Structural composition of NaCT
The structure of either human or the mouse NaCTs has not been
crystallized. The current knowledge for the structural basis of the
NaCTs is based upon Vibrio cholerae bacterial dicarboxylate
transporter VcINDY. The structure VcINDY greatly differs from the human
NaCT but still acts as the best option for the modeling studies
[11]. It has a total of 11 transmembrane (TM) helices hairpin loops
marked as HPin (hairpin in) and HPout(hairpin out). The TM 4, 5, 9 and 10 are each broken into two parts as a
and b. The HPin towards the cytosolic side is connected
to helix H4c to one side and TM5 on the other side. Similarly,
HPout coming out from the periplasm is connected to the
TM10 and H9c. The hairpin along with the broken helix (intramembrane
loop) plays a chief role in membrane transport. The NaCT (VcINDY) has
two repeated folds with 26% similarity in amino acid sequence from the
N-and C-terminal side i.e., TM2-TM6 from the N terminal side is related
with TM7-TM11 of C terminal side. The binding pocket for the citrate is
formed from the residues HPin, TM5,
HPout and TM10. The citrate molecule binds between Na1
(N terminal side hairpin tip-capping loop motif formed by
HPin tip, TM5a and TM5b) and Na2 sites (C terminal side
hairpin tip-capping loop motif formed by HPout tip,
TM10a and TM10b)[12] Figure 2 . The zebrafish
Na+ coupled citrate transporter was cloned showing
61% identical and 77% similarity to the human NaCT. The cloned human
NaCT showed 77% identity with rat NaCT and the gene is located on human
chromosome 17 at p12-13 [13]. Khamaysi et al., studied the
slc13 transporter H4c as a dynamic anchor domain which controls the
metabolite transport (succinate and citrate). It was observed that
intracellular determinants interact with the H4c domain and control the
transport. The interaction to both the intracellular determinants and
the membrane phospholipids is attained by a sole basic residuei.e ., Arg108. The conclusions were drawn by carrying out several
experimental studies in Xenopus oocytes and fluorescent
microscopy of mammalian cells. The Arg108 present in highly conserved
H4c was found to be critical for the metabolite transport.
Neutralization of this basic residues inhibited the transport functions
[14].
Figure 2: A NaCT representation of a VcINDY in which transport
domain is represented by the binding sites hairpin loops and unwounded
helices 5 and 10. The N terminal end is related to the C terminal end
through an inverted repeat symmetry (shown in red and blue color box)
except the TM1[12,15].
The receptors of the VcINDY can be expressed in two forms - the outward
facing state and the inward-facing state for the transport mechanism to
occur. The substrate binds to the outward facing state formed by the
transport domain (shown in orange) which slides 15Å downward
(translocate) with 43o rotation to form the inward
facing states (as such elevator is sliding downwards). The elevator is
formed by the transport domain and the hydrophobic barrier provided by
transmembrane 4 and 9 (oligomerization domain) and scaffold domain. This
leads to the release of the substrate to the cytoplasm. After releasing
the substrate, the transporter again returns to it outward facing state
to start the next cycle [15] (Figure 3) . It was also
observed that crosslinking VcINDY to either inward or outward facing
state inhibit the transport activity. The test was done by
reconstituting the double cysteine mutants in proteoliposomes and access
the transport abilities of the transporter. The crosslink was induced
with excess HgCl2 in both cases of outward and inward
stabilizing mutants. The transport activity was restored when the
crosslinking was reduced. The transport activity was strong in
HgCl2 treated cysteine less proteins [15].
Figure 3: Substrate transport by elevator type mechanism. The
substrate binds to the outward facing of the transport domain,
translocation inhibited by the hydrophobic barrier (scaffold and
oligomerization domain). The transport domain translocated to 15Å
downward rotating 43o to the inward facing state
releasing the substrate to the cytoplasm [16].
Although the receptors functional properties are extensively discussed
but the different analogues bindings within these receptors for the
treatment of various metabolic disorders has not been covered in detail
in previously published reviews. This article encompasses different key
intermediates analogues responsible for the blocking of these SLC family
receptors for promising results.
- Distribution and functionThe sodium-coupled with anionic transporters includes three
transporters of dicarboxylates and tricarboxylates (NaDC1, NaDC3 and
NaCT). The metabolic intermediates of TCA cycle such as citrate,
α-ketoglutarate and succinate are the substrate for these transporters
which maintains the levels of these intermediates in plasma, urine,
and tissue levels [17].
- SLC13A5 (NaCT): These receptors are widely distributed.
NaCT found in the liver, brain, and salivary glands primarily
transport citrate into these tissues. It is major carrier of citrate
in the liver [18]. The NaCT (SLC13A5) is mammalian homolog of
the Drosophila INDY. The partial loss in the citrate transport
causes increase in lifespan in Drosophila [11]. The rat NaCT
(Na+ coupled citrate transporter) has 77%
similarity with the cloned human NaCT. It mediates the
Na+ coupled citrate transport and is the first
transporter recognized in the mammalian cells that showed higher
affinity for citrates [19]. It is mostly expressed in liver but
also found in brain and testis. The transport is electrogenic
whereas its activation showed sigmoidal curve (more involvement of
the Na+ ions in activation). The citrates are
found in high concentration in the human blood and are involved in
the metabolic energy and synthesis of fatty acids and cholesterol
[20]. NaCT is more selective to citrate than other
dicarboxylates or tricarboxylates [11]. The primary neurons
culture of mouse cerebral cortex showed the NaCT transport system,
but it was found to be absent in the astrocytes. It is
Na+-dependent, Li+-sensitive
inhibited by the unlabeled tricarboxylate citrate and other
dicarboxylates e.g., malate, fumarate, succinate, and
α-ketoglutarate [21]. The mutation in SLC13A5 gene codes for
Na+/citrate co-transporter results in loss of
function and causes neurological disorders that includes neonatal
epilepsy, delayed brain development and tooth dysplasia in children.
Targeting the NaCT deletion in mice has beneficial effect against
diet induced obesity with no brain dysfunction. Mutation different
from the brain located in SLC13A5 may have beneficial effect. Thus,
there is greater emphasis in designing inhibitors which specifically
cause the NaCT loss in the liver rather than NaCT loss in the brain
[11]. The SLC13A5 mutation in nine epilepsy patients were
identified. The drug acetazolamide (carbonic anhydrase inhibitors)
and the atypical anti-seizures drugs decreases the seizures in four
patients. Also, it was noticed that the ketogenic diet and fasting
worsens the symptoms. The mutations of NaCT transport function and
protein expression were further studied by transient transfection
against COS-7 cells. It was observed that there was no transport
activity from the mutant transporter proteins affecting helix
packing or substrate bindings. Other treatment such as chaperones
and low temperature were not able to improve the transport functions
of mutated NaCT. A decreased protein expression and activity in
wild-type transporter have been observed when both the NaCT and
mutant NaCT are co-expressed [22]. Selch et al., studied
the mutation associated with the SLC13A5 gene NaCT mediate transport
functions. Taking HEK293 as wild type and eight mutation NaCT
proteins, it was observed that proteins were synthesized identical
to the wild types but for the mutants pG219R, pG219E, pT227M, pL420P
and pL488P the citrate transport was completely inhibited. These
conserved amino acids are present in the transmembrane pore and are
important for the NaCT-mediated transport [23]. The VcINDY
homolog of SLC13A5 represent the receptors for information on
substrate binding sites and mechanism. It has large open cavity
towards the cytosolic sides for sodium driven succinate transport
and is inhibited by the dicarboxylates such as malate and fumarate.
In this it requires four sodium to couple with the substrate (4:1).
The NaCT has many functions to perform such as energy production
(mitochondria Kreb’s cycle), glycolysis, gluconeogenesis, fatty
acid, and cholesterol synthesis in the cytosol. The SLC13A5 also
regulates fibroblast growth factor receptors (FGF23) function
leading to the overproduction of the FGF23 and abnormal
mineralization of the extracellular matrix leading to
hypophosphatemic rickets disease. The NaCT activity is stimulated by
the presence of Li+ ions [10].
- Citrate transporter of Lactic acid bacteriaThe human sodium-dependent citrate transporters (NaCT) and
succinate/dicarboxylate from Lactobacillus acidophilus has
great similarity in the structure as VcINDY. All these results
support for the VcINDY protein structure representing the overall
protein structure in ion transporter superfamily[8]. The
citrate transporter and the malate transporter of lactic acid
bacteria Leuconostoc mesenteroides and Lactococcus
lactis were analyzed and both showed transport of substrate
containing 2-hydroxycarboxylates. The structural changes either of
the OH and COO- drastically reduced the affinity
to the transporters. The dicarboxylate with the formula
OH-CR-(COO-)2, theS -enantiomer of which binds efficiently and translocated
whereas the R -enantiomer has no affinity. The binding order
magnitude of citrate or S -enantiomer is one order higher
than other uncharged R groups containing compounds including
lactate; this shows the importance of second carboxylate in
greater affinity with the proteins. The binding preference for theR -enantiomer is more in Leuconostoc mesenteroideswhen R groups are hydrophobic whereas onlyS -enantiomer is translocated in Lactococcus lactis[24]. The 46 residues at the C-terminal region containing
C-terminal putative transmembrane XI was analyzed for the
substrate recognition through chimeric transporters. The
replacement of the C-terminal region in Leuconostoc
mesenteroides with Lactococcus lactis and vice
versa did not alter the transport characteristics of the di and
tri carboxylate i.e ., malate and citrate whereas
monocarboxylate substrate mandelate and 2-hydroxyvalerate
interactions with the proteins were changed. The incorporation of
C-terminal residues of one bacterium into another and vice
versa led to higher affinity. The interchanging of the C-termini
has more complex effect on R enantiomers. It was indicated
that binding pocket is located in other transmembrane and
transmembrane XI [25]. The substrate transported by citrate
transporter of Leuconostoc mesenteroides and malate
transporter of Lactococcus lactis are shown inFigure 4 .Figure 4: The substrate transported with citrate
transporter of Leuconostoc mesenteroides and malate
transporter of Lactococcus lactis
- Citrate Transporter of Klebsiella pneumoniae
The loops of the transmembrane segments VIII and IX (AH loop) represent
the amphipathic surface helix of Klebsiella pneumoniae . The
mutation of the cysteine residues especially G324C, F331C, and F332C has
very low citrate transport activity whereas other mutants I321C and
S333C showed decreased activity with membrane permeable thiol reagentN -ethylmaleimide (NEM) but not with membrane impermeable
4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid and
[2-(trimethylammonium)ethyl]methanethiosulfonate. The impermeability
of the reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid
supports for the localization of the AH loop in the cytoplasmic region
[26].
Inhibitors of NaCT
Higuchi et al., showed that deletion of the genes that encode for
SLC13A5 in mice protects against obesity and other metabolic disorders
such as diabetes. Suppressing the transporter also suppresses the
hepatocellular carcinoma. The author reported one irreversible and
non-competitive inhibitor of human NaCT, and which does not affect the
mouse NaCT. The compound 1 inhibits the constitutively
transporter in HepG2 cells and ectopically expressed in HEK-293 cells.
The inhibition is in nanomolar range of ⁓100 nM (Figure 5 ). The
molecular modelling studies generated human NaCT and mouse NaCT taking
humanized variant of VcINDY as the template. The compound and the
citrate showed different inhibitors to human NaCT versus mouse
NaCT [27].
Figure 5: The compound inhibits HepG2 cell transporter and
ectopically expressed HEK-293 cells.
It was further evidenced that HepG2 cells represent the excellent cell
models to study the regulatory mechanisms of endogenous di and
tricarboxylates. Also, it was found that there is higher rate of
succinate transport as compared to citrate. The human retinal pigments
epithelial cells transfected with hNaDC-1, hNaDC-3 and hNaCT were
compared with citrate and succinate in HepG2 cells and the results
showed that HepG2 cells transport were constant with hNaDC-3 and hNaCT
expressed in human retinal pigments epithelial cells. The high affinity
transport for succinate is consistent with hNaDC-3 [2]. The blockade
of hepatic extracellular citrate uptake through the blockade of NaCT may
be regarded as potential treatment of metabolic disorders. This led
Huard et al., in 2015 to identify and characterize some novel
small dicarboxylate molecules especially the active
(R )-enantiomer 2 to be highly selective for NaCT
compared to NaDC1 and NaDC3. The (S )-enantiomer was found to be
inactive. The mechanism for the compound 2 was found to be
competitive to the citrate, specific, stereo sensitive and direct to the
NaCT receptors. The compound acts a substrate for active cellular uptake
and inhibits both in vitro and in vivo by directly binding
the compound to NaCT receptors. The IC50 of the compound2 against HEKNaCT was found to be 0.41 µM as
compared to the racemate having IC50 value of 0.80
µM[18] Figure 6 .
Figure 6: Compound 2 with their IC50value against HEKNaCT
The inhibition of sodium citrate transporter NaCT (SLC13A5) is a major
target for metabolic disease. As discussed above citrate acts as
signaling molecules that regulates fatty acid and glucose metabolizing
enzymes. Two compounds 3 and 4 inhibits the human NaCT
with very high affinity. Compound 3 is very specific for hNaCT
whereas, compound 4 inhibits all the three transporters (NaCT,
NaDC1 and NaDC3) but NaCT with greater affinity. Compound 3inhibited the citrate uptake (hNaCT) with no inhibition of citrate C14
transport activity in NaDC1 and NaDC3 whereas 4 inhibited all
the three transporters at 100 µM. The citrate uptake blockade by
compound 3 in the liver inhibits the conversion of
extracellular citrate to tricarboxylic acid cycle (TCA) intermediates.
This results in reduction of high plasma glucose concentration in mice
fed with high fat (Figure 7 ). Pajor et al ., 2016
explored the molecular modelling inside the citrate receptors and
analyzed the amino acids involved in the binding sites. The amino acid
residue (G228, V231, V232, and G409) near the citrate binding sites
affect both the transport as well as inhibition of citrate uptake. The
amino acid V231 distinguish between the compound 3 and4 as inhibitors. Some of the residue outside the binding sites
such as Q77 and T86 are important in NaCT inhibition[28].
Figure 7: Sodium citrate transporter NaCT (SLC13A5) inhibitors
One of the studies carried out by Huard et al., optimized the
earlier series of synthesized compound to more potent compound5 . The compound showed potent inhibition of the
[14C] citrate uptake in the liver and kidney. The
compound showed more suitable in vivo pharmacokinetic profile for
better in vivo pharmacodynamics. The compound also reduced the
plasma glucose concentrations[18] (Figure 8 ).
Figure 8: Optimization of the compound 1 to more
active isomer 5 against NaCT receptors
It was demonstrated that metformin inhibits the NaCT plasma membrane
citrate transporter in HepG2 cells with decrease in the levels of the
citrate and decreases mRNA levels. It inhibits gluconeogenesis and thus
has anti-diabetic effects. An 5’ adenosine monophosphate-activated
protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide
ribonucleotide (AICAR) has similar effect. Activating AMPK inhibits mTOR
which further inhibits rapamycin and thus decreases NaCT expression. The
decrease in the cellular levels of the citrate stimulate glycolysis and
inhibits gluconeogenesis (citrate suppresses glycolysis by inhibiting
phosphofructokinase-1 and activate gluconeogenesis by activating
fructose 1,6-biphosphate) [29] (Figure 9 ).
Figure 9: Flow diagram showing new mechanism of action of the
metformin acting on SLC13A5 citrate receptor
The cytosolic citrate activates fatty acid synthesis and downregulates
the glycolysis and β-oxidation of fatty acids. The cytosolic
concentration of the citrate controls rate of synthesis of fatty acids
in the liver and adipose tissue which in turn is controlled by the
transport through NaCT transporter. It was evidenced that mutation in
homologous fly gene results in reduced fat storage. The study in the
mice (NaCT knockout) further confirms reduced lipogenesis, higher lipid
oxidation and energy expenditure. This led to protection of mice from
obesity and insulin resistance [12]. The divalent anion sodium
symporter (DASS) includes Staphylococcus aureusNa+/dicarboxylate symporter SdcS, mammalian SLC13
Na+/dicarboxylate cotransporters NaDC1 and NaCT. The
anthranilic acid derivatives such as N -(p-amylcinnamoyl)
anthranilic acid 6 is a slow inhibitor of the mammalian
members. The SdcS was also inhibited by the N -(p-amylcinnamoyl)
anthranilic acid and nonsteroidal anti-inflammatory drugs (NSAIDs) such
as flufenamate and niflumate as allosteric inhibitors. The
IC50 was calculated as 55 µM forN -(p -amylcinnamoyl) anthranilic acid (Figure 10 ).
With succinate the graph was sigmoidal with K0.5 of 9 µM
and Hill coefficient 1.5. When given withN -(p -amylcinnamoyl) anthranilic acid K0.5remains to be 1.5 but there was decrease in the V max and
increased in Hill coefficient. Also, the mutants (N108C) reactivity was
not affected by the N -(p -amylcinnamoyl) anthranilic acid
inhibition [30].
Figure 10 : The N -(p-amylcinnamoyl) anthranilic acid
inhibitors of both mammalian and Staphylococcus aureusNa+/dicarboxylate symporter
The classical inhibitor benzene tricarboxylate for the mitochondrial
citrate transporter protein led Aluvila et al., to identify
competitive inhibitors against the mitochondrial citrate transporter
protein through in silico screening of the ZINC database. A total
of 10 compounds were identified which showed more than 50% inhibitions
at the concentration of the 1 mM. One of the compounds showed the
highest percentage inhibition of 85% as compared to the benzene
tricarboxylate (% inhibition = 79%). Figure 11 shows
the compound 7 identified by the in-silico database
screening taking benzene tricarboxylate as the reference. One of the
compound 8 acts as potent inhibitors against both the bacterial
citrate transporter protein as well as plasma membrane citrate
transporter [31].
Figure 11: Competitive mitochondrial citrate inhibitors byin silico screening of the ZINC database.
Na+/Dicarboxylate co-transporter, SLC13A2
(NaDC1) : The hNaDC1 found in renal proximal tubule and small
intestine and has low affinity for both the di and tri-carboxylate
(oxaloacetate, fumarate, and malate). The main substrate is succinate
(Km value of 0.9 mM), α-ketoglutarate and citrate
(main physiological substrate). In renal proximal tubule and small
intestine, it reabsorbs the intermediates of the TCA from urine and
diet, maintains the urinary levels of citrate and plays a crucial role
in the development of kidney stone [10,17]. Citrate can chelate
the calcium (Ca2+) and avoid its precipitation.
Thus, low urinary citrate promotes the development of the kidney
stones [3]. It involves bindings of three sodium ions and then the
dicarboxylate ions results in passage of one positive ions. The ion
lithium competitively inhibits the transport after binding at one of
the sodium bindings sites, but the sensitivity varies with
species-to-species [10]. Human NaCT is activated by the presence
of Li+ whereas rodents NaCT showed
inhibition[13]. The stimulation of NaDC1 by metabolic acidosis
through physical exercise, clinical treatments led to decrease in the
citrate concentration [10]. The transporter of SLC-13 family
transport di or tricarboxylate (succinate or citrate) along with two
to four Na+ ions. Colas et al., in the year
2017 modeled outward facing conformation of
Na+/dicarboxylate co-transporters of both human (h)
and rabbit (rb) using outward facing model of bacterial homolog VcINDY
template. It was observed that mutagenesis in rbNaDC1 of T474 with
cysteine results in inactive protein. The cysteine substitution in
M539C results in low affinity to both sodium and lithium ions,
indicates for the hNaDC3 sites. The mutants of Y432C and T86C led to
increased Km value for succinate suggesting for these
amino acids in outward-facing substrate binding sites[32].
- Inhibitors of NaDC1Pajor et al., 2010., showed that the transmembrane helix (TM11)
is important for sodium and lithium bindings and citrate affinity. The
amino acid sequence of the TM11 is between 543 and 563, where glycine
543 resides inside of the membrane and valine 563 to the outside. The
amino acid was mutated to see whether it was affecting the transport
activity and was observed that in many of them it decreases the
transport activity. Some of the mutants were inactive such as G550C,
W561C and L568C. The receptor was inhibited by low concentration of
lithium in the presence of sodium. Thus, the work showed that Ile554
is somewhat located in the middle of the helix and is determinants of
the bindings of the lithium[33].
- SLC13A3 (NaDC3): The hNaDC3 has higher affinity for both
the di-carboxylate and tri-carboxylate and binds to succinates
(Km value of 20-102 µM), α-ketoglutarate (important
physiologically substrate for NaDC3), and citrate and inhibited by
other TCA intermediates such as fumarate, oxaloacetate, and malate.
Others physiological metabolites substrate for NaDC3 of Kreb’s cycle
includes glutarate and N -acetyl-L-aspartate
[10]. It is primarily found in brain, placenta, kidney, liver,
and pancreas [2]. Citrate transport by NaDC3 is also stimulated
by acidic pH. Some of the oral chelators such asmeso -2,3-dimercaptosuccinate (succimer) and
dimercaptopropane-1-sulfonate (Dimaval) interact with NaDC3, others
are antibacterial benzylpenicillin, NSAID flufenazine and
immunosuppressive drug mycophenolic acid [10]. The sodium
dicarboxylate cotransporters NaDC1 located in the brush bordered and
NaDC3 in basolateral cell membranes transport three sodium ions and
one dicarboxylates in case of succinates and glutarate generating
inward currents whereas, oxalate and malonate cannot. Thetrans dicarboxylate maleate generated current like the
succinate as compared to the cis substrate. Both glutarate
and α-ketoglutarate showed larger current as compared to succinates
whereas folate and glutamate failed to do so. At 60 mV, the kinetic
studies showed the value of K0.5 to be 45 ± 13µM for
α-ketoglutarate and 25 ± 12µM for succinate. At concentration of up
to 5 mM lithium concentration do not inhibit rat and flounder
orthologs hNaDC3 instead it mediates succinate dependent current in
the absence of sodium [34].
- Inhibitors of NaDC3
Colas et al ., 2015 uses homology model for human hNaDC1, mouse
mNaDC1 and mouse mNaDC3 and human hNaCT and predicted small molecules
inhibitors by the virtual screening. The compound 9 was
selected for generating the most potent hit. The compound 9inhibited [14C] transport activity in all the
transporters except for NaCT. All the five compounds (10-14 )
inhibited one or other SLC13 members, compound 11 being the
most potent inhibitors in mNaDC1 (IC50 = 72 µM in one
experiment and 82 µM in second experiment) and the compound 15most selective against hNaDC3 inhibition (cis -inhibition assay).
The IC50 value of 11 against mNaDC1 was 85 µM
(one experiment) and 100 µM in second experiment whereas it poorly
inhibited the hNaDC3 (⁓2 mM). The compound 11 was found to show
the bindings with amino acid sidechains Thr236 of mNaDC1 and was
specific to this transporter. The hNaCT binds with citrate and four
sodium ions inhibited by the dicarboxylate 13 . The newly
compounds showed inhibition at IC50 value in µM
range[1] (Figure 11 ).
Figure 11: Compounds representing the SLC13 members inhibitors;15 being the most selective against hNaDC3
SLC25A1/Citrate/isocitrate Inhibitors
Cytosol citrates provide the source of acetyl CoA for fatty acid and
sterol biosynthesis and act as regulators controlling gluconeogenesis,
glycolysis, and lipogenesis. Conversely citrate/isocitrate carrier
promotes citrate to the mitochondria from the cytoplasm thus causing TCA
and oxidative phosphorylation with the generation of NADPH. This reverse
effect led to the cancer cell expansion and growth of the tumor cells.
Undoubtedly citrate/isocitrate carrier utilizes the resources to meet
the energetic demand whereas the loss of it is pathogenic. Benzene
tricarboxylate 16 (1st generation; analogs of
citrate) interferes with the citrate bindings proteins at high
concentration (5 mM) acts as citrate transporter protein inhibitors
(CTPI). The second inhibitors CTPI-1; 17 was effective also at
higher concentration of 1-2 mM and acts as competitive inhibitor. There
is 20-fold improvement in the activity with CTPI-2; 18 as
compared to CTPI-1; 17 and inhibits the CIC at a lower dose of
10-50 µM. Also, CTPI-2; 18 reverts steatosis, reduces obesity,
glucose intolerance, triglycerides, and cholesterol levels [35].Figure 12 showed the structure of first, second and third
generation of citrate/isocitrate inhibitors. High levels of SLC25A1
showed enhanced levels of immune checkpoints inhibitors for antibody
targeting and pro-inflammatory response for tumor killings. The
inhibition of SLC25A1 reduced gluconeogenesis (involved in regulation of
blood glucose levels) and thereby normalizes the hyperglycemia and
glucose tolerance [36]. The SLC25A1 specific CTPI-2 inhibitors
reduces macrophage infiltration, preventing steatohepatitis, revert
symptoms of inflammatory steatohepatitis and also modify obesity related
with high fat. Inhibition of SLC25A1 inhibits peroxisome
proliferator-activated receptor gamma (PPARγ) which inhibits the
gluconeogenic genes and thus normalizes the hyperglycemia and glucose
intolerance [37].
Figure 12: First, second, and third generation citrate and
isocitrate inhibitors
Citrate role in Cancer
The citrate exists as an essential molecule in the metabolism and its
excess availability by the citrate transporters causes high growth and
proliferation of cancer cells. Inhibiting the mitochondrial and the
cytoplasm citrate transporters may provide a viable strategy to develop
chemotherapeutic agents [38]. The plasma membrane citrate
transporter may act as tumor marker and can help in diagnosis of cancer.
Glutamine is a major source of citrate in the cancer cells as the
citrate produced either by the Krebs cycle in mitochondria or by
reductive carboxylation of glutamine. The mitochondria activity is
reduced in cancer, so the major source of citrate synthesis is through
increase reverse activity of the Kreb’s cycle. The plasma membrane
citrate transporter may be considered as a novel target for the cancer
identification and treatment [39]. The extracellular citrate
promotes the cancer metabolism by reversing the Kreb’s cycle from
glutamine. It is very much possible to be observed in citrate rich
organs such as liver, brain and bones where it may enhance the secondary
tumor growth, metastasis and in colonizing the cancer cells survival
[40]. The cancer cells have increased levels of the ATP citrate
lyase than normal cells such as colorectal, breast, non-small lung cell
and hepatocellular cancers, etc. Overexpression of the ATP citrate lyase
may be inhibited by either chemical inhibitors or knockdown of ATP
citrate lyase [41]. Studies have demonstrated that increase in the
citrate or inhibition of ATP citrate lyase stop the tumor growth,
inhibit the key anti-apoptotic Mcl-1, and increase in cisplatin
sensitivity. Reduced formation of acetyl CoA from decreased citrate
concentration causes deacetylation of the proteins and inhibition of
apoptosis and epigenetic changes thereby resulting in cancer
aggressiveness [42]. The decreased acetyl CoA (reduced intracellular
lipid levels and histone acetylation) was also observed viacaspase-10-mediated cleavage of ATP citrate lyase. The decreased histone
acetylation downregulates metastatic and proliferative genes [43].In vitro studies showed that cancer cells deprived of citrate
with specific inhibitor gluconate (plasma membrane citrate transporter)
inhibit the metastases and reduces the growth of human pancreatic cancer
cells and angiogenesis in vivo . The above findings provide
evidence that citrate metabolite is an important support for the
progression of cancer [44]. Results of some preclinical studies
showed that high doses of citrate exhibit anticancer effects that
includes inhibition of signaling pathway (IGF-1R/AKT), inhibition of
growth of tumors, and promotion of apoptosis [45]. The high dose of
citrate inhibited the A549 lung cancer growth. The increased citrate
concentration makes tumor cell differentiation, higher infiltrating
T-cells, increased cytokines and inhibited IGF-1R-AKT-PTEN-elF2a
phosphorylation. Both the glycolysis and the TCA cycle were found to be
suppressed in vitro [46]. The cancer cells have more citrate
oxidizing potential as compared to the normal prostate cells. The level
of the citrate gets decrease while there is an increase in the levels of
the choline moving from normal cells to the cancerous prostate analyzed
by magnetic resonance spectral imaging [3]. Cancer cells development
causes extracellular acidity which in turn makes many anticancer drugs
ineffective as acidity promotes protonation and decreases
internalization. The phosphofructokinase-1 (PFK1) and PFK2 (regulator of
the glycolysis) may be inhibited by high concentration of citrate as
these kinases enhances the glycolysis, signaling cascade and cell cycle
progression against in vitro studies in HCC cell lines. These
inhibition decreases the ATP in the cells, acts against the
HIF-1α and PI3K/AKT signaling. This promotes apoptosis,
increase in the action of cisplatin treatment, and inhibits the cancer
cell growth [47].
- Citrate role in InflammationCitrate/isocitrate may increase inflammation and thus inhibiting the
citrate/isocitrate reduces the citrate transport and hence decrease
levels of pro-inflammatory prostaglandins E2 (PGE2) and nitric oxide
(NO; mediators of the inflammation response) [35]. Citrate is
considered to produce pro-inflammatory molecules. Excess citrate gets
cleaved into acetyl-CoA and oxaloacetate by ATP citrate lyase in the
cytosol after translocating from the matrix via mitochondrial
citrate carrier. Acetyl CoA produces NADPH for NO and ROS production
by using PGE2 and oxaloacetate. Itaconate derived through citrate
results in the synthesis of inflammatory mediators and thus inhibiting
either citrate or the ATP citrate lyase can results in reduction of
inflammatory mediators [48]. Infantino et al ., in a study
showed that citrate carrier has a role in the inflammation as citrate
carrier mRNA and protein increase the lipopolysaccharide
(LPS)-activated immune cells thus citrate carrier gene silencing and
its inhibition significantly reduce nitric oxide and ROS and
prostaglandins [49]. The inhibitors of ATP citrate lyase;
tricarballylic acid could reduce the citrate induced TNF-α as ATP
citrate lyase increased the citrate metabolism. The tricarballylic
acid analogue of citrate has lower affinity to chelate with calcium
and thus able to block ATP citrate lyase more effectively thereby
reducing the augmentation efficiency of the citrate to LPS induced
TNF-α inflammatory response [50]. In LPS stimulated RAW 264.7
macrophages calcium citrate treated cells showed reduced intracellular
ROS and increased in activities of antioxidant enzymes. It also
inhibits the NO production and other pro-inflammatory markers such as
iNOS, COX-2 and NF-kB. The pro-inflammatory cytokines
(IL-1β, IL-6 and TNF-α) were also reduced indicating that calcium
citrate has anti-inflammatory properties [51]. There was higher
expression of SLC25A1 (mitochondrial citrate carrier) and ATP citrate
lyase in Bechet’s syndrome patients (multisystemic inflammatory
disorder). Mitochondrial citrate or its metabolites such as acetyl-CoA
and OAA are important mediators of inflammation [52]. Citrate
metabolism into acetyl-coenzyme A is utilized for fatty acid synthesis
and protein acetylation which in turn are linked with macrophage and
active dendritic cells activation. One of the inhibitors itaconate or
methylene succinate possesses antibacterial and anti-inflammatory
agents by inhibiting succinate dehydrogenase [53]. Figure
13 showed the structure of itaconic acid or methylene succinic acid.Figure 13 : Structure of itaconic acid or methylene succinic
acid
- Citrate role in CNS physiologyThe astrocytes in the CNS produce a large amount of citrate. It can be
reflected by the high concentration of the citrates in cerebrospinal
fluids (CSF). The human SLC13A5 loss causes severe epilepsy and
encephalopathy. This is not obligatory as SLC13A5-knockout mice do not
show any evidence of epilepsy or encephalopathy [54]. In another
study however it was confirmed by Henke et al ., that SLC13A5
deleted mouse showed increased frequency of seizure (observed in the
50% of the mice and low frequency in other 50%), alteration in the
citrate concentration in CSF (lower than in the plasma but mutant mice
citrate is higher than wild types) and brain tissue and
pro-epileptogenic excitability changes in the hippocampus. The
generalized seizure is mainly associated with the knockout mice. The
histological changes in hippocampus and para hippocampus do not show
any neuronal changes in either wild or knockout mice [55]. The
overexpression of the SLC13A5 neuron-specific receptors in mice showed
autistic-like behaviors. It also showed the disrupted white matter
integrity and different synaptic functions and structures. The
hippocampus and the cortex area showed exceptional adaptation in the
complete sets of proteome and acetyl proteome. The study showed that
there is clear link between the aberrant citrate/acetyl CoA flux and
autistic-like behaviors [56]. Citrate has a role to chelate the
divalent cations including Ca, Mg, and Zn and thus regulate the
extracellular concentration of these ions for excitation in the
neurons and further acts as modulator of glutamate receptors and NMDA
subtypes of receptors in CNS. An increase in citrate or decrease in
the glutamine concentration was confirmed both in vivo andin vitro with use of fluorocitrate which led to the inhibition
of the TCA cycle and loss of GABA synthesis. There is reported
correlation of increased CSF glutamine and Mg2+concentration in depressed patient [57]. It also showed
hyperfunction of NMDA receptor signaling through some literature
studies. This can be better understood by the zinc chelation
hypothesis. The SLC13A5 mutation causes extracellular citrate to rise
which then start chelating zinc. Negative allosteric regulation of the
NMDA receptors is lost with large Ca2+ influx and
that leads to enhance NMDA receptor mediated synaptic transmission and
pro-convulsant effect [54] Figure 14.Figure 14: Zinc chelation hypothesis; the normal SLC13A5
receptor transport of extracellular citrate to cytoplasm takes place,
the NR2A subunits of NMDA receptors occupied by the zinc ion regulate
the Ca2+ influx. Mutated SLC13A5 led to increase in
the extracellular citrate, chelates the zinc and allosteric inhibitors
to NMDA receptors is freed led to increased Ca2+influx and enhanced NMDA receptor-mediated synaptic transmission.
The SLC13A5 deficiency dysregulate the hepatic metabolism and initiate
the pediatric epilepsy. Kumar et al., used the13C isotopes to study the pathophysiology of the
disease against SLC13A5 deficient hepatocellular carcinoma cells and
primary rat cortical neurons and found that hypoxic HCC cells use
extracellular citrate for fatty acids and TCA intermediates. Under
limited conditions of glutamine/other nutrients and oxygen conditions,
the citrate supports the growth and lipid synthesis of HCC cell lines.
Also, it was evident that citrate uptake in Huh7 cells is protective
against Zn2+ cytotoxicity [58]. The congenital
disorder 22q11.2 deletion syndrome was studied by Napoli et al .
and found to have serious role in the mitochondrial citrate
transporter SLC25A1. There were metabolic differences with
dysregulation in energy homeostasis in 22q11.2 deletion syndrome
children. The children showed elevated levels of cholesterol and
2-hydroxyglutaric acid (2HG) and less than half were also associated
with hyperprolinemia. It was also observed that there was a shift from
oxidative phosphorylation to the glycolysis in 22q11.2 deletion
syndrome children and increase in reductive metabolism of
α-ketoglutarate and glucose as carbon source for TCA cycle [59].
- Role of citrate in fatty liver disease
The SLC13A5 receptors present in the sinusoidal membrane of the
hepatocyte’s uptakes the circulating citrate for the metabolic use
[54]. It was evident that dysregulated expression of ATP citrate
lyase was found in the patient with non-alcoholic fatty liver disease
(NAFLD; associated with lipid accumulation). The lipid accumulation
increases the endoplasmic reticulum (ER) stress and upregulated ATP
citrate lyase expression through internal ribosome entry site mediated
translation. The translation of mRNA is Cap independent and stimulatede novo lipogenesis [60]. Guo et al., showed that
acetylation of the ATP citrate lyase at Lys-540, Lys-546 and Lys-554
(3K) increases protein stability and promotes cell proliferation and
lipid synthesis in the lung cancer cells. The increased acetylation of
3K and decreased SIRT2 levels were also found in both livers of the mice
and the human with NAFLD. Overexpression of hepatic SIRT2 decreased the
acetylation of 3K and protein levels and thereby lessen the hepatic
steatosis in high fat/high sugar diet-fed mice[61]. Dysregulation of
hepatic ATP citrate lyase led to hepatic steatosis, insulin resistance
and hyperglycemia. It was evaluated in leptin-deficient db/dbmice and was found that ATP citrate lyase is selectively elevated in the
liver and not in white adipose tissues. Abolition of ATP citrate lyase
reduced the hepatic content of acetyl CoA and malonyl CoA and inhibited
the de novo lipogenesis in hepatic steatosis in db/dbmice. It also inhibited nuclear hormone proliferator-activated
receptor-gamma coactivator (PPAR) γ receptors. In hepatic abrogated ATP
citrate lyase improves systemic glucose metabolism by down regulated
expression of gluconeogenic genes in the liver and enhanced insulin
sensitivity [62]. Increased citrate levels were observed in the
NAFLD may be due to increased fatty acids. The excess citrate excites
hydrogen peroxide (hydroxy radicals) induced oxidative stress in HepG2
cells [63]. The cytoplasmic citrate by plasma citrate transporter
(SLC13A5), ATP dependent lyase (ACLY), and mitochondrial citrate
regulate de novo lipogenesis. The chronic liver disease caused by
NAFLD progress due to upregulation of the de novo lipogenesis.
Curcumin was investigated against NAFLD induced by oleic acid and
palmitic acid in primary mouse hepatocyte and with high-fat and
high-fructose diet. Curcumin was found to reduce oleic acid/palmitic
acid or high fat and high fructose induced NAFLD (hyperlipidemia and
hepatic lipid deposition). Curcumin may block the transporter SLC13A5,
SLC25A1 and enzyme activity of ACLY and improves the metabolic lipid
disorder. The mechanism by which curcumin acts is by blocking the
citrate transport (improves dysregulated SLC13A5) and inhibitory effect
of ACLY (cleaves citrate into acetyl-CoA and oxaloacetic acid) in oleic
acid and palmitic acid stimulated HepG2 cells via AMPK-mTOR
signaling pathway. Curcumin has significant lipid lowering effect and
inhibit the overexpression of SLC13A5 and ACLY. This further reduces the
upregulation of de novo lipogenesis and regulates the lipid
accumulation in NAFLD in mice [64]. The hydroxy citric acid extract
from the Garcinia cambogia was evaluated for the inflammatory,
atherogenic and metabolic biomarkers in 40 obese women with NAFLD. The
results showed decrease in the macronutrient intake and energy in both
the hydroxy citric acid group and the control groups. There was also
reduction in the levels of the triglycerides, total cholesterol, fasting
blood sugar, low density lipoproteins cholesterol (LDL-C) and increase
in high density lipoprotein cholesterol (HDL-C) in the hydroxy citric
acid. The ratio of TG/HDL-C ratio was decreased as compared to the
control. The inflammation factors were not reduced as such in the
hydroxy citric group but it improved metabolic factors [65].
Role of citrate in the treatment of obesity and type 2
diabetes mellitus (T2DM)The ATP citrate lyase, the one converting citrate to acetyl CoA, is
highly induced in the obese patient with chronic kidney disease. The
increase activity of ATP citrate lyase leads to the ectopic
accumulation of lipid, glomerulosclerosis, and albuminuria. It also
upregulates many rate-limiting lipogenic enzymes and promotes de
novo lipogenesis for lipid accumulation. The raised acetyl CoA also
causes hyperacetylation at the sites of H3K9/14 and H3K27. The citrate
inhibitor completely blocked the hyperacetylation as well as induction
of lipogenic enzymes. The ATP citrate lyase promotes renal lipid
accumulation, fibrogenesis and renal injury in obesity [66]. A
clinical study was conducted for 3 months in 100 obese patients to
evaluate the effects of hydroxy citric acid and one of the major
ingredients of Garcinia cambogia extract. Study results showed
that there was significant reduction in the body weight, serum
triglycerides, HDL, and LDL levels. The hydroxy citric acid treatment
may reduce the body weight gain and fat deposits in obese patients
[67]. The enzymatic activities and proteins levels of the citrate
synthase were reduced in obese patients in another study of human
omental adipose tissue [68]. The increased activity of the ATP
citrate lyase in the livers of the hereditary obese mice is two to
four times more than non-obese siblings and the activity was observed
to be reduced in starvation and increased by refeeding. The increased
in activity is also found to be increased after the onset of lactation
[69]. The hypothesis that exogenously taken citrate in the form of
processed foods and drinks contribute to the weight gain was evaluated
in mice fed with citrate with or without sucrose. The results showed
that neither it increased the weight gain nor change the lipid pattern
in any of the tested groups (citrate or citrate + sucrose). The group
with citrate + sucrose enhanced the pro-inflammatory cytokines (TNF-α,
IL-6, IL-10 and IL-1β), fasting glycemia and glucose intolerance.
Thus, citrate consumption may act as contributing agent for the
complication associated with obesity such as altered glucose
metabolism or adipose tissue inflammation [70]. Patients with
obesity and diabetes mellitus are more likely to be associated with
low urine citrate excretion among nephrolithiasis (15% more than 7%
control). Uric stone and calcium oxalate monohydrate are two most
common stone in citrate wasting groups (low urine citrate excretion).
It was evident that citrate wasting groups showed higher uric acid,
calcium, oxalate, and sodium [71]. In diabetes patients, the
enzymatic activity was reduced rather than proteins levels. The key
enzymes of the TCA cycle (citrate synthase) were affected. The
compounds shown in the Figure 15 with the general formula19 claims for treating diseases related with the citrate
transporter. The compound is used for treating diabetes especially
T2DM, obesity, and other age-related disorders. Among them the
compound 20 and 21 showed highest functional
activity against citrate INDY in HEK293 and HepG2 cell lines [72].
The low citrate synthase activity as tested in A/J strain variant of
H55N polymorphism accumulated more fats and was tolerant to glucose.
The knockdown citrate synthase activity accelerated palmitate induced
apoptosis in 50% of the tested in C2C12 mouse muscle cells [73].
Citrate administration could also decrease diabetic adverse cardiac
changes. It decreased mean glucose levels whereas glucose levels are
less affected in streptozotocin induced diabetic rats. Citrate
application with the antioxidants decreased blood glucose levels
protecting the β-cells of the pancreas from damage. The citrate groups
reduced the harmful effect on the heart tissue of the diabetes. Also,
caspase 3 immunoreaction optical density was decreased in citrate
group and integrity of the intercalated discs was found to be
maintained as compared to the diabetic groups [74]. The rate of
citrate transport was reduced to 35% in diabetic mitochondria. There
was also an increase in cholesterol contents and ratio of
cholesterol/phospholipid ratio. The analysis confirmed the
accumulation of hepatic citrate carrier mRNA, and decreased levels of
the protein. The lower contents of both the mitochondrial citrate
carrier and protein is because of decrease citrate carrier in diabetes
[75].Figure 15: General formula and most potent compound for
treating type 2 diabetes, obesity, and age-related disorders