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Dithymoquinone as a novel inhibitor for 3-carboxy-4-
methyl-
5-propyl-2-furanpropanoic acid (CMPF) to prevent renal
Failure
(that we are currently working on)
Muniba Faiza1, Tariq Abdullah2, Prof. Yonghua Wang1*
1School of Food Science and Engineering, South China University of Technology, Guangzhou
510640, China; Email: muniba.faiza@gmail.com
2IQL Bioinformatics, Idea Quotient Labs, New Delhi, India; Email: tariq@ideaquotient.in
*Corresponding Author
Yonghua Wang, Ph.D.
Vice dean in the College of Light Industry and Food Sciences
School of Food Science and Engineering,
South China University of Technology,
Guangzhou, Guangdong Province, China 450001
Tel and Fax: 86-020-87113842
E-mail: yonghwang@scut.edu.cn
ABSTRACT
3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) is a major endogenous ligand found
in the human serum albumin (HSA) of renal failure patients. It gets accumulated in the HSA and
its concentration in sera of patients may reflect the chronicity of renal failure [1-4]. It is
considered uremic toxin due to its damaging effect to the renal cells. The high concentrations of
CMPF inhibit the binding of other ligands to HSA . Removal of CMPF is difficult through
conventional hemodialysis due to i ts strong binding affinity. We hypothesized that the
competitive inhibition may be helpful in removal of CMPF binding to HSA. A compound with
higher HSA binding affinity than CMPF could be useful to prevent CMPF from binding so that
CMPF could be excreted by the body through the urine. We studied an active compound
dihydrothymoquinone/ dithymoquinone (DTQ) found in black cumin seed (Nigella sativa),
which has higher binding affinity for HSA. Molecular docking simulations were performed to
find the binding affinity of CMPF and DTQ with HSA. DTQ was found to have higher binding
affinity possessing more interactions with the binding residues than the CMPF. We studied the
binding pocket flexibility of CMPF and DTQ to analyze the binding abilities of both the
compounds. We have also predicted the ADME properties for DTQ which shows higher
lipophilicity, higher gastrointestinal (GI) absorption, and blood-brain barrier (BBB) permeability.
We discovered that DTQ has potential to act as an inhibitor of CMPF and can be considered as
a candidate for the formation of the therapeutic drug against CMPF.
KEYWORDS
CMPF, renal failure, black cumin seed (Nigella sativa), thymoquinone, dithymoquinone,
molecular docking, binding affinity.
INTRODUCTION
CMPF has been linked to the acute renal failure [1-3,5]. CMPF is a major endogenous toxin
retained in uremic serum and major drug-binding inhibitor [6]. The accumulation of CMPF
is caused by binding of CMPF to HSA, which cannot be removed through conventional
hemodialysis [7]. Currently, there is no known inhibitor for CMPF which can reduce its
accumulation.
Our Hypothesis
CMPF accumulation is due to its high binding affinity towards HSA among uremic toxins [8-
10]. We hypothesized that another compound with a higher binding affinity towards HSA than
CMPF should be able to bind with HSA. Thus, effectively blocking CMPF from binding. This
could have beneficial effects on human health apart from the effects on kidneys. The binding
of CMPF could be reduced leading to the decreased chances of acute renal failure. Assuming
this, we hypothetically considered a compound which has been earlier proved to be beneficial for
human health, such as cancer, tumor, and more [11-17]. This compound is dithymoquinone
(DTQ), one of the active compounds found in black cumin seed (Nigella sativa), which is
well known for its positive effects on human in various conditions such as tumor, cancer,
clearance of hepato-renal toxicity, diabetes, [8-18].and found to possessrelatively higher
binding affinity than CMPFWe predicted the inhibitory effect of DTQ against CMPF using in
silico docking performed by AutoDock tools [19]. In order to determine the capability of DTQ
as the potential drug candidate against CMPF, we predicted its ADME properties using
SwissADME [20]. The crucial role of CMPF in renal failure, binding with HSA, and DTQ
is discussed in detail in the following sections.
HSA- receptor for endogenous substances and drugs
HSA is an abundant plasma protein that belongs to a multigene family of proteins including α-
fetoprotein, vitamin-D binding protein, and human group specific component [21]. HSA binds to
a wide range of drugs and/ or endogenous ligands including bilirubin, thyroxine, non-esterified
fatty acids, and hemin [21], all of them being lipophilic, acidic compounds, in multiple sites [22-
27]. HSA protein consists of 585 amino acids with 17 disulphide bridges, one tryptophan residue
(Trp 214), and a single free-thiol (Cys 34) [28]. According to Sudlow et al., (1975) [29], there
are two binding sites in HSA: Site I and Site II. He & Carter (1992) [28] observed the binding
pockets of HSA lying in two subdomains: IIA and IIIA, which were the same sites but they
used different nomenclature to denote them. HSA is considered a silent receptor for drugs [30]
because it serves as a repository for a wide variety of compounds as the negative charge on HSA
facilitates the electrostatic binding of various ligands and acts as a depot and carrier for many
drug compounds [31-34]. Due to the presence of limited number of high- affinity binding
sites in HSA, detailed molecular information about these sites could be helpful in studying the
effects of other drugs or ligands and the structural information of both low- and high- affinity
binding sites is useful in designing new drugs with the aim either avoiding binding to HSA or to
make use of its functions [35].
CMPF is a metabolite of F-acids [36-40] which was first detected in human urine [37] and then
later in human blood [38]. CMPF is considered as one of the major drug- binding inhibitor [1,6,41-
43]. The drug-binding effect observed in vivo that the CMPF levels rise in kidney patients are due
to the specific steric blocking of drug site 1 by this compound [43,44]. Sakai et al., (1995) [44]
studied the interactions of CMPF in comparison of other three uremic toxins: indoxyl sulphate,
indole acetic acid, and hippuric acid using fluorescent probe displacement, ultrafiltration, and
equilibrium dialysis. The results showed that CMPF had the highest binding affinity (10-7 M-1)
among the three uremic toxins. As compared with the other uremic toxins, CMPF consists of
least rate of elimination and the most potent inhibitor [10,44-47]. Several studies have
demonstrated that uremic toxins are involved in the production of reactive oxygen species
(ROS) [48-50]. In a study conducted by Miyamoto et al., (2012) [50], it was shown that
CMPF induces the renal cell damage by enhancing the production of ROS in the presence of
Angiotensin-II (A-II), which is an inducer of oxygen free radicals. Also, in the presence of iron,
CMPF and A-II induce the Fenton's reaction leading to an increase in ROS production. The
subsequent interaction of CMPF with dissolved oxygen leads to the overproduction of oxygen
free radicals.
All the experiments mentioned above are sufficient enough to prove the damaging role of
CMPF in renal failure because they have used reliable techniques (GC-MS, HPLC, etc.) to study
the levels of CMPF in uremic/ dialyzed patients. On the basis of these findings, we can conclude
that CMPF is responsible for renal failure by getting accumulated in the renal cells and due to
its prooxidant nature, it increases the ROS production in the endothelial cells, which ultimately
leads to the renal cellular damage. Another role of CMPF in renal damage which may not be
denied is its ability to bind very strongly to HSA making itself a strong potent inhibitor resulting
in accumulation and ultimately to renal failure. It may be of great importance to remove
accumulated CMPF from HSA to prevent renal failure due to its inhibiting properties. Other
compounds cannot easily replace CMPF which has a higher binding affinity. Addressing this
major problem, we have shown that, DTQ - a compound which is already well- known for its
beneficial effects on human health, and an active component of black cumin seed (Nigella
sativa) has higher binding affinity towards HSA than the CMPF and higher lipophilicity due to
which it may be proven as a potential drug candidate in curing renal failure caused by CMPF.
Black Cumin Seed (Nigella sativa)
CMPF being the major drug-binding inhibitor, its binding with HSA could be restricted using any
other compound with higher binding affinity and high therapeutic efficacy. Black cumin seed
(Nigella sativa) has been known for its therapeutic efficiencies since many centuries. It has
been referred in Islamic tradition as having healing powers [51]. It is a spice grown in
Mediterranean region and in Western Asian countries such as India, Afghanistan, and Pakistan.
The historical references to the seeds are also found in some of the oldest religious medical
texts such as it is referred by Hippocrates and Dioscorides as ‘Melanthion’, the Bible describes it
as the curative black cumin (Isaiah 28:25, 27 NKJV), and according to hadith, Islamic messenger
Prophet Muhammad (PBUH) said “In the black seed is healing for every disease except death”
(Sahih Bukhari).
Black cumin seeds have also been used in traditional medicines for many past years for the
treatment of a wide range of diseases including a headache, bronchial asthma, infections,
dysentery, back pain, hypertension, and gastrointestinal problems [52]. The pharmacological
investigations of black cumin seed extracts have revealed its potential broad spectrum activities
as antihistaminic [53], antimicrobial [54], anti-diabetic [55], anti-hypertensive [56],
immunopotentiation [57], and anti-inflammatory [58]. It has also been proved to be consisting
of anti-tumor [12] and anticancer activities [59-61].
DTQ- a solution to CMPF accumulation problem
The bioactive constituents of the volatile black seed oil were first identified by Al-Dakhakhany
(1963) [62] showing thymoquinone (TQ) as the most active constituent (about 54%) among the
others. Later on, other active compounds: DTQ, thymol (THY), and thymohydroquinone (THQ)
were identified by Ghosheh et al., (1999) [11] using HPLC. TQ (2-isopropyl-5-methyl-
1,4-benzoquinone) has been shown to exhibit anti-inflammatory, anti-oxidant, anti-neoplastic
activities both in-vivo and in-vitro [63]. DTQ, THY, and THQ are more likely the metabolites
of TQ as under physiological conditions, TQ gets slowly reduced to DTQ and THY [64].
TQ has been shown to possess antioxidant properties through different mechanisms such as it
inhibits the formation of 5-hydroxyeicosa-tetraenaoic as well as 5-lipoxygenase products [65]
which are required for the viability of colon cancer cells. Most importantly, in the context of this
study, TQ has been shown to act as a scavenger to various ROS [66-68]. The molecular surfaces
of TQ and DTQ possess significant amount of positively charged electron-deficit regions so that
they may be subjected to nucleophilic attacks by glutathione and nucleotide bases in DNA,
thereby, causing cellular toxicity due to the depletion of glutathione and damage of DNA due to
the oxidation of nucleotide bases.
Badary et al., (1997) [69] studied the effects of TQ on cisplatin-induced nephrotoxicity and
concluded that TQ ameliorates the nephrotoxicity in rodents and induce the anti-tumor activity
of cisplatin. Due to the antioxidative and anti-inflammatory properties of TQ, it acts against
renal injuries [15]. Fouda et al., (2008) [70] studied the effects of TQ on renal oxidative damage
and proliferative response induced by mercuric chloride in rats and based on their results it was
concluded that it may be a clinically valuable agent in the prevention of acute renal failure
caused by inorganic mercury intoxication. Similarly, Hamed et al., (2013) [17] studied the
effects of black cumin seed oil on hepato-renal injury induced by bromobenzene exposure.
They concluded that the treatment with black seed oil significantly enhanced the hepato-renal
protection mechanism, reduced disease complications, and delayed its progression [17].
All these investigations done earlier supports that renal failure/damage can be
prevented/treated with black cumin seed due to the presence of anti-oxidative component TQ
and/ DTQ. TQ and DTQ both are similar in a way that they both have oxygen atoms on 1,4-
positions of the benzene ring (Fig. 1), which contribute to their anti-oxidative properties. In this
study, we performed docking experiments on HSA with DTQ (having same properties as TQ)
and HSA with CMPF and found that DTQ was able to bind more effectively than CMPF.
Therapeutics available for uremic ailments
Numerous uremic toxins such as indoles and phenols were detected for the first time in uremic
plasma between the 1940s and 1990s [71-73]. It has been proved that a majority of uremic
toxins originate endogenously through mammalian metabolism and therefore, it is being
increasingly recognized that intestinal microbial metabolism has a large effect on mammalian
blood metabolites resulting in the formation of various uremic toxins such as indoxyl sulphate,
p-cresol sulphate, etc. [74-76]. CMPF, which is also a uremic toxin, is said to a metabolite of F-
acids [36-40] and there are still no evidence to locate the place of its metabolization in humans.
There are two main existing therapeutic approaches to treat ailment caused by uremic toxins:
1) interventions that modulate bacterial growth such as probiotics, prebiotics, and dietary
modifications. 2) adsorbents that bind uremic toxins to reduce their absorption by the host.
Both of these approaches are based on the fact that these uremic toxins are metabolized by the
gut microbiota, but in the case of CMPF, it is still rendered questionable. However, the
adsorbent approach is sometimes not helpful in retarding the progression of chronic kidney
disease, for example, Sevelamer hydrochloride (Renagel; Genzyme, Cambridge, MA, USA) has
been shown to bind some of the uremic toxins such as indoxyl sulphate (10-15%) and p-cresol
(40-50%, depending on pH) [77], but it did not result in decreased serum concentrations of
these uremic toxins in a mouse model of chronic kidney disease. However, the treatment of
sevelamer in humans for studying the alterations in serum concentrations of microbial
metabolites remains to be investigated.
METHODS
Retrieval of the structures
The 3D structures of DTQ and CMPF were downloaded from PubChem (PubChem CID: 398941
and PubChem CID: 123979, respectively). The protein crystal structure of HSA was downloaded
from Protein Data Bank (PDB ID: 2BXA) [78].
Molecular docking simulations
As revealed by Ghuman et al., (2005) [78], CMPF binds to the Y-150, R-257, H-242, R-222, and
K-199 in the site I of HSA. Molecular docking simulations were done by AutoDock Vina [19].
CMPF and DTQ were docked with HSA by having default x, y, and z coordinates, i.e., 40 for
each, and the center coordinates were set accordingly. The docking results were analyzed using
PyMol [79].
Binding Analysis
Some of the factors which were influenced by binding of CMPF and DTQ with HSA were
analyzed by BINANA software [80]. BINANA (BINding ANAlyzer) is a python-
implemented algorithm to analyze ligand binding. This software analyzes the key binding
characteristics such as pi-pi interactions, hydrogen bonds, salt bridges, etc. In this study, we
used BINANA to identify the active-site flexibility of CMPF and DTQ with HSA. The results
are shown in Table 1.
ADME properties analysis
The ADME properties of DTQ were predicted by SwissADME software [20]. The
ADME properties predicted by the SwissADME include physicochemical properties,
lipophilicity, water solubility, pharmacokinetics, drug-likeness, and medicinal chemistry.
These properties are shown in Table 2.
RESULTS
Docking simulation results
Docking results showed that DTQ bound more effectively than CMPF showing the higher
binding affinity (-8.1 kcal/mol) than the later (-7.0 kcal/mol). DTQ was able to occupy the
binding pocket more efficiently than the CMPF (Fig. 2A and 2B). Docking simulations of
CMPF with HSA showed the interaction with four residues of HSA: Y150, K199, R222, and
R257 having the bond lengths equal to 2.9 A, 3.0 A, 3.0 A, and 3.2 A respectively (Fig. 3A).
Similarly, DTQ bound with Y150, K199, R222, and R257 residues of HSA having the bond
lengths equal to 3.0 A, 2.7 A, 3.0 A, and 3.2 A respectively (Fig. 3B). The bond lengths
formed by DTQ are comparatively smaller than that of CMPF, this is the another parameter to
support that DTQ is capable of binding more efficiently than CMPF and since the former bound
at the same position as the later one, we can conclude that DTQ may be a potent inhibitor of CMPF.
Fig. 2 Binding pocket of HSA A) CMPF B) DTQ. DTQ bind in the same pocket as CMPF in
HSA and covers the binding site more efficiently than the CMPF.
Fig. 3 Representing the interaction and bond lengths of A) CMPF and B) DTQ with HSA.
DTQ showed shorter bond lengths with the interacting residues than that of CMPF.
Binding Analysis results
According to Koshland, the active-site residues usually adjust to allow the binding of a specific
substrate [81]. The induced-fit model assumes that the active site is flexible and changes shape
to allow the complete binding of a substrate [82], which is a case here for CMPF and DTQ
binding with HSA. BINANA categorizes each atom into six possible characterizations: alpha‐
sidechain, alpha‐backbone, beta‐ sidechain, beta‐ backbone, other‐sidechain, and other‐
backbone. The number of close‐ contact receptor atoms falling under each of these six
categories is tallied as a metric of binding‐site flexibility. According to the predicted results of
BINANA, DTQ showed more flexibility than CMPF towards the backbone and the side-
chain (Table 1). These results suggest that the binding residues of HSA have to undergo
more conformational changes for DTQ which may be due to the more complex structure than that
of CMPF, and it may also contribute to the higher binding affinity of the former than the later.
Table 1 Binding pocket flexibility properties of CMPF and DTQ.
Ligand
Side-chain/
Backbone
Secondary structure
Count
CMPF
Side-chain
Alpha
153
Backbone
Alpha
111
DTQ
Side-chain
Alpha
208
Backbone
Alpha
153
Side-chain
Other
4
ADME properties
The ADME properties of DTQ were predicted in order to determine its lipophilicity. Lipophilicity
is one of the important parameters which is considered for drug candidates metabolism,
greater the lipophilicity, more is the absorption. The ADME results are shown in Table 2.
Table 2 ADME properties of CMPF and DTQ predicted by SwissADME.
ADME properties
DTQ
Molecular weight
328.40 g/mol
Lipophilicity (WLogP)
2.71
Consensus Log Po/w
2.62
Water Solubility (ESOL)
-3.05
GI absorption
High
BBB permeant
Yes
Log Kp (skin permeation)
-6.83 cm/s
Lipinski rule
Yes
Leadlikeness
Yes
Synthetic accessibility
4.65
Lipophilicity is the ability of the compound to dissolve in lipophilic solutions to permeate
through the various biological membranes. It is measured the distribution of the compound
between the non-aqueous (octanol) and the aqueous (water) phase, and the result is expressed
as the logarithm of the concentration ratios termed as logP. A desired logP value is less than 5.0
[83]. In the case of DTQ, the estimated logP value is 2.62, which shows its higher lipophilicity.
The BBB permeability is not considered good for the compounds as it may cause other harmful
effects to the brain, but in the case of black cumin seed, it has been found that it causes beneficial
neuropsychiatric effects and helps in to improve memory, and mood [84-86].
BOILED-Egg results predicted by SwissADME
Brain Or IntestinaL EstimateD permeation (BOILED-Egg) method predicts an accurate model
to analyze the gastrointestinal (GI) absorption and blood-brain barrier (BBB) of the candidate
drugs [87]. It works by calculating the lipophilicity and polarity of the molecules. The BOILED-
Egg representation of DTQ is shown in Fig. 4. The white region represents the physicochemical
space of the molecule possessing the highest probability of absorption by the GI tract, whereas
the yellow region represents the physicochemical space of the molecule having the highest
probability to permeate the brain. According to the BOILED-Egg representation generated by
the SwissADME, DTQ has high GI absorption and the BBB permeability (Fig. 4).
CONCLUSION AND FUTURE DEVELOPMENTS
In this study we attempted to provide an insight into the role of CMPF in renal failure as implicated
by many studies since quiet a time. CMPF has been found to be involved in causing type 2 diabetes
by disrupting the beta-cell function [88-91], but it stands contradictory as some of the research
does not correlate CMPF with diabetes [92,93] and also due to the lack of evidences CMPF has
not been positively correlated with diabetes [94]. Therefore, we have addressed only the problem
of CMPF binding with HSA leading to its accumulation and causing renal failure. According to
the studies mentioned above, the capability of CMPF to bind strongly with HSA has posed a
difficult task to remove the accumulated CMPF from the renal tubular cells. In this paper, we have
proposed a suitable solution for the situation through in silico studies. We conclude that DTQ is
capable to bind more strongly to HSA than the CMPF and shows inhibitory effects, which requires
further in vitro studies to elucidate its actual mechanism of inhibition. The past publications
provide sufficient evidences regarding the health benefits of active components of black cumin
seed (Nigella sativa), therefore, we can conclude that DTQ possess many health benefits for
humans due to its capability to bind with HSA more efficiently than CMPF, making it the best
possible solution for inhibition of CMPF binding to HSA. Also, as CMPF is a strong lipophilic
uremic solute (Depner, 1981), therefore, we comparatively analyzed the ADME properties of DTQ
and CMPF in silico. According to the predicted ADME properties, DTQ could be considered as
the potential drug candidate against the action of CMPF and can be further utilised to make
therapeutic drugs to treat renal failure caused by the accumulation of CMPF in renal cells.
Conflict of Interest
Authors declare that there is no conflict of interest whatsoever.
Declaration
MF and TA conceived the idea and performed the experiments. MF, TA and YW wrote the
manuscript.
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Figure legends
Fig.1 2D structure of a) TQ b) DTQ
Fig. 2 Binding pocket of HSA A) CMPF B) DTQ. DTQ bind in the same pocket as CMPF in
HSA and covers the binding site more efficiently than the CMPF.
Fig. 3 Representing the interaction and bond lengths of A) CMPF and B) DTQ with HSA.
DTQ showed shorter bond lengths with the interacting residues than that of CMPF.
Fig. 4 BOILED-Egg representation of DTQ. Yellow region represents the BBB and the white
region represents the GI absorption, and red circle represents the molecule (i.e., DTQ).
Fig.1 2D structure of a) TQ b) DTQ
Fig. 2 Binding pocket of HSA A) CMPF B) DTQ. DTQ bind in the same pocket as
CMPF in HSA and covers the binding site more efficiently than the CMPF.
Fig. 3 Representing the interaction and bond lengths of A) CMPF and B) DTQ with HSA.
DTQ showed shorter bond lengths with the interacting residues than that of CMPF.
Fig. 4 BOILED-Egg representation of DTQ. Yellow region represents the BBB and the
white region represents the GI absorption, and red circle represents the molecule (i.e., DTQ).