate
Deoxycholic acid is one of the secondary bile acids that
are metabolic byproducts of intestinal bacteria. Eriyamremu
et al. reported that sodium deoxycholatecould can significantly reduce ACL activity in the experimental rat liver
[108].
5. CURRENT & FUTURE DEVELOPMENTS
Cancer is a potentially fatal disease with poor responses
to current therapies. Recent new insights on tumor metabolism alterations contribute to the concept of targeting tumor
bioenergetics as an anticancer strategy. Based on the marked
elevation of expression and activity of ACL in many immortalized cells and tumors and the effect on cancer cell proliferation or tumor growth induced by ACL downregulation,
various ACL inhibitors with different structures and targeting sites have been designed, synthesized and evaluated on
anticancer activity. Of these discussed compounds Table 1,
most inhibitors, such as hydroxycitrate, vinylcitrates, thiocitrates, fluorocitrates, play a competitive inhibition against
citrate, which are characterized by a common citrate-featured
head. Others are competitive inhibitors to CoA, such as
MEDICA 16. Some of them exert a multiple inhibition simultaneously, like SB-201076 which inhibits ACL activity
through competitive mechanism with respect to citrate and
noncompetitive manner to CoA. This class of inhibitors may
hold stronger promises due to their dual targets.
4.9.4. Polychlorinated Biphenyls
Polychlorinated biphenyls (PCBs), a molecule composed
of two benzene rings, are a class of organic compounds with
1 to 10 chlorine atoms attached to biphenyl. It is well known
that PCBs have great toxicity due to its structural similarities
to dioxin and certain hormones, such as thyroxine and estradiol. Recently, Kling et al. [109] found that PCBs administered in diet (0.01%, w/v) inhibits citrate cleavage independent of citrate concentrations. Hereby, PCBs are probably
noncompetitive inhibitors of ACL.
4.9.5. Vanadate
Vanadium is a trace element essential for the growth and
normal existence of living cells. Vanadium affects many
biochemical processes, such as protein phosphorylation by
its biologically active forms, pcntavalenr vanadate (VO3-,
H2VO4-) or tetravalent vanadyl cation (VO2+) [110]. However, investigators reported that vanadate at 1 mM inhibits
Table 1.
Common Inhibitors of ATP Citrate Lyase.
Name
Inhibotor Type and
Study
Feature
In
Vitro
Ki /IC50 Value
Hydroxycitrate
Competitive inhibitor with Ki
of 0.15 and 50μM for rat
enzyme and Ki of 300μM for
human enzyme.
In
Vivo
Clinical
Trial
1) Decreased plasma cholesterol and triglyceride levels
2) Suppressed platelet aggregation
Tumor Type
Reference
[67, 72-75,
78, 81]
Yes
Yes
Yes
Glioblastoma;
bladder carcinoma;
colon carcinoma;
melanoma; lung
carcinoma; adenocarcinoma
3) Suppressed cancer growth,
migration and invasion
Sulfoximinecontaining citric
acid
Reversible inhibitior with Ki
of 250μM for rat enzyme
No evidence
Yes
No
No
No evidence
[6, 83-84]
Epoxidecontaining citric
acid
Reversible inhibitior with Ki
of 18μM to 400μM for rat
enzyme
No evidence
Yes
No
No
No evidence
[85, 87]
Chlorocontaining citric
acid
Reversible inhibitior with Ki
of 29μM to 340μM for rat
enzyme
No evidence
Yes
No
No
No evidence
[85-86]
Thiol-containing
citric acid
Reversible inhibitior with Ki
of 58μM to 480μM for rat
enzyme
No evidence
Yes
No
No
No evidence
[85]
Fluorocontaining citric
acid
Competitive inhibitor with Ki
of 0.7μM to 192μM for rat
enzyme
No evidence
Yes
No
No
No evidence
[88-90]
163
164 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
Zu et al.
(Table 1) Contd….
Name
Inhibotor Type and
Study
Feature
Ki /IC50 Value
In
Vitro
In
Vivo
Clinical
Trial
Tumor Type
Reference
Noncompetitive inhibitor
with Ki of 13μM for citrate
and 7 μM for ATP
1) Decreased insulin secretion
Radicicol
2) Suppressed cancer cells
migration and invasion
Yes
No
No
Glioblastoma
[91-94]
Antimycin
With Ki of 4.0 to 64.8μM
No evidence
Yes
No
No
No evidence
[96-97]
Butanedioic acid
derivatives
Competitive inhibitor with
respect to citrate and noncompetitive inhibitor with
respect to CoA; Ki= 1-3μM
Yes
Yes
No
Lung carcinoma;
prostate carcinoma;
ovarian cancer
[2, 6, 98]
1) Decreased plasma cholesterol and triglyceride
2) Suppressed platelet aggregation
3) Reduced tumor growth and
induced tumor differentiation
Dicarboxylic acid
derivatives
A citrate competitive inhibitor
of ACL with a Ki of 16 μM
and a competitive inhibitor
with respect to CoA (Ki =
3μM)
Reduced cholesterol level
Yes
Yes
No
No evidence
[99-102]
2-Hydroxy-Narylbenzenesulfonamide derivatives
With IC50 of 0.13 to 10.9μM
Lowered plasma cholesterol,
triglyceride and glucose levels
Yes
Yes
No
No evidence
[103-104]
Purpurone
With IC50 of 0.7μM
Reduced fatty acid synthesis
Yes
No
No
No evidence
[105]
2, 3-Butanedione
Noncompetitive inhibitor
No evidence
Yes
No
No
No evidence
[38]
Phenylglyoxal
Noncompetitive inhibitor
No evidence
Yes
No
No
No evidence
[38]
L-Glutamate
Indirect competitive inhibitor
with Ki of 0.3 mM
No evidence
Yes
No
No
No evidence
[34, 108]
Deoxycholate
No evidence
No evidence
No
Yes
No
No evidence
[108]
Polychlorinated
biphenyls*
Noncompetitive inbibitor
No evidence
No
Yes
No
No evidence
[109]
Vanadium
No evidence
No evidence
Yes
No
No
No evidence
[110, 111]
* Such compounds are toxic substances for body.
A couple of concerns need to be taken into consideration
in developing ACL inhibitors as antitumor agents. It is noted
that some chemicals possess promising inhibitory activity to
ACL, but the cell-permeability limit their uses in organisms,
such as SB-201076. Therefore, relevant chemical modifications may be required, such as SB-204490, a cell-penetrant
derivative of SB-201076. Second, some inhibitors show efficient ACL inhibition, reducing plasma cholesterol and
triglyceride levels, but cytotoxicity is limited. These inhibitors may hold the promise as treatment agents for metabolic
syndromes, but their potential as antitumor agents need to be
carefully evaluated, or combinational therapies are required.
Third, clinical trials are required to document the safety and
efficacy of ACL inhibitors as antitumor agents. Currently,
although some ACL inhibitors present efficacy of inhibiting
ACL and suppressing cell proliferation and tumor growth in
laboratory studies, clinical data are limited and relevant studies need to be strengthened. In summary, in spite of the limit
in ACL investigation and application, it is convincing that
targeting ACL as a therapeutic strategy of human cancer
holds the promisea and is under active investigation.
ACKNOWLEDGEMENTS
This work is supported by projects from Hunan Provincial Natural Science Foundation of China (11JJ4068) and
ACL as Novel Cancer Therapeutic Target
from Medical Scientific Research Foundation of Hunan
Province, China (B2010-045).
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
[20]
[21]
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
Arends J. Metabolism in cancer patients. Anticancer Res 2010; 30:
1863-8.
Pearce NJ, Yates JW, Berkhout TA, Jackson B, Tew D, Boyd H.
The role of ATP citrate-lyase in the metabolic regulation of plasma
lipids. Hypolipidaemic effects of SB-204990, a lactone prodrug of
the potent ATP citrate-lyase inhibitor SB-201076. Biochem J 1998;
334: 113-9.
Medes G, Thomas A, Weinhouse S. Metabolism of neoplastic
tissue. IV. A study of lipid synthesis in neoplastic tissue slices
in vitro. Cancer Res 1953; 13: 27-9.
Kutlu B, Cardozo AK, Darville MI, Kruhøffer M, Magnusson N,
Ørntoft T, et al. Discovery of gene networks regulating cytokineinduced dysfunction and apoptosis in insulin-producing INS-1
cells. Diabetes 2003; 52: 2701-19.
Kuhajda FP. Fatty-acid synthase and human cancer: New
perspectives on its role in tumor biology. Nutrition 2000; 16: 2028.
Gribble AD, Dolle RE, Shaw A, McNair D, Novelli R, Novelli CE,
et al. ATP-citrate lyase as a target for hypolipidemic intervention.
Design and synthesis of 2-substituted butanedioic acids as novel,
potent inhibitors of the enzyme. J Med Chem 1996; 39: 3569-84.
Melnick JZ, Srere PA, Elshourbagy NA, Moe OW, Preisig PA,
Alpern RJ. Adenosine triphosphate citrate lyase mediates
hypocitraturia in rats. J Clin Invest 1996; 98: 2381-7.
Kanao T, Fukui T, Atomi H, Imanaka T. Kinetic and biochemical
analyses on the reaction mechanism of a bacterial ATP-citrate
lyase. Eur J Biochem 2002; 269: 3409-16.
Lim K, Ryu S, Suh H, Ishihara K, Fushiki T. (-)-Hydroxycitrate
ingestion and endurance exercise performance. J Nutr Sci
Vitaminol 2005; 51: 1-7.
Chu KY, Lin Y, Hendel A, Kulpa JE, Brownsey RW, Johnson JD.
ATP-citrate lyase reduction mediates palmitate-induced apoptosis
in pancreatic beta cells. J Biol Chem 2010; 285: 32606-15.
Szutowicz A, Kwiatkowski J, Angielski S. Lipogenetic and
glycolytic enzyme activities in carcinoma and nonmalignant
diseases of the human breast. Br J Cancer 1979; 39: 681-7.
Turyn J, Schlichtholz B, Dettlaff-Pokora A, Presler M, Goyke E,
Matuszewski M, et al. Increased activity of glycerol 3-phosphate
dehydroge-nase and other lipogenic enzymes in human bladder
cancer. Horm Metab Res 2003; 35: 565-9.
Hatzivassiliou G, Zhao F, Bauer DE, Andreadis C, Shaw AN,
Dhanak D, et al. ATP citrate lyase inhibition can suppress tumor
cell growth. Cancer Cell 2005; 8: 311-21.
Yahagi N, Shimano H, Hasegawa K, Ohashi K, Matsuzaka T,
Najima Y, et al. Co-ordinate activation of lipogenic enzymes in
hepatocellular carcinoma. Eur J Cancer 2005; 41: 1316-22.
Yancy HF, Mason JA, Peters S, Thompson CE, Littleton GK, Jett
M. Metastatic progression and gene expression between breast
cancer cell lines from African American and Caucasian women. J
Carcinog 2007; 6: 8.
Beckner ME, Fellows-Mayle W, Zhang Z, Agostino NR, Kant JA,
Day BW, et al. Identification of ATP citrate lyase as a positive
regulator of glycolytic function in glioblastomas. Int J Cancer
2010; 126: 2282-95.
Varis A, Wolf M, Monni O, Vakkari ML, Kokkola A, Moskaluk C,
et al. Targets of gene amplification and overexpression at 17q in
gastric cancer. Cancer Res 2002; 62: 2625-9.
Elshourbagy NA, Near JC, Kmetz PJ, Sathe GM, Southan C,
Strickler JE, et al. Rat ATP citrate-lyase. Molecular cloning and
sequence analysis of a full-length cDNA and mRNA abundance as
a function of diet, organ, and age. J Biol Chem 1990; 265: 1430-5.
Singh M, Richards EG, Mukherjee A, Srere PA. Structure of ATP
citrate lyase from rat liver. Physicochemical studies and proteolytic
modification. J Biol Chem 1976; 251: 5242-50.
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
165
Elshourbagy NA, Near JC, Kmetz PJ, Wells TN, Groot PH, Saxty
BA, et al. Cloning and expression of a human ATP-citrate lyase
cDNA. Eur J Biochem 1992; 204: 491-9.
Joyce MA, Fraser ME, James MN, Bridger WA, Wolodko WT.
ADP-binding site of Escherichia coli succinyl-CoA synthetase
revealed by x-ray crystallography. Biochemistry 2000; 39: 17-25.
Joyce MA, Fraser ME, Brownie ER, James MN, Bridger WA,
Wolodko WT. Probing the nucleotide-binding site of Escherichia
coli succinyl-CoA synthetase. Biochemistry 1999; 38: 7273-83.
Wolodko WT, Fraser ME, James MN, Bridger WA. The crystal
structure of succinyl-CoA synthetase from Escherichia coli at 2.5A resolution. J Biol Chem 1994; 269: 10883-90.
Fraser ME, Hayakawa K, Hume MS, Ryan DG, Brownie ER.
Interactions of GTP with the ATP-grasp Domain of GTP-specific
Succinyl-CoA Synthetase. J Biol Chem 2006; 281: 11058-65.
Fraser ME, James MN, Bridger WA, Wolodko WT.
Phosphorylated and dephosphorylated structures of pig heart, GTPspecific succinyl-CoA synthetase. J Mol Biol 2000; 299: 1325-39.
Pierce MW, Palmer JL, Keutmann HT, Avruch J. ATP-citrate
lyase. Structure of a tryptic peptide containing the phosphorylation
site directed by glucagon and the cAMP-dependent protein kinase.
J Biol Chem 1981; 256: 8867-70.
Ramakrishna S, D'Angelo G, Benjamin WB. Sequence of sites on
ATP-citrate lyase and phosphatase inhibitor 2 phosphorylated by
multifunctional protein kinase (a glycogen synthase kinase 3 like
kinase). Biochemistry 1990; 29: 7617-24.
Berwick DC, Hers I, Heesom KJ, Moule SK, Tavare JM. The
identification of ATP-citrate lyase as a protein kinase B (Akt)
substrate in primary adipocytes. J Biol Chem 2002; 277: 33895900.
Nakashima RA, Paggi MG, Pedersen PL. Contributions of
glycolysis and oxidative phosphorylation to adenosine 5'triphosphate production in AS-30D hepatoma cells. Cancer Res
1984; 44: 5702-6.
Baggetto LG. Deviant energetic metabolism of glycolytic cancer
cells. Biochimie 1992; 74: 959-74.
Kaplan RS, Mayor JA, Wood DO. The mitochondrial tricarboxylate transport rotein. cDNA cloning, primary structure, and
com-parison with other mitochondrial transport proteins. J Biol
Chem 1993; 268: 13682-90.
Sun T, Hayakawa K, Bateman KS, Fraser ME. Identification of the
citrate-binding site of human ATP-citrate lyase using X-ray
crystallography. J Biol Chem 2010; 285: 27418-28.
Sun T, Hayakawa K, Fraser ME. ADP-Mg(2+) bound to the ATPgrasp domain of ATP-citrate lyase. Acta Crystallogr Sect F Struct
Biol Cryst Commun 2011; 67: 1168-72.
Cascales C, Martin-Sanz P, Pittner RA, Hopewell R, Brindley DN,
Cascales M, et al. Effects of an antitumoural rhodium complex on
thioacetamide-induced liver tumor in rats. Changes in the activities
of ornithine decarboxylase, tyrosine minotransferase and of
enzymes involved in fatty acid and glycerolipid synthesis. Biochem
Pharmacol 1986; 35: 2655-61.
MacDonald MJ, Longacre MJ, Langberg EC, Tibell A, Kendrick
MA, Fukao T, et al. Decreased levels of metabolic enzymes in
pancreatic islets of patients with type 2 diabetes. Diabetologia
2009; 52: 1087-91.
Michno A, Skibowska A, Raszeja-Specht A, Cwikowska J,
Szutowicz A. The role of adenosine triphosphate citrate lyase in the
metabolism of acetyl coenzyme a and function of blood platelets in
diabetes mellitus. Metabolism 2004; 53: 66-72.
Sjoberg ER, Varki A. Kinetic and spatial interrelationships between
ganglioside glyco-syltransferases and O-acetyltransferase(s) in
human melanoma cells. J Biol Chem 1993; 268: 10185-96.
Ramakrishna S, Benjamin WB. Evidence for an essential arginine
residue at the active site of ATP citrate lyase from rat liver.
Biochem J 1981; 195: 735-43.
Houston B, Nimmo HG. Purification and some kinetic properties of
rat liver ATP citrate lyase. Bio chem J 1984; 224: 437-43.
Pfitzner A, Kubicek CP, Rohr M. Presence and regulation of ATP:
Citrate lyase from the citric acid producing fungus Aspergillus
niger. Arch Microbiol 1987; 147: 88-91.
Potapova IA, El-Maghrabi MR, Doronin SV, Benjamin WB.
Phosphorylation of recombinant human ATP: Citrate lyase by
cAMP-dependent protein kinase abolishes homotropic allosteric
166 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
regulation of the enzyme by citrate and increases the enzyme
activity. Allosteric activation of ATP: Citrate lyase by
phosphorylated sugars. Biochemistry 2000; 39: 1169-79.
Shashi K, Bachhawat AK, Joseph R. ATP: Citrate lyase of
Rhodotorula gracilis: Purification and properties. Biochim Biophys
Acta 1990; 1033: 23-30.
Rangasamy D, Ratledge C. Compartmentation of ATP: Citrate
lyase in plants. Plant Physiol 2000; 122: 1225-30.
Sato R, Okamoto A, Inoue J, Miyamoto W, Sakai Y, Emoto N,
et al. Transcriptional regulation of the ATP citrate-lyase
genebysterol regulatory element-binding proteins. J Biol Chem
2000; 275: 12497-502.
Kim KS, Park SW, Kim YS. Regulation of ATP-citrate lyase at
transcriptional and post-transcriptional levels in rat liver. Biochem
Biophys Res Commun 1992; 189: 264-71.
Park SW, Kim KS, Whang SK, Kim JS, Kim YS. Induction of
hepatic ATP-citrate lyase by insulin in diabetic rat--effects of
insulin on the contents of enzyme and its mRNA in cytosol, and the
transcriptional activity in nuclei. Yonsei Med J 1994; 35: 25-33.
Kim KS, Kang JG, Moon YA, Park SW, Kim YS. Regulation of
ATP-citrate lyase gene transcription. Yonsei Med J 1996; 37: 21424.
Fukuda H, Noguchi T, Iritani N. ranscriptional regulation of fatty
acid synthase gene and ATP citrate-lyase gene by Sp1 and Sp3 in
rat hepatocytes. FEBS Lett 1999; 464: 113-7
Fukuda H, Iritani N, Katsurada A, Noguchi T. Insulin- and
polyunsaturated fatty acid-responsive region(s) of rat ATP citrate
lyase gene promoter. FEBS Lett 1996; 12: 204-7.
Fukuda H, Iritani N, Noguchi T. Transcriptional regulatory region
for expression of the rat ATP citrate-lyase gene. Eur J Biochem
1997; 247: 497-502.
Towle HC, Kaytor EN, Shih HM. Regulation of the expression of
lipogenic enzyme genes by carbohydrate. Annu Rev Nutr 1997; 17:
405-33.
Yoshiro, K., Tomohisa, O., Akira, M., Akira, O. Pharmaceutical
composition for suppression of the expression of ATP citrate lyase
and use thereof. US20090176835 (2009).
Katz NR, Giffhorn S. Glucose- and insulin-dependent induction of
ATP citrate lyase in primary cultures of rat hepatocytes. Biochem J
1983; 212: 65-71.
Swinnen JV, Brusselmans K, Verhoeven G. Increased lipogenesis
in cancer cells: New players, novel targets. Curr Opin Clin Nutr
Metab Care 2006; 9: 358-65.
Tuhácková Z, Krivánek J. GTP, a nonsubstrate of ATP citrate
lyase, is a phosphodonor for the enzyme histidine autophosphorylation. Biochem Biophys Res Commun 1996; 218: 61-6.
Wagner PD, Vu ND. Phosphorylation of ATP-citrate lyase by
nucleoside diphosphate kinase. J Biol Chem 1995; 270: 21758-64.
Alexander MC, Kowaloff EM, Witters LA, Dennihy DT, Avruch J.
Purification of a hepatic 123000- dalton hormone-stimulated 32Ppeptide and its identification as ATP-citrate lyase. J Biol Chem
1979; 254: 8052-6.
Yu KT, Benjamin WB, Ramakrishna S, Khalaf N, Czech MP. An
insulin-sensitive cytosolic protein kinase accounts for the
regulation of ATP citrate-lyase phosphorylation. Biochem J 1990;
268: 539-45.
Bauer DE, Hatzivassiliou G, Zhao F, Andreadis C, Thompson CB.
ATP citrate lyase is an important component of cell growth and
transformation. Oncogene 2005; 24: 6314-22.
Migita T, Narita T, Nomura K, Miyagi E, Inazuka F, Matsuura M,
et al. ATP citrate lyase: Activation and therapeutic implications in
non-small cell lung cancer. Cancer Res 2008; 68: 8547-54.
Mashima T, Seimiya H, Tsuruo T. De novo fatty-acid synthesis and
related pathways as molecular targets for cancer therapy. Br J
Cancer 2009; 100: 1369-72.
Garber K. Energy deregulation: Licensing tumors to grow. Science
2006; 312: 1158-9.
Schwartz L, Abolhassani M, Guais A, Sanders E, Steyaert JM,
Campion F, et al. A combination of alpha lipoic acid and calcium
hydroxycitrate is efficient against mouse cancer models:
Preliminary results. Oncol Rep 2010; 23: 1407-16.
Knowles LM, Yang C, Osterman A, Smith JW. Inhibition of fattyacid synthase induces caspase-8-mediated tumor cell apoptosis by
up-regulating DDIT4. J Biol Chem 2008; 283: 31378-84.
Zu et al.
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui TV, Cross JR,
Thompson CB. ATP-citrate lyase links cellular metabolism to
histone acetylation. Science 2009; 324: 1076-80.
Thompson, C.B., Bauer, D., Hatzivassiliou, G. Compositions and
methods for treating cancer. US20070259956 (2007).
Scott, A.M., Ashok, K.B., Bhagavathula, R. Hydroxycitric acid
concentrate and food products prepared therefrom. US5536516
(1996).
Jena BS, Jayaprakasha GK, Singh RP, Sakariah KK. Chemistry and
biochemistry of (-)-hydroxycitric acid from Garcinia. J Agric Food
Chem 2002; 50: 10-22.
van Loon LJ, van Rooijen JJ, Niesen B, Verhagen H, Saris WH,
Wagenmakers AJ. Effects of acute (-)hydroxycitrate supplementation on substrate metabolism at rest and during exercise in
humans. Am J Clin Nutr 2000; 72: 1445-50.
William, G.R., Wightman, K.R. Hydroxycitric acid derivatives.
US4006166 (1977).
Yamada T, Hida H, Yamada Y. Chemistry, physiological
properties, and microbial production of hydroxycitric acid. Appl
Microbiol Biotechnol 2007; 75: 977-82.
Watson JA, Fang M, Lowenstein JM. Tricarballylate and
hydroxycitrate: Substrate and inhibitor of ATP: Citrateoxaloacetate lyase. Arch Biochem Biophys 1969; 135: 209-17.
Cheema-Dhadli S, Halperin ML, Leznoff CC. Inhibition of
enzymes which interact with citrate by (-)-hydroxycitrate and 1,2,3tricarboxybenzene. Eur J Biochem 1973; 38: 98-102.
Sullivan AC, Triscari J, Hamilton JG. Hypolipidemic activity of
(-)-hydroxy-citrate. Lipids 1977; 12: 1-9.
Sullivan AC, Singh M, Srere PA, Glusker JP. Reactivity and
inhibitor potential of hydroxycitrate isomers with citrate synthase,
citrate lyase, and ATP citrate lyase. J BiolChem 1977; 252: 758390.
Zambell KL, Fitch MD, Fleming SE. Acetate and butyrate are the
major substrates for de novo lipogenesis in rat colonic epithelial
cells. J Nutr Sci Vitaminol 2003; 133: 3509-15.
Berkhout TA, Havekes LM, Pearce NJ, Groot PH. The effect of (-)hydroxycitrate on the activity of the low-density-lipoprotein
receptor and 3-hydroxy-3-methylglutaryl-CoA reductase levels in
the human hepatoma cell line HepG2. Biochem J 1990;272: 181-6.
Guais A, Baronzio G, Sanders E, Campion F, Mainini C, Fiorentini
G, et al. Adding a combination of hydroxycitrate and lipoic acid
(METABLOC™) to chemotherapy improves effectiveness against
tumor development: Experimental results and case report. Invest
New Drugs 2012; 30: 200-11.
Laurent, C., Adeline, G.V. Pharmaceutical association containing
lipoic acid and hydroxycitric acid as active ingredients.
US20110236506 (2011).
Brunengraber H, Sabine JR, Boutry M, Lowenstein JM. 3-Hydroxysterol synthesis by the liver. Arch Biochem Biophys 1972;
150: 392-6.
Sullivan AC, Triscari J, Spiegel JE. Metabolic regulation as a
control for lipid disorders. II. Influence of (--)-hydroxycitrate on
genetically and experimentally induced hypertriglyceridemia in the
rat. Am J Clin Nutr 1977; 30: 777-84.
Sugden MC, Steare SE, Watts DI, Palmer TN. Interactions of
between insulin and thyroid hormone in the control of lipogenesis.
Biochem J 1983; 210: 937-44.
William, L.A., Joseph, R.M. Process for the preparation of
methionine sulfoximine. US3607928 (1971).
Dolle RE, McNair D, Hughes MJ, Kruse LI, Eggelston D, Saxty
BA, et al. ATP-citrate lyase as a target for hypolipidemic
intervention. Sulfoximine and 3-hydroxy-beta-lactam containing
analogues of citric acid as potential tight-binding inhibitors. J Med
Chem 1992; 35: 4875-84.
Dolle RE, Gribble A, Wilkes T, Kruse LI, Eggleston D, Saxty BA,
et al. Synthesis of novel thiol-containing citric acid analogues.
Kinetic evaluation of these and other potential active-site-directed
and mechanism-based inhibitors of ATP citrate lyase. J Med Chem
1995; 38: 537-43.
Robert, W.G., Richard, W.K., Francis, A.M., Ann, C.S.
Chlorocitric acids. US4312885 (1982).
Clark, W.P., Sidney, T. Optical resolution of organic carboxylic
acids. US3901915 (1975).
ACL as Novel Cancer Therapeutic Target
[88]
[89]
[90]
[91]
[92]
[93]
[94]
[95]
[96]
[97]
[98]
Marletta MA, Srere PA, Walsh C. Stereochemical outcome of
processing of fluorinated substrates by ATP citrate lyase and
malate synthase. Biochemistry 1981; 20: 3719-23.
Dolle RE, Novelli R, Saxty BA. Preparation of (+)-erythro)and (+)(threo)-2-vinyl citric acids as potential mechanism-based inhibitors
of ATP-citrate lyase. Tetrahedron Lett 1991; 32: 4587-90.
Saxty BA, Novelli R, Dolle RE, Kruse LI, Reid DG, Camilleri P,
et al. Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as
inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.
Eur J Biochem 1991; 202: 889-96.
Yukio, S., Kimio, I., Yoshio, T., Yoko, S., Tomowo, K., Takeshi,
K. Radicicol derivatives, their preparation and their anti-tumour
activity. EP0460950 (1991).
Ki SW, Ishigami K, Kitahara T, Kasahara K, Yoshida M,
Horinouchi S. Radicicol binds and inhibits mammalian ATP citrate
lyase. J Biol Chem 2000; 275: 39231-6.
Schulte TW, Akinaga S, Soga S, Sullivan W, Stensgard B, Toft D,
et al. Antibiotic radicicol binds to the N-terminal domain of Hsp90
and shares important biologic activities with geldanamycin. Cell
Stress Chaperones 1998; 3: 100-8.
Sharma SV, Agatsuma T, Nakano H. Targeting of the protein
chaperone, HSP90, by the transformation suppressing agent,
radicicol. Oncogene 1998; 16: 2639-45.
Guay C, Madiraju SR, Aumais A, Joly E, Prentki M. A role for
ATP-citrate lyase, malic enzyme, and pyruvate/citrate cycling in
glucose-induced insulin secretion. J Biol Chem 2007; 282: 3565765.
Claude, V., Rene, S., Surendra, N.S. Process for the preparation of
Antimycin A. US3869346 (1975).
Barrow CJ, Oleynek JJ, Marinelli V, Sun HH, Kaplita P, Sedlock
DM. Antimycins, inhibitors of ATP-citrate lyase, from a
Streptomyces sp. J Antibiot 1997; 50: 729-33.
Gribble AD, Ife RJ, Shaw A, McNair D, Novelli CE, Bakewell S,
et al. ATP-Citrate lyase as a target for hypolipidemic intervention.
2. Synthesis and evaluation of (3R,5S)-omega-substituted-3carboxy-3, 5-dihydroxyalkanoic acids and their gamma-lactone
prodrugs as inhibitors of the enzyme in vitro and in vivo. J Med
Chem 1998; 41: 3582-95.
Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 2
[99]
[100]
[101]
[102]
[103]
[104]
[105]
[106]
[107]
[108]
[109]
[110]
[111]
167
Jacob, B.-t. Long-chain ,-dicarboxylic acids and derivatives
thereof and pharmaceutical compositions containing them.
US4689344 (1987).
Atkinson LL, Kelly SE, Russell JC, Bar-Tana J, Lopaschuk GD.
MEDICA 16 inhibits hepatic acetyl-CoA carboxylase and reduces
plasma triacylglycerol levels in insulin-resistant JCR: LA-cp rats.
Diabetes 2002; 51: 1548-55.
Rose-Kahn G, Bar-Tana J. Inhibition of lipid synthesis by beta
beta'-tetramethyl-substituted, C14-C22, alpha, omega-dicarboxylic
acids in cultured rat hepatocytes. J Biol Chem 1985; 260: 8411-5.
al-Shurbaji A, Skorve J, Berge RK, Rudling M, Björkhem I,
Berglund L, et al. Effect of 3-thiadicarboxylic acid on lipid
metabolism in experimental nephrosis. Arterioscler Thromb 1993;
13: 1580-6.
Li JJ, Wang H, Tino JA, Robl JA, Herpin TF, Lawrence RM, et al.
2-Hydroxy-N-arylbenzenesulfonamides as ATP-citrate lyase
inhibitors. Bioorg Med Chem Lett 2007; 17: 3208-11.
Patrick, C., Pierre, R., Dominique, O. Arylbenzenesulfonamide
containing pharmaceutical compositions. US4761430 (1988).
Chan GW, Francis T, Thureen DR, Offen PH, Pierce NJ, Westley
JW, et al. Purpurone, an inhibitor of ATP-citrate lyase: A novel
alkaloid from the marine sponge Iotrochota sp. J Org Chem 1993;
58: 2544-6.
Li QJ, Fan AL, Lu ZY, Cu YX , Lin WH, Jia YX. One-Pot
AgOAc-mediated synthesis of polysubstituted pyrroles from
primaryamines and aldehydes: Application to the total synthesis of
purpurone. Org Lett 2010; 12: 4066-9.
Szutowicz A, Stepie M, Angielski S. The inhibition of rat brain
ATP: Citrate oxaloacetate-lyase by L-glutamate. J Neurochem
1974; 22: 85-91.
Eriyamremu GE, Adamson I. Early changes in energy metabolism
in rats exposed to an acute level of deoxycholate and fed a
Nigerian-like diet. Ann Nutr Metab 1994; 38: 174-83.
Kling D, Gamble W. In vivo inhibition of citrate cleavage enzyme
by polychlorinated biphenyls. Experientia 1981; 37: 1258-9.
Nechay BR, Nanninga LB, Nechay PSE. Role of vanadium in
biology. Symposium summary. Fed Proc 1986; 45: 123-32.
Krivanek J, Novakova L. ATP-citrate lyase is another enzyme the
histidine phosphorylation of which is inhibited by vanadate. FEBS
Lett 1991; 282: 32-4.