Polyoxometalates: Study of inhibitory kinetics and mechanism against α-glucosidase

Article abstract of DOI:10.1016/j.jinorgbio.2019.110784

Alpha-glucosidase is considered to be an important target for the treatment of noninsulin-dependent diabetes. In this work, the inhibitory effects of polyoxometalates (POMs) affected by three different factors (heteroatom, transition metal substitution el

Full text of DOI:10.1016/j.jinorgbio.2019.110784

Accepted Manuscript
Polyoxometalates: Study of inhibitory kinetics and mechanism
against α-glucosidase
Guoxiang Chi, Li Wang, Bingnian Chen, Jian Li, Jingjing Hu,
Shuxia Liu, Meijuan Zhao, Xiaomei Ding, Yue Li
PII:
S0162-0134(19)30292-2
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110784
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Reference:
110784
JIB 110784
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Journal of Inorganic Biochemistry
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Accepted date:
11 May 2019
13 July 2019
15 July 2019
Please cite this article as: G. Chi, L. Wang, B. Chen, et al., Polyoxometalates: Study
of inhibitory kinetics and mechanism against α-glucosidase, Journal of Inorganic
Biochemistry, https://doi.org/10.1016/j.jinorgbio.2019.110784
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Polyoxometalates: Study of Inhibitory Kinetics and
Mechanism Against α-Glucosidase
Guoxiang Chi a, Li Wang a,†, Bingnian Chen b,†, Jian Li a, Jingjing Hu a, Shuxia Liu c,†,
Meijuan Zhao a, Xiaomei Ding a, Yue Li a
a College of Food and Biological Engineering, Jimei University, Xiamen, 361021, P.R. China
b Xiamen University Hospital, Xiamen 361005, P.R. China
c Key Laboratory of Polyoxometalate Science of Ministry of Education, College of Chemistry,
Northeast Normal University, Changchun, Jilin 130024, P.R. China
Abstract
Alpha-glucosidase is considered to be an important target for the treatment of
noninsulin-dependent diabetes. In this work, the inhibitory effects of polyoxometalates (POMs)
affected by three different factors (heteroatom, transition metal substitution element and vanadium
substitution number) on α-glucosidase were studied. We found that POMs with Keggin-type and
vanadium-substituted Dawson-type structures act as effective and mostly competitive inhibitors
for α-glucosidase (IC50 values around 40-160 μM), and most compounds can compete with the
substrate for the active site of α-glucosidase. By analyzing and comparing the inhibitory effects of
each series of POMs on α-glucosidase, the results demonstrated that the structure and composition
of the POMs themselves may indirect influence on their inhibitory capabilities. Moreover, we
gained initial information about the structure-inhibition relationship of different POMs. More
intriguingly, molecular docking simulation suggested that all compounds bind into the active site
of α-glucosidase by multiple van-der-Waals and hydrogen bond interactions. Our kinetic data
† Corresponding author. Tel.: +86-592-6181487; Fax.: +86-592-6181487
E-mail address: wanglimerry@jmu.edu.cn (Li Wang); 3421836578@qq.com (Bingnian Chen);
liusx@nenu.edu.cn (Shuxia Liu)
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demonstrate the considerable potential of POMs for the development of clinically valuable
α-glucosidase inhibitors.
Keywords: Polyoxometalates; α-Glucosidase inhibitors; Enzyme kinetics; Molecular docking
1. Introduction
Type 2 diabetes (non-insulin-dependent diabetes) is a type of a chronic hyperglycemia
disorder caused by insulin deficiency or insulin resistance [1, 2]. α-Glucosidase is a carbohydrate
hydrolase which is considered to be an important target for the treatment of non-insulin-dependent
diabetes mellitus [3]. In recent years, the development of safe and effective α-glucosidase
inhibitors has become a hotspot of research for the treatment of non-insulin-dependent diabetes
mellitus. Peng et al. [4] reported that kaempferol showed a notable inhibition activity on
α-glucosidase. Taha et al. [5] designed and synthesized seventeen Coumarin based derivatives that
showed outstanding α-glucosidase inhibitory potential many folds better than the standard
acarbose. However, the reported research on α-glucosidase inhibitors mainly focuses on natural
extracts and organic compounds, and few inorganic agents have been reported that can inhibit
α-glucosidase. Moreover, the current therapeutic drugs (acarbose [6], miglitol [7] and voglibose
[8]) on the market are incompletely satisfactory due to the high cost and toxic side effects (Table
S1). Therefore, the design of high efficiency, low cytotoxicity and low cost α-glucosidase
inhibitors is an attractive goal in the field of medicinal chemistry.
Polyoxometalates (abbreviated as POMs), due to their incomparable structural diversity,
novel functional properties and multifaceted bioactivities, have shown good application prospects
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that encompasses anticancer [9-12], antiviral [10, 13], antibacterial [14,15], against Alzheimer's
disease [16-18] and enzyme inhibition [19-22] in the field of medicine. Many studies have
validated that POMs possess significant inhibitory effects on the activities of kinase, nucleotidase,
phosphatase, sialyltransferase, nuclease, sulfotransferase, acetylase and protease [23]. Gumerova
et al. [24] studied the inhibitory effects of nine different polyoxotungstates (POTs) on P-type
ATPases in vitro (Ca2+-ATPase) and ex vivo (Na+/K+-ATPase), and the results reveal some POTs
are potent ATPase inhibitors, with K9(C2H8N)5[H10Se2W29O103] showing high selectivity towards
Ca2+-ATPase. Moreover, the information about POMs as anti-diabetic agents is rapidly increasing
in the last 30 years thereby creating many highlights (See Scheme S1). We found that the research
of scientists is mainly focused on the anti-diabetic effect of vanadate [25,26] and tungstate [25,27].
Most recently, our group has reported that the H3PMo12O40 and three transition metal-substituted
Keggin-type phosphomolybdic acids can be excellent inhibitors to α-glucosidase activity, and the
H3PMo12O40 possess the strongest inhibition effect [28]. However, the structure-activity
relationship of POMs and fundamental questions regarding the binding mode of POMs inhibitors
on enzymes is largely unknown, especially for α-glucosidase. In addition, POMs are characterized
by species diversity, structural modification, and extensive inhibition of enzymes. Therefore, the
screening of a large number of POMs, especially of the Keggin and Dawson-type with superior
inhibitory properties and lower cytotoxicities [29-32] may result in clinically valuable
α-glucosidase inhibitor drugs.
Herein, the primary focus of this paper is to present the Keggin-type and
vanadium-substituted Dawson-type POMs (Fig. S1) with potential pharmacological activity on
α-glucosidase. Three structural crucial factors of POMs - heteroatom, transition metal substitution
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element and vanadium substitution number - have been varied and the corresponding inhibition
kinetics performed. Importantly, we initially analyzed the structure-inhibition relationship of
POMs and fundamental questions regarding the mechanisms of the inhibitory capabilities of
POMs on α-glucosidase. This work provides an enlightened strategy for the in-depth study of
polyoxometalate medicinal chemistry and designing novel, safe and effective α-glucosidase
inhibitors.
2. Materials and methods
2.1. Reagents
H5GaMo12O40 [33], H4SiMo12O40 [34], Na7PMo11MO40 (M=Ni, Mn, Zn, Cr) [35, 36] and
H6+nP2Mo18-nVnO62 (n=1~5) [37, 38] (abbreviated as GaMo12, SiMo12, PMo11M and P2Mo18-nVn,
respectively) were synthesized as previously described with slight modifications. Next, the
structure of the synthetic compounds were characterized by IR (Jasco FT/IR-480) and UV/Vis
(Cary-50) spectroscopy. Moreover, the structure of P2Mo18-nVn (n=1~5) were further characterized
by solid nuclear magnetic resonance spectrometry (UNITY+-300). α-Glucosidase of S. cerevisiae
(EC 3.2.1.20), p-Nitrophenyl-α-D-glucopyranoside (pNPG), p-Nitrophenol (pNP) and Dimethyl
sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemical Co. (USA). All other reagents
were locally sourced and of analytical grade, and the ultra-pure water was used throughout.
2.2. Experiment procedure
2.2.1. Enzyme kinetics analysis
In this experiment, pNPG was used as a substrate to generate a yellow product (pNP) with
catalysis by α-glucosidase, and the OD value after the reaction of the system was monitored by the
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microplate reader (Synergy H1) at a wavelength of 405nm [28]. Briefly, the reaction system was
sequentially supplemented with 133 μL of 0.1M phosphate buffer (Na2HPO4-NaH2PO4 buffer, pH
6.8), 7 μL of different concentrations of inhibitors completely dissolved in DMSO solution (the
final concentration of DMSO in the test solution was 5%), and 10 μL of α-glucosidase solution
that was added immediately. Next, the mixture was incubated at 37.0 °C for 10 min, and 20 μL of
the substrate (pNPG) preheated at 37.0 °C for 10 min was added to the mixture. The reaction was
carried out at 37.0 °C for 20 min, and then the reaction was terminated by the addition of an equal
volume of Na2CO3 solution (1 M). The OD value of the 200 μL reaction solution was monitored in
a 96-well plate by using the microplate reader at 405 nm [28]. As previously described [28], the
inhibitory effect (IC50), inhibition mechanism and inhibition type of three different factors
(heteroatom, transition metal substitution element and vanadium substitution number) of POMs on
α-glucosidase were studied.
2.2.2. Molecular docking study
To predict the binding mode of each series of synthesized compounds with α-glucosidase, a
molecular docking study was carried out using MOE (Molecular Operating Environment)
software package. According to our previous report [28, 39], the three-dimensional structures of
the synthesized compounds were generated via using the builder tool in MOE. The generated
compound was 3D protonated and energy minimized using the default parameters of the MOE
(gradient: 0.05, Force Field: MMFF94X, pH 6.8). These compounds were then saved into an mdb
file for further evaluation.
However, the 3D structure for α-glucosidase of S. cerevisiae has not been solved yet. The
template structure; Herein, the crystal structure of isomaltase from S. cerevisiae (PDB 3AJ7) share
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82% homology with the S. cerevisiae α-glucosidase, which is generally as considered excellent
value for the selection of template [40]. Moreover, the homology modeling process of
α-glucosidase and the details, and parameters of molecular docking in this paper, are the same as
those of previously described [28, 41].
2.2.3. Statistical analysis
The experimental data were repeated five times under parallel determination by using SPSS
version 17.0 and GraphPad Prism version 7.0 for analysis. Experimental drawings were completed
using Origin 8.0 professional software. Error bars indicate ± s.d.
3. Results and discussion
3.1. Analysis of Keggin-type POMs with different kinds of central atoms (heteroatom)
3.1.1. Spectroscopic characterization
Table S2 listed the IR spectra and the UV-Vis spectra data of the compounds GaMo12 and
SiMo12. In the IR spectra, the two compounds showed most characteristic bands at 700-1100 cm-1
due to molybdenum-oxygen stretching vibrations. In the UV-Vis spectra, the compounds showed
two characteristic absorption bands at 195 nm and 310 nm, respectively. The data in the table
strongly indicated that these two complexes have Keggin-type structures, which is consistent with
the results of references [33, 34].
3.1.2. The inhibitory effect of compounds on α-glucosidase
To compare the inhibitory effects of POMs with different central atoms on α-glucosidase, we
selected compounds GaMo12 and SiMo12 as inhibitors to study the effect of POMs on the activity
of α-glucosidase-catalyzed pNPG. As shown in Fig. S2, the α-glucosidase activity revealed a
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significant decrease with increased the concentrations of the inhibitors (GaMo12 and SiMo12). The
concentrations of GaMo12 and SiMo12 when the α-glucosidase activity was decreased by 50%
(IC50) were 615.10 ± 17.00 μM and 33.71 ± 0.76 μM, respectively. Compared with our previously
reported PMo12 [28], the inhibitory effects of these three Keggin-type POMs are GaMo12 < SiMo12
< PMo12. From the above results, the IC50 revealed a significant decrease with increased the
heteropolyanions charge (Ga: -3, Si: -4 and P: -5), which indicated a significant indirect influence
of the central atom charge on the inhibition behaviour. In addition, the acid strength of POMs were
determined by alkaline method as GaMo12 < SiMo12 < PMo12, and studies have shown that the
oxidizability of Keggin-type POMs with the same structure are mainly affected by the electrostatic
effect of the anionic charge, which decreases with the oxidation state of the central atom, and its
oxidizability decreases as the negative charge of the POMs increases [42,43]. Based on the above
theory, we found that the different central atomic species of POMs change their own redox
property and acidity, which may cause significant differences in the inhibition behaviour of the
three POMs on α-glucosidase. We also found that the inhibitory effects of GaMo12 and SiMo12 on
α-glucosidase were less effective than those of PMo12, but the inhibitory effects of these two
inhibitors were more significant than those of standard commercial acarbose (IC50=750 ± 1.5 μM)
[44]. Among them, the inhibitory strength of SiMo12 is approximately 23 times higher than that of
acarbose.
In addition, we also studied the inhibitory mechanism and inhibitory type of inhibitors on
α-glucosidase activity. The results are shown in Table 1. From Fig. S3, in the reaction system, the
plots of the remaining enzyme activity versus the concentrations of the enzyme produced several
adjacent straight lines, which all passed through the origin. Next, a linear decrease with increasing
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concentrations of inhibitors indicated that the inhibition mechanisms of these two inhibitors on
α-glucosidase were reversible [28, 45]. Therefore, these two inhibitors are non-covalently attached
to the active center of the enzyme by multiple polar and non-polar interactions. Moreover, the
inhibition type of the GaMo12 and SiMo12 on the α-glucosidase activity was determined using the
Lineweaver-Burk (double-reciprocal) plots of the reciprocal of the reaction rate versus the
reciprocal of the substrate concentration. The inhibitory constant (KI or KIS) of the inhibitors was
further calculated through secondary mapping, and the results are shown in Fig. S4. As shown in
Fig. S4, the graph of 1/v versus 1/[s] represented a set of straight lines which all intersected at or
extremely close to the Y-axis. Next, the linear slope increased gradually as the concentrations of
the GaMo12 and SiMo12 inhibitors increased, while the vertical axis intercept remained unchanged,
which suggested that the inhibitors compete with the substrate for the active site of the
α-glucosidase [45]. Furthermore, the inhibitory constants KI of the GaMo12 and SiMo12 were
calculated by further plotting the linear slopes versus the inhibitor concentrations of 0.630 mM
and 0.032 mM, respectively.
<Table 1.>
3.1.3. Analysis of molecular docking results
Different inhibitory effects on the target protein may occur due to differences in the chemical
structure or the mechanism of action of the inhibitor itself. To vividly represent the binding forms
and interactions of the ligands (GaMo12 and SiMo12) in the active site of α-glucosidase, the
experimental results were analyzed by the MOE (Fig. 1AB) and the PyMOL (Fig. 1CD) software.
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As shown in Fig. 1AB, the amino acid residues surrounding the compound are represented by pink
and green circles. GaMo12 and SiMo12 accurately fit into the deep pocket of the active site and is
largely surrounded by the polypeptide. After docking scoring, the scores for GaMo12 and SiMo12
docking with α-glucosidase were -11.68 and -13.62 kcal/mol, respectively. The score meant that
the docking of these two compounds with α-glucosidase was successful. In addition, the smaller
the numerical value is, the better is the ability of the compound to bind to the biomacromolecule.
Therefore, in combination with the previous report [28], the binding ability of the compound to
α-glucosidase is GaMo12 < SiMo12 < PMo12 (Table S3). Further molecular docking study validated
the experimental results of the enzyme kinetics.
<Fig. 1. (A, B, C and D)>
Furthermore, as shown in Fig. 1, SiMo12 forms five hydrogen bonds with the amino acid
residues (Ser240, His280, Pro312 and Arg315) in the active region, while GaMo12 forms only
three hydrogen bonds with the amino acid residues (Ser240 and Asp242) in the active region
(Table S3), so the inhibition of SiMo12 is greater than that of GaMo12. Similarly, the inhibition of
PMo12 is greater than that of SiMo12 and GaMo12. In addition to the hydrogen bonding interactions
with the enzyme, the compounds also form several van der Waals interactions which significantly
contributes to the strong binding of the compounds. However, we found no significant difference
in the number of amino acid residues involved in van der Waals interactions among the three
compounds. According to the molecular simulation, the SiMo12 and GaMo12 does bind to the
enzyme active groups in the form of hydrogen bonding and van der Waals interactions. Therefore,
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the catalytic group of the enzyme is generally not destroyed, and the enzyme is not inactivated,
which proves that both compounds are a reversible inhibitor [28].
3.2. Analysis of different transition metal-substituted Keggin-type POMs
3.2.1. Spectroscopic characterization
Table S4 listed the IR spectra (four characteristic bands at 700-1100 cm-1) and the UV-Vis
spectra (two characteristic absorption bands at 200 nm and 260 nm) data of the compounds
PMo11M (M=Ni, Mn, Zn, Cr). These data in the table strongly indicated that these complexes have
Keggin-type structures, which is consistent with the results of references [35, 36]. Among them,
the peak positions of the compounds in the table were shifted, which may be due to the
substitution of transition metals.
3.2.2. The inhibitory effect of compounds on α-glucosidase
To compare the effects of different transition metal-substituted Keggin-type POMs on
α-glucosidase, four transition metal-substituted Keggin-type POMs were synthesized and used as
inhibitors to study their inhibitory effects on α-glucosidase. As shown in Fig. S5, the
α-glucosidase activity gradually decreased as the concentration of the inhibitors PMo11M (M=Ni,
Mn, Zn, Cr) increased. Compared with the previously studied inhibitors (PMo11V, PMo11Co and
PMo11Fe) [28], the inhibitory effects of the seven Keggin-type POMs on α-glucosidase were
PMo11Fe ＜ PMo11Cr ＜ PMo11Co ＜ PMo11Zn ＜ PMo11V ＜ PMo11Mn ＜ PMo11Ni. Next,
compared with standard acarbose (IC50=750 ± 1.5 μM) [44], the inhibition potency of PMo11Ni
(IC50=37.29 ± 1.72 μM) was approximately 20 times higher than that of acarbose, so the
synthesized inhibitors in our laboratory possess significant inhibitory effects on α-glucosidase. It
was revealed that, as compared to the IC50 of each compound, we found that the inhibitory effects
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of the three compounds (PMo11Ni, PMo11Mn and PMo11V) on α-glucosidase were generally much
stronger than the other four, which may be mainly due to the substitution of metal elements of
POMs to α-glucosidase has different inhibition behaviour. The substitution of different transition
metal elements may also change their own redox properties, which may indirect influence the
inhibition behaviour of the POMs on α-glucosidase, and the need for further research is
emphasized. Moreover, the reason why the inhibitory effect of PMo11Fe on α-glucosidase is worse
than other compounds may be that its inhibition type is different from others. Similarly, the
inhibition mechanism of the four compounds we synthesized were all reversible (Fig. S6). Except
that PMo11Ni showed mixed-type inhibition, the inhibition types of the other three compounds
were all competitive (Fig. S7). In the mixed-type inhibition, the inhibitor PMo11Ni can affect the
affinity of the enzyme to the substrate and affect the catalytic action of the enzyme. PMo11Ni can
bind to both the free enzyme and the enzyme-substrate complex, but this affects its inhibitory
effect on α-glucosidase. Furthermore, Table 1 lists the inhibition constants (KI and KIS) of the four
compounds for α-glucosidase.
3.2.3. Analysis of molecular docking results
In this part, we selected the best inhibitory compound PMo11Ni for α-glucosidase, and further
researched the binding forms and interactions between PMo11Ni and α-glucosidase at the
molecular level by molecular simulation, as shown in Fig. 2. The interactions between the ligand
PMo11Ni and the most important amino acids generated by MOE are visualized in Fig. 2AC. The
inhibitor perfectly matches to the active site pocket of α-glucosidase (Fig. 2AB), and the docking
score was -13.35 kcal/mol after docking. The low score can be interpreted as measure for a strong
enzyme-inhibitor binding and therefore as a reliable docking event.
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<Fig. 2. (A, B and C)>
In addition, PMo11Ni forms five hydrogen-bond interactions with the amino acid residues
(Ser240, His280, Ser311, Pro312 and Arg315) in the active region (Fig. 2AC). PMo11Ni forms van
der Waals interactions with the amino acid residues (Tyr158, Asp242, Leu246, Gln279, Asp307,
Thr310, Leu313 and Phe314) around the active region (Fig. 2AC). In the docking interaction
residue analysis, the four polar amino acids around the active binding site of the enzyme are
Tyr158, Gln279, Asp307 and Thr310, respectively. These polar amino acid residues significantly
contributes to the strong binding of the compound.
3.3. Analysis of vanadium-substituted Dawson-type POMs
3.3.1. Spectroscopic characterization
Elemental analysis (%): Anal. Calc. (Found) for P2Mo17V: P 1.80 (1.77), Mo 47.34 (47.23), V
1.48 (1.47); Anal. Calc. (Found) for P2Mo16V2: P 1.80 (1.77), Mo 44.66 (44.54), V 2.91 (2.95);
Anal. Calc. (Found) for P2Mo15V3: P 1.74 (1.73), Mo 40.28 (40.17), V 4.28 (4.33); Anal. Calc.
(Found) for P2Mo14V4: P 1.81 (1.76), Mo 39.26 (39.29), V 5.96 (5.94) and Anal. Calc. (Found) for
P2Mo13V5: P 1.88 (1.83), Mo 37.73 (37.56), V 7.72 (7.70). These data results were consistent with
the results of reference [37].
In addition, Table S5 listed the IR spectra (four characteristic bands at 700-1100 cm-1) and the
UV-Vis spectra (two characteristic absorption bands at 210 nm and 310 nm, repectively) data of
31
the compounds P2Mo18-nVn (n=1~5), and the P-NMR spectra was shown in Fig. S8. These data
strongly indicated that these compounds were Dawson-type vanadium-substituted POMs, which
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was consistent with the results of references [37, 38].
3.3.2. The inhibitory effect of compounds on α-glucosidase
Compared with Keggin-type structure, Dawson-type structure of POMs are relatively less
studied and their redox ability is stronger. Therefore, a series of vanadium-substituted
Dawson-type POMs were synthesized and used as inhibitors to study their inhibitory effects on
α-glucosidase. As shown in Fig. S9, the α-glucosidase activity revealed a significant decrease with
increased the concentrations of the inhibitors P2Mo18-nVn (n=1~5). Comparing the IC50 of each
compound (Table 1), it was found that the inhibitory effects of the five Dawson-type POMs on
α-glucosidase were P2Mo17V < P2Mo14V4 < P2Mo16V2 < P2Mo13V5 < P2Mo15V3. It was revealed
that, as compared to the acarbose (IC50=750 ± 1.5 μM) [44], the inhibition potency of P2Mo15V3
(IC50=57.01 ± 2.11 μM) was approximately 13 times higher than that of acarbose, so the
synthesized inhibitors possess significant inhibitory effects on α-glucosidase. Moreover, the V is
easier reduced than the Mo, so that the Mo in the Dawson-type POMs is substituted by the V, and
its oxidizability is markedly enhanced [37]. Therefore, we analyzed that the inhibitory effect of
vanadium-substituted POMs on α-glucosidase is P2Mo17V < P2Mo16V2 < P2Mo15V3, because the
increase in the number of vanadium substitutions may enhance the oxidizability of the compounds,
which may indirect influence their binding affinities to the active site of the α-glucosidase. From
this, it is speculated further that the inhibition of α-glucosidase is related to the elemental
composition of the POMs, and the more the V-containing component (not more than three), the
stronger the inhibitory effect on α-glucosidase. Similarly, we also found that the inhibition
mechanisms of these five vanadium-substituted Dawson-type POMs are reversible (Fig. S10), and
their inhibition types are all competitive (Fig. S11). In addition, Table 1 lists the inhibition
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constants (KI) of these five compounds for α-glucosidase.
3.3.3. Analysis of molecular docking results
The above enzyme kinetics showed that the compound P2Mo15V3 had the best inhibitory
effect on α-glucosidase. Therefore, we further researched the binding forms and interactions of
P2Mo15V3 and α-glucosidase at the molecular level by the MOE (Fig. 3AB) and the PyMOL (Fig.
3C) software. As shown in Fig. 3AB, the P2Mo15V3 accurately fit into the deep pocket of the
active site and is largely surrounded by the polypeptide, and the docking score was -19.55
kcal/mol after docking. The low score can be interpreted as measure for a strong enzyme-inhibitor
binding and therefore as a reliable docking event. Furthermore, P2Mo15V3 forms six hydrogen
bonds with the amino acid residues (Tyr158, Asp242, His280 and Ser311) in the active region (Fig.
3AC). In addition to the hydrogen-bond interactions with the enzyme, the compound also forms
several van der Waals interactions which significantly contributes to the strong binding of the
compound. P2Mo15V3 forms van der Waals interactions with the amino acid residues (Val232,
Ser240, Pro243, Leu246, Pro312, Leu313, Phe314 and Arg315) around the active region (Fig.
3AC). Similarly, in the docking interaction residue analysis, these polar amino acid residues
(Ser240 and Arg315) significantly contributes to the strong binding of the compound.
<Fig. 3. (A, B and C)>
4. Conclusions
In summary, to gain initial information about the structure-inhibition relationship of different
POMs, three structural crucial factors of POMs - heteroatom, transition metal substitution element
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and vanadium substitution number - have been varied and the corresponding inhibition kinetics
and molecular simulation performed. Enzyme kinetics demonstrated that our synthetic compounds
PMo12 (6.41 ± 0.38 μM) [28], PMo11Ni (IC50=37.29 ± 1.72 μM) and P2Mo15V3 (IC50=57.01 ± 2.11
μM) has the strongest inhibitory effect on α-glucosidase in their respective series, and their
inhibitory effect was approximately 117, 20 and 13 times higher than that of standard acarbose,
respectively. In addition, we found that all synthetic compounds except PMo11Ni (reversible
mixed-type) and PMo11Fe (reversible noncompetitive) exhibited reversible competitive inhibition.
Table 1 listed the inhibition constants (KI and KIS) of all compounds for α-glucosidase. More
intriguingly, we found that the IC50 revealed a significant decrease with increased the
heteropolyanions charge (Ga: -3, Si: -4 and P: -5), which indicated a significant indirect influence
of the central atom charge on the inhibition behaviour. The structure and composition of POMs
may change their own oxidation-reduction, acidity, and electrostatic effects of anion charge, which
may also indirect influence the inhibition behaviour of POMs on α-glucosidase. Moreover,
molecular docking simulation demonstrated that the compounds we synthesized competitively
bind to the active site of α-glucosidase mainly through multiple van-der-Waals and hydrogen bond
interactions. The involvement of more polar amino acid residues significantly contributes to the
strong binding of POMs on α-glucosidase. To some extent, molecular simulations validated the
experimental results of enzyme kinetics, and further expound upon the mechanisms of action at
the molecular level. Through future experiments, we will further study the effects of factors such
as counter cations and lattice water on the structure-activity relationship of POMs, and further
expand our investigation with the human α-glucosidase and the model enzyme isomaltase.
Furthermore, we will determine the X-ray structure of α-glucosidase-POMs complex for
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comparison and for evaluating the accuracy of the modelling data. In this sense, this work further
advances our understanding of the design and synthesis of POMs as therapeutic agents for type 2
diabetes. Also, this work opens up a window toward the biomedical applications of
polyoxometalate chemistry.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No.
21871110) and the National Key Research and Development Program of China (Grant No.
2018YFD0901004).
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Figure captions
Figure 1. Docking conformations of ligand GaMo12 and SiMo12 in α-glucosidase.
Two-dimensional ligand interaction map of compounds GaMo12 (A) and SiMo12 (B) docked into
the binding site of α-glucosidase; Three-dimensional complex interaction of α-glucosidase and the
ligands GaMo12 (C) and SiMo12 (D).
Figure 2. Binding mode of the ligand PMo11Ni with α-glucosidase residues. (A) Two-dimensional
ligand interaction map of compound PMo11Ni docked into the binding site of α-glucosidase. (B)
The ligand PMo11Ni in the active site of α-glucosidase. (C) Three-dimensional complex
interaction of ligand PMo11Ni with α-glucosidase residues.
Figure 3. Binding mode of the ligand P2Mo15V3 with α-glucosidase residues. (A)
Two-dimensional ligand interaction map of compound P2Mo15V3 docked into the binding site of
α-glucosidase. (B) The ligand P2Mo15V3 in the active site of α-glucosidase. (C) Three-dimensional
complex interaction of ligand P2Mo15V3 with α-glucosidase residues.
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Table 1 Enzyme kinetic results of all synthetic compounds on α-glucosidase. Error bars indicate ±
s.d.
Inhibitory constant
KI/(mmol·L-1) KIS/(mmol·L-1)
Inhibitory
mechanism
Inhibitory
type
Compound (POMs) IC50/(μmol·L-1)
Ref.
H5GaMo12O40
H4SiMo12O40
615.10 ± 17.00
33.71 ± 0.76
6.41 ± 0.38
reversible
reversible
reversible
reversible
reversible
reversible
competitive
competitive
competitive
mixed-type
competitive
competitive
0.630
0.032
0.018
H3PMo12O40
[19]
[19]
Na7PMo11NiO40
Na7PMo11MnO40
Na4PMo11VO40
37.29 ± 1.72
47.66 ± 1.15
52.33 ± 1.41
0.049
0.079
0.146
0.109
0.412
Na7PMo11ZnO40
Na7PMo11CoO40
Na7PMo11CrO40
Na6PMo11FeO40
H7[P2Mo17VO62]
99.23 ± 2.24
103.10 ± 2.88
126.20 ± 3.70
161.90 ± 7.68
164.20 ± 14.20
reversible
reversible
reversible
reversible
reversible
competitive
competitive
competitive
noncompetitive
competitive
0.059
0.121
0.278
0.312
0.328
[19]
[19]
H8[P2Mo16V2O62]
H9[P2Mo15V3O62]
H8[P2Mo14V4O62H2]
H9[P2Mo13V5O62H2]
117.40 ± 3.64
57.01 ± 2.11
127.13 ± 4.01
99.65 ± 2.45
reversible
reversible
reversible
reversible
competitive
competitive
competitive
competitive
0.278
0.024
0.110
0.053
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Highlights



Keggin-type and vanadium-substituted Dawson-type polyoxometalates were synthesized.
The structure-inhibition relationship of polyoxometalates were initially analyzed.
Molecular modeling revealed the polyoxometalates binding mode.
26

Figure 1

Figure 2

Figure 3