The medical field now needs more novel drugs to treat obesity and type-2 diabetes mellitus (T2D) than ever before. Obesity and T2D are both characterized by resistance to the hormones leptin and insulin. PTP-1B is a promising target for drug growth as strong genetic, pharmacological and biochemical evidence points to the possibility of treating diabetes and obesity by blocking the PTP-1B enzyme. Studies have also found that PTP-1B is over expressed in patients with diabetes and obesity, suggesting that inhibiting PTP-1B may be a useful technique in their care. There aren't any clinically used PTP-1B inhibitors, despite the fact that numerous naturally occurring PTP-1B inhibitors demonstrated great therapeutic promise. This is most likely because of their low activity or lack of selectivity. It is still important to look for more effective and focused PTP-1B inhibitors. A few organo vanadium metal complexes were synthesized, characterized, and binding studies on vanadium complexes with PTP-B were also performed using Fluorescence Emission Spectroscopy. Additionally, we theoretically (molecular modeling) and experimentally (enzyme kinetics) examined the PTP-1B inhibitory effects of these vanadium metal complexes and found that they have excellent PTP-1B inhibitory properties.
Research Article
Enzyme PTP-1B Inhibition Studies by Vanadium Metal Complexes: A Kinetic Approach
https://doi.org/10.21203/rs.3.rs-2333030/v1
This work is licensed under a CC BY 4.0 License
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In the treatment of diabetes, vanadium has been shown to mimic insulin. When compared to inorganic vanadium salts, complexes of vanadium with appropriate organic ligands can improve [1] permeation, tissue uptake, effectiveness, and reduce toxicity. An organic vanadyl derivative [bis(maltolato)oxovanadium(IV)] is currently being tested as an anti-diabetic drug in phase II clinical trials[2]. The mechanism suggests that vanadium complexes inhibit the enzyme protein tyrosine phosphatase-1B (PTP-1B) [3–12]. Various studies on diabetic patients revealed that the PTP activity increases in type II diabetic (T2D) patients along with increased levels of PTP-1B protein. Inhibition on the activity of PTP-1B enzyme is thought to be of great promise for lowering insulin and leptin resistance. Therefore, PTP-1B inhibition may represent a novel therapeutic strategy for the treatment of patients who are obese and at risk for type II diabetes. Major current pharmacotherapy for T2D includes thiazolidinediones, gliptins, sulfonylureas, metformin and liragutide etc.
PTP-1B is an active player in T2D patients. It is a unique enzyme which is included in protein tyrosine phosphatase (PTP) family [13]. It is encoded by the PTPN1 gene in humans. It is a negative regulator of insulin [14, 15] signaling pathway and it is considered to be an important therapeutic target for treatment of T2D patients.
In view of inhibitory action of vanadium metal complexes, many research groups are in the process of synthesizing novel vanadium complexes with a hope to get an ideal metal complex to inhibit the PTP-1B, and our group is one among them. As a part of our project, several binary and ternary complexes have been synthesized and characterized in previous papers [16, 17], from vanadium metal and acetylacetone(4,4,4-trifluoro-1-(2-theonyl)-1,3-butanedione). Imidazole, 2-Methyl-imidazole, and 2-Ethyl-imidazole auxiliary ligands were employed. Using analytical techniques metal complexes were characterized.
Before conducting inhibitory studies on PTP-1B enzyme, we thought it would be a good idea to investigate the interaction of these complexes with PTP-1B and determine whether these metal complexes bind with protein (PTP-1B), how strongly they bind, and the order of binding with different metal complexes. It has also been established that interactions between proteins and metal complexes are thought to be relatively strong if the Stern-Volmer constant (Ksv) and binding constant (Kb) between protein and drug are found to be in the order of 105M−1 and 104-106M−1, respectively [18–20]. The experimentally determined binding constant (Kb) for the various vanadyl metal complexes under study is of the order of 106 M− 1, indicating that PTP-1B is a powerful binder of these vanadyl metal complexes in vivo[19, 21]. Understanding the inhibition of PTP-1B overexpression by vanadium complexes will be aided by the binding constant (Kb) obtained for PTP-1B with various vanadium metal complexes, which will aid in explaining the Vanadium complexes' glucose (serum) reducing properties. Binding and enzyme kinetic studies are performed on PTP-1B enzyme with these vanadium metal complexes using Spectrophotometer and Fluorescence spectrometry [22–25].
Materials and Reagents
In our lab, vanadium metal complexes (abbreviated as [6-B], [6-imi], [6-me-imi], and [6-et-imi]) are prepared and characterized, as shown in Fig. 1 Enzyme PTP-1B was obtained from Cell Sciences, Newburyport, MA 01950, USA. Solvents like DMSO, Methanol, Finar's analytical reagent grade tris buffer was used without further purification. SRL provided analytical reagent grade p-nitrophenylphosphate (pNPP).
Instruments
Spectrophotometer (EPOCH-BioTek, SN 1701311) 96 microwell plate reader.
Methods
PTP-1B binding studies: protocol used [23]
PTP-1B's fluorescence spectra were taken in both the absence and presence of increasingly concentrated vanadium metal complexes. The buffer solution contained PTP-1B (10 mM tris at pH 7.5). The PTP-1B was excited at 282 nm with an excitation and emission slit width of 5 nm, and the emission spectra of fluorescence were recorded in the wavelength range 292–400 nm, and maximum emission found at 338 nm. By manually adding different concentrations of vanadium metal complexes to the 2 ml of 0.05 µM PTP-1B solution, the fluorescence titrations were completed.
Table 1 shows the experimental data for [6-me-imi] with PTP-1B as a representative case. Tables S1 and S2 (SI) show the remaining experimental data. Using the Orgin software, a plot is drawn between the change in intensities (I) and the wavelengths (λ). For different concentrations of metal complexes, similar graphs are generated and overlapped for better comparison (Fig. 2). The remaining figures are shown in Figures S1 and S3 (SI).
|
S.No |
Concentration of Enzyme (µM) |
Conc of metal complex[C] (µM) |
Log[C] |
Intensity(I)* From experiment |
I0 /I |
I0-I / I |
Log I0-I / I |
|---|---|---|---|---|---|---|---|
|
1 |
0.05 |
0.0049 |
-2.3042 |
702.510 |
1.1917 |
0.1917 |
-0.71722 |
|
2 |
0.05 |
0.0098 |
-2.0054 |
660.919 |
1.2667 |
0.2667 |
-0.57387 |
|
3 |
0.05 |
0.0147 |
-1.8314 |
641.028 |
1.3060 |
0.3060 |
-0.51417 |
|
4 |
0.05 |
0.0195 |
-1.7086 |
584.021 |
1.4335 |
0.4335 |
-0.36295 |
|
5 |
0.05 |
0.0243 |
-1.6138 |
514.359 |
1.6277 |
0.6277 |
-0.20224 |
|
6 |
0.05 |
0.0290 |
-1.5367 |
491.712 |
1.7026 |
0.7026 |
-0.15324 |
Note: Free PTP-1B has an intensity (I 0 ) of 837.27, which is constant at a specific volume of PTP-1B. (~ 5 µL) at λmax = 338nm *Intensities observed at λmax = 338nm alone are given in Table 1.
The graph reveals that pure enzyme (represented by the broken line) exhibits the highest intensity. When metal complex is added, the intensity decreases (is quenched). When a metal complex is added in greater concentration, the intensity is reduced more. This type of experimental behavior suggests that enzymes and metal complexes interact. The shift in the position of λmax (Red shift / Blue shift) corresponds to chromospheres molecule changes [26].
Enzyme Kinetics
Experimental verification of the inhibitory activities of vanadium metal complexes prepared for the purpose of the inhibiting the overactive PTP-1B enzyme. Inorganic phosphate is liberated from the rest of the organic molecule by the hydrolysis of organic-phosphate compounds, which is catalysed by phosphatases. (Fig. 3)
In this study, the synthetic substrate used is Para nitro phenyl phosphate (pNPP). The pNPP hydrolysis in the presence of PTP-1B (PTPase) will be studied and the corresponding reaction is shown in Fig. 4. pNPP is colorless substrate that hydrolyses to p-Nitro phenol. The phenolate ion (pKa 7.2) is soluble and yellow in color. This color is conveniently measured at 405nm on a spectrophotometer.
The purpose of this experiment is to evaluate the reaction rate. Since both compounds (reactants and products) are colorless, we can’t measure directly. Established procedure is to add NaOH. The benefits of adding NaOH is twofold, i) it stops enzyme activity (reaction termination) ii) it generate yellow color (due to p-Nitro phenolate formation) which has maximum absorption at 405nm. Measuring the A405 of the product (p-Nitro phenolate) is as good as measuring the quantity or concentration of p-Nitro phenol produced in the reaction before addition of NaOH.
Reaction conditions optimization
In order to create an effective enzyme assay, the optimal conditions for the specific reaction must be evaluated in order to yield the most products. Maximum absorption λmax, maximum temperature, pH, the concentration of reactants, the concentration of enzyme, the concentration of inhibitor, and so on have all been used to determine various optimal conditions for the reaction "hydrolysis of pNPP by PTP-1B enzyme" in order to obtain a maximum product of maximum OD. The optimal conditions for "hydrolysis of pNPP by PTP-1B enzyme" are as follows: λmax = 405nm, pH = 7.5, T = 27oC, [E] = 40nM, [S] = 15mM, [I] = 10µm. (The detailed procedure for optimization is given in supplementary file) Once the optimal conditions for pNPP catalytic activity have been determined, the enzyme kinetics can be investigated. The protocol was carried out exactly as described in previous publications [27–29].
The procedure used to hydrolyze pNPP in the presence of PTP-1B.
The substrate (pNPP) was prepared and placed in various test tubes at different concentrations (5, 7.5, 15, 30, and 50 mM). Each test tube contained a fixed amount of the enzyme (40 nM), which was then diluted in a buffer (tris pH = 7.5) and added. After that, each test tube was incubated for 30 minutes at 27°C. The assays were terminated by the addition of 5µL of 10 M NaOH. The absorbance at 405 nm was measured using a 96-well microplate reader, and the recorded values were used as "soft data" (Table 2). The same soft data was generated twice more in the same manner. Soft data produced by the microplate reader includes the absorbance or the optical density at various time intervals for various substrate concentrations. A straight line was obtained from plotting absorbance versus time using the Origin programme. Reactions with various substrate concentrations also produced similar types of lines, which were all plotted on a single graph (Fig. 5). The rates of the reaction at various substrate concentrations were calculated from these plots. Table 3 lists the calculated rate constants. Figure 6 shows Michaelis-Menten (M-M) plots (rectangular hyperbolas) created with these various rate constants for various substrate concentrations. This graph was used to calculate the values of Vmax and Km. Figure 7 depicts the Line Weaver-Burk (L-B) plots (1/rate Vs 1/[S]) generated from Table 4 data. L-B plots, also known as double reciprocal plots, are the most commonly used linearization method and provide the most accurate values for Vmax and Km [30, 31].
Table 2 Values of absorbance for the PTP-1B enzyme's hydrolysis of the substrate (pNPP) were acquired by a 96-well plate. (Buffer= Tris pH=7.5, T = 270C, λmax=405nm, E=40nM)
|
Time (minutes) |
Substrate concentration (pNPP) 5mM 7.5mM 15mM 30mM 50mM |
||||||||||||||||||||||||||||||||||||
|
|
|||||||||||||||||||||||||||||||||||||
|
|
|
[pNPP] (mM) |
Reaction rate (V) (milliMole minute− 1) |
|---|---|
|
0 |
0 |
|
5 |
0.0127 |
|
7.5 |
0.0175 |
|
15 |
0.0286 |
|
30 |
0.0331 |
|
50 |
0.0356 |
Values are obtained from the graph (Fig. 2)
Table 4 [S] and [V] reciprocal values* for pNPP hydrolysis by PTP-1B
|
1/[S] |
1/V |
|
0.20 |
78.94 |
|
0.13 |
57.69 |
|
0.06 |
37.39 |
|
0.03 |
30.06 |
|
0.02 |
28.03 |
Table 3 was used to get the values for [S] and V.
*These values are used for plotting L-B plots.
The procedure used to hydrolyze pNPP in the presence of an inhibitor and PTP-1B
While maintaining the other conditions mentioned in section above, this reaction was conducted in the presence of an inhibitor at a fixed concentration (5 µM) (Fig. 8). Table 5 contains a list of the obtained OD values with time intervals. To calculate the rate of reaction as previously described, a Time Vs OD graph is drawn (Fig. 9). The M-M and L-B plots, which were also produced from Tables 6 and 7, are represented in Figs. 10 and 11, respectively. The same experiment was repeated with various inhibitor concentrations. ([6-me-imi] = 0.5µM, 2.5µM, and 5µM). Table 8 shows the OD values obtained over time at various inhibitor concentrations. Figures 12 and 13 displayed the obtained M-M and L-B plots.
After approximately 30 minutes of incubation at 27°C, the assays were terminated by the addition of 5µL of 10 M NaOH. At 405nm, absorbance was measured using a microplate reader. Table 5 shows the obtained OD values with time intervals.
Table 5 Values of absorbance for the PTP-1B enzyme's hydrolysis of the substrate in the presence of the inhibitor [6-me imi] were recorded using a microplate reader. (Buffer = tris pH=7.5, T = 270C, E=0.4μl, [I] = 10μM)
|
Time (minutes) |
substrate concentration(pNPP) 5mM 7.5mM 15mM 30mM 50mM |
||||||||||||||||||||||||
|
|
|||||||||||||||||||||||||
|
|
|
[pNPP] (mM) |
Rate of reaction (milliMoles minutes− 1) |
|---|---|
|
0 |
0 |
|
5 |
0.005 |
|
7.5 |
0.006 |
|
15 |
0.011 |
|
30 |
0.019 |
|
50 |
0.026 |
Table 7 Rate reciprocal values at different substrate reciprocal values in presence of Inhibitor
|
1 / S |
1 / V |
|
0.20 |
187.5 |
|
0.133 |
146.34 |
|
0.066 |
87.46 |
|
0.033 |
50.08 |
|
0.02 |
38.56 |
|
[I] |
[S] = 5 mM |
7.5 mM |
15 mM |
30 mM |
50 mM |
|---|---|---|---|---|---|
|
0.1 |
0.102 |
0.122 |
0.196 |
0.298 |
0.33 |
|
0.5 |
0.1 |
0.113 |
0.183 |
0.248 |
0.298 |
|
2.5 |
0.085 |
0.101 |
0.187 |
0.194 |
0.271 |
|
5 |
0.053 |
0.066 |
0.134 |
0.174 |
0.228 |
| Termination of 20 minutes of interval | |||||
|
[I] |
[S] = 5 mM |
7.5 mM |
15 mM |
30 mM |
50 mM |
|---|---|---|---|---|---|
|
0.1 |
0.233 |
0.269 |
0.441 |
0.595 |
0.601 |
|
0.5 |
0.223 |
0.246 |
0.402 |
0.585 |
0.595 |
|
2.5 |
0.168 |
0.234 |
0.368 |
0.441 |
0.54 |
|
5 |
0.103 |
0.144 |
0.245 |
0.355 |
0.495 |
| Termination of 30 minutes of interval | |||||
|
[I] |
[S] = 5 mM |
7.5 mM |
15 mM |
30 mM |
50 mM |
|---|---|---|---|---|---|
|
0.1 |
0.33 |
0.47 |
0.75 |
0.92 |
1.05 |
|
0.5 |
0.30 |
0.392 |
0.618 |
0.86 |
1.02 |
|
2.5 |
0.25 |
0.34 |
0.582 |
0.74 |
0.96 |
|
5 |
0.16 |
0.205 |
0.343 |
0.599 |
0.78 |
PTP-1B Enzyme Molecular Docking Studies on Vanadium Metal Complexes
Theoretical confirmation of inhibitors' interactions with the active site of the PTP-1B enzyme (as represented by the vanadium complexes under investigation). Additionally, to ascertain the degree of order of their preferential binding to the active site. All of the vanadium complexes were docked into the protein binding site to determine their potential binding affinity and mode of interaction. The crystal structure of the PTP-1B enzyme was discovered using the Protein Data Bank (PDB) of the Research Collaboration for Structural Bioinformatics (RCSB, http://www.rcsb.org).
Fluorescence studies of PTP-1B binding
A series of parameters (such as Stern-Volmer Quenching constant, quenching rate constant, binding constant, and number of binding sites) are evaluated using PTP-1B fluorescence intensities at 338 nm to define the interaction between PTP-1B and metal complexes.
The quenching rate constant (Kq) is calculated as follows
Using experimental data, a graph is plotted between [Io/I] Vs [Q]. Figure 14 depicts the interaction of [6-me- imi] with PTP-1B. The remaining figures are shown in Fig. S2 and S5 (SI).
The stern-volmer [32] equation describes fluorescence quenching.
Io / I = 1 + Ksv [Q] = 1 + Kq Ʈo [Q]
Where Io = enzyme fluorescence intensity
I = Fluorescence intensity of an enzyme in the presence of a metal complex
Ksv = quenching stern volmer Constant
[Q] = quencher concentration (metal complexes)
kq = enzyme bimolecular quenching rate constant
Ʈo = In the absence of a quencher, the average life time of biomolecules is 10− 8s[33].
A straight line is obtained by plotting data from Table1 on a graph between I0 / I Vs [C].
The slope of the graph, known as Ksv, is calculated (stern-volmer constant). In this case, the slope is 26.12, the intercept is 1.18, and the Ksv is 26.12 × 106 M− 1.
(Kq) = Ksv / Ʈ0, where (Ʈ0 = 10− 8 = 10− 8 is the average life of PTP-1B in the absence of a quencher; Thus, Kq = 26.12 ×1014M-1 S-1
Calculation of the number of binding sites and the binding constants (Kb)
The interactions between the [6-me-imi] complex and PTP-1B are depicted in Fig. 15 on a graph between log (Io-I / I) and log [Q]. The remaining figures are shown in Figures S3 and S
Using the data from Table 1, double logarithmic plots of log (complex) and log (Io-I) / I were generated. The slope and intercept values are computed. The slope is used to calculate the number of enzyme binding sites, and the intercept is used to calculate the binding constant (Kb)
Log of Kb = 0.87
Kb=8.7 × 106 M− 1
Slope = 0. 60
As a result, the number of binding sites ~ 1. The number of binding sites [34] indicates the number of independent binding sites for complexes on PTP-1B.
Similar experimental results were obtained for different metal complexes, and Table 9 shows the associated Kq, Kb, and n with their interaction with PTP-1B.
Conclusion: These findings indicate that a variety of metal complexes do bind to the PTP-1B enzyme. However the binding ability differs and the order of binding found to be as follows.
[6-etimi] > [6-meimi] > [6-imi] > [6-B]
|
S.No |
Complex |
Kq (1014M−1) |
Kb (106M−1) |
n |
|---|---|---|---|---|
|
1 |
[6-B] |
26.36 |
7.58 |
0.616 |
|
2 |
[6-imi] |
17.33 |
7.76 |
0.89 |
|
3 |
[6-me-imi] |
22.12 |
8.7 |
0.74 |
|
4 |
[6-et-imi] |
18.58 |
9.09 |
0.77 |
Kinetics of Enzyme
Determination of Vmax and Km for hydrolysis of pNPP in the presence of PTP-1B
From M-M plots
The reaction rates are plotted against [S] to obtain the well-known M-M plots of the rectangular hyperbolic curve. The rate of reaction increases with an increase in [S] up to a certain concentration, after which the rate of reaction remains constant even with an increase in [S]. The rates are taken on the y-axis while different [S] are on the x-axis. The Vmax is the point on the y-axis where the reaction rate becomes saturated, as shown in Fig. 2. Vmax denotes the rate at which the total enzyme is in the [E-S] complex form. It is the maximum experimentally achievable rate. Km is defined as the concentration of the substrate at half of the Vmax. The reaction velocity and km are related in the following way.
V= \(\frac{{\text{V}}_{\text{m}\text{a}\text{x}}}{1+\frac{{\text{K}}_{\text{m}}}{\left[\text{S}\right]}}\) (1)
The Lineweaver-Burk (double reciprocal) plot, shown in equation-1, is one of the most common methods for precisely fitting experimental data.
From L-B plots
The L-B plot's straight line corresponds to the following equation. When a graph is drawn between \(\frac{1}{\text{V}}\) Vs\(\frac{1}{\left[\text{S}\right]}\)
The above equation is written as Y = mX + C, where the Y-intercept equal to\(\frac{1}{{\text{V}}_{\text{m}\text{a}\text{x}}}\), the X-intercept equal to \(\frac{-1}{{\text{K}}_{\text{m}}}\)and, slope equal to \(\frac{{\text{K}}_{\text{m}}}{{\text{V}}_{\text{m}\text{a}\text{x}}}\). Table 10 shows the Vmax and Km values obtained from Michaelis-Menten and Lineweaver-Burk plots.
pNPP hydrolysis Vmax(app) and Km(app) calculations in the presence of PTP-1B and inhibitor
In the presence of inhibitors, Vmax and Km are referred to as Vmax(apparent) and Km (apparent). The steps for calculating Vmax(apparent) and Km(apparent) using M-M and L-B plots are the same as in the previous section. The measured Vmax(apparent) and Km(apparent) values are shown in Table 11. When the values in Table 12 are compared, it is clear that Vmax is unaffected while Km values are. As a result, because the Vanadium complex [6-meimi] influences the rate of the reaction, it is an inhibitor, and the type of inhibition is competitive. Table 13 shows the measured values of Km(app) and Vmax(app) for various Vanadium complexes. All of the Vanadium complexes studied have unaffected Vmax and affected Km values, indicating competitive inhibition.
Calculation of IC50
A substance's half-maximal inhibitory concentration (IC50) is a measurement of its ability to inhibit a specific biological or biochemical function. The amount of a specific drug or inhibitor required to inhibit an enzyme process by half is indicated by this quantitative measure. To calculate IC50, a series of dose-response data were generated. The OD and percentage inhibition were calculated using various inhibitor concentrations (Table 14). A graph is created by plotting Log [I] versus % Inhibition (on the x-axis) (on the y-axis). The graph is used to calculate 50% maximal inhibition (IC50) using the following equation (Fig. 16).
IC50 = Maximum inhibition – 50% (Maximum inhibition - Minimum inhibition)
% Inhibition= \(\frac{\text{O}\text{D} \text{W}\text{i}\text{t}\text{h}\text{o}\text{u}\text{t} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{o}\text{r} –\text{W}\text{i}\text{t}\text{h} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{r}}{\text{W}\text{i}\text{t}\text{h}\text{o}\text{u}\text{t} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{o}\text{r}}\)×100 (3)
Inhibition constant calculation (Ki)
The inhibition constants using the formula, Ki was calculated for each inhibitor [35].
Where Km is the Michaelis-Menten constant without the inhibitor, [S] is the substrate concentration and Ki is the inhibition constant (dissociation constant), IC50 is the half maximal inhibitory concentration,
We obtained Ki = 4.37 \(\times\)10− 6M or Ki = 4.37 µM.
In the presence of I, reaction rates are slower than in the absence of I. This finding (Table 15) indicates that the vanadium metal complex [6-meimi] has an inhibitory effect. When the values of Vmax(apparent) and Km(apparent) are compared to Vmax and Km, it is discovered that Vmax and Vmax(app) are the same, but Km and Km(app) are not. This behavior suggests that binding is competitive [36]. It is well known that in the presence of a competitive inhibitor, the reaction can achieve a Vmax(app) value equal to the Vmax value, but this necessitates a higher substrate concentration. In other words, Vmax and Vmax(app) should be the same. Km(app) differs from Km in that it requires a greater concentration of substrate. As a result, as observed in our current investigation, Km(app) values will always be higher [37] than Km values. According to these theoretical equations, a competitive inhibitor influences only Km and not Vmax. In other words, whether I is present or not, Vmax values are the same. Km, on the other hand, is sensitive in the presence of I. The experimental values for pNPP hydrolysis by PTP-1B enzyme in the presence or absence of I show nearly identical Vmax values but different Km values, implying competitive inhibition for all vanadium complexes under consideration.
Use of Ration of\(\frac{{\text{K}}_{\text{i}}}{{\text{K}}_{\text{m}}}\)
The \(\frac{{\text{K}}_{\text{i}}}{{\text{K}}_{\text{m}}}\) m ratio directly corresponds to the binding constant for I and S towards E.
It is a well known fact that if
- \(\frac{{\text{K}}_{\text{i}}}{{\text{K}}_{\text{m}}}\) > 1 (S tightly binds to E, and I has little effect on kinetics.)
- \(\frac{{\text{K}}_{\text{i}}}{{\text{K}}_{\text{m}}}\) = 1 (I and S have the same affinity for E.)
- \(\frac{{\text{K}}_{\text{i}}}{{\text{K}}_{\text{m}}}\) < 1 (I tightly binds to E, and I will have a greater influence on the kinetics.)
In the current studies, all of the inhibitors tested had a \(\frac{{\text{K}}_{\text{i}}}{{\text{K}}_{\text{m}}}\) ratio less than one. All other inhibitors produced similar graphs, and their Ki values are listed in Table 16.
Type of Inhibition exerted on PTP-1B by inhibitor
The following experiments were carried out to learn more about the type of inhibition [38]. Different inhibitor concentrations (0.5M, 2.5M, 5M, 10M, and 25M) were prepared, as were various S concentrations (5mM, 7.5mM, 15mM 30mM and 50mM). The concentration of enzyme was held constant at 40nM. A grid of solutions at each substrate concentration was created by varying inhibitor concentrations at a constant (40nM) enzyme concentration. Absorbencies were measured after adding NaOH to reaction wells at 0min, 10min, 20min, and 30min intervals. A microplate reader was used to measure absorbance at 405nm.
L-B plots were created and are depicted in Fig. 13. A family of lines intersected on the 1/V axis in this plot, indicating competitive inhibition.
|
Source |
Vmax(mM sec− 1) |
Km(mM) |
|---|---|---|
|
M-M plot |
0.045 |
11.0 |
|
L-B plot |
0.05 |
14.5 |
|
Source |
Vmax(apparent) mM sec− 1) |
Km (apparent) (mM) |
|---|---|---|
|
M-M plot |
0.0521 |
50.207 |
|
L-B plot |
0.038 |
55.55 |
|
Complex |
Vmax(app) (Vmax) (mM sec-1) |
Km (app) (Km) (mM) |
|---|---|---|
|
[6-meimi] |
0.038 (0.050) |
55.55 (14.5) |
| (Values in parenthesis are without inhibitor) | ||
|
Complex |
Vmax(mM sec-1) |
Vmax(app) (mM sec-1) |
Km(mM) |
Km(app) (mM) |
|---|---|---|---|---|
|
Pure enzyme |
0.05 |
14.5 |
||
|
[6-B] |
- |
0.039 |
- |
55.55 |
|
[6-imi] |
- |
0.037 |
- |
32.25 |
|
[6-me-imi] |
- |
0.038 |
- |
55.55 |
|
[6-et-imi] |
- |
0.050 |
- |
45.45 |
|
[I]µM |
log10[I] |
OD |
% Inhibitiion |
|---|---|---|---|
|
0.5 |
0.301 |
0.75 |
11.55 |
|
2.5 |
0.397 |
0.61 |
27.12 |
|
5 |
0.698 |
0.58 |
31.60 |
|
10 |
1 |
0.34 |
59.55 |
|
25 |
1.397 |
0.05 |
94.10 |
|
[pNPP] (mM) |
Rate of the reaction (mM sec− 1) |
|
|---|---|---|
|
In absence of I |
In presence of I |
|
|
0 |
0 |
0 |
|
5 |
0.0127 |
0.0049 |
|
7.5 |
0.0175 |
0.0076 |
|
15 |
0.0286 |
0.0110 |
|
30 |
0.0331 |
0.02035 |
|
50 |
0.0356 |
0.0289 |
|
S.No. |
Inhibitors |
Vmax(app) |
Km(app) |
dG(KJ) |
Ki(µM) |
\(\frac{{\text{K}}_{\text{i}}}{{\text{K}}_{\text{m}}}\) |
IC50 |
Mode of inhibition |
|---|---|---|---|---|---|---|---|---|
|
1 |
[6-B] |
0.039 |
55.55 |
-7.769 |
4.68 |
0.32 |
9.54 |
Competitive |
|
2 |
[6-imi] |
0.037 |
32.25 |
-7.342 |
4.48 |
0.30 |
9.12 |
Competitive |
|
3 |
[6-me-imi] |
0.038 |
55.55 |
-6.915 |
4.37 |
0.30 |
8.91 |
Competitive |
|
4 |
[6-et-imi] |
0.050 |
45.45 |
-7.471 |
3.47 |
0.23 |
7.07 |
Competitive |
Ptp-1b Molecular Modelling Studies
The Molegro virtual Docker [MVD] was used to dock the various metal complexes into the active site of the PTP-1B[39]. MVD has a higher accuracy than other stock softwares such as Glide, FlexX and Sorflex [40, 41]. The structure of a protein loaded onto an MVD platform for the purpose of identifying potential active sites or cavities [42]. With a grid resolution of 0.8A, five binding cavities were detected.
An examination of the cavities displayed (Fig. 17) revealed that the binding pocket-1 is the primary cavity surrounded by the catalytic triad (Lys 116,Lys 120 and Arg 221). Protein tyrosine phosphatase-1B (PTP-1B) is a monomer with 321 amino acids and one unique protein chain A composed of alpha helix, beta strands, and coils[43, 44]. Almost 4 vanadium derivatives were synthesised and tested for bioinformatics and in vivo research. The scoring functions were reported using five binding cavities obtained from the site map. Using scoring mechanisms like the Mol Dock score, the lead targets were located [45]. Plant score functionality was used to further examine the data's accuracy. The optimal binding mode of a molecule (compound) to the active site of a macromolecule is interpreted in order to obtain more potent compounds as inhibitors. Figure 18 depicts the binding pose of each compound in their highly bound cavity. Figure 19 depicts images of selected compound ([6-meimi]) secondary structures, hydrogen bond interactions, and detected cavities after docking.
According to the dock score analysis for 2HNP, the molecules have a stronger affinity for cavity site-1 (Vol = 84.48). Compounds at site-1's mol dock and plant scores are displayed in Table 17. Three hydrogen bonds were created between the 2HNP molecule and the amino acid residues Lys 116, Lys 120, and Arg 221 in the presence of [6-meimi]. As a result, [6-meimi] had a higher binding capacity of 2HNP during docking than the remaining vanadium complexes.
The following is the decreasing order of inhibitor preferential binding
[6-et-imi] > [6-me-imi] > [6-imi] > [6-B]
The top discovered vanadium complex ([6-et-imi]) had a binding affinity of -79.00 based on a PLANTS score equal to -129.87Mol dock score. Based on this model of potential inhibition, this study suggests that the [6-meimi] complex discovered in this study can be used for future laboratory studies.
|
S.No. |
Complex |
Plants Score |
MolDock Score |
Rerank Score |
|---|---|---|---|---|
|
1 |
[6-B] |
-60.93 |
-122.59 |
-63.77 |
|
2 |
[6-imi] |
-73.35 |
-146.49 |
-50.27 |
|
3 |
[6-me-imi] |
-77.16 |
-156.14 |
-110.16 |
|
4 |
[6-et-imi] |
-79.00 |
-129.87 |
-94.32 |
In conclusion one can say that enzyme PTP-1B enhances the rate of hydrolysis of p-nitrophenylphosphate. But in the presence of inhibitors, (V-Metal complexes) the enzyme PTP-1B is not effective in increasing the rate of hydrolysis of p-nitrophenylphosphate. In other words, the activity of enzyme is inhibited by the inhibitor. Further, the inhibition is competitive in nature, i.e. the inhibitor binds to the same active site where substrate binds as shown below. There is a competition between S and I for active site of enzyme. An earlier study on PTP-1B in presence of different vanadium complexes also reveals the mechanism of competitive inhibition for hydrolysis of pNPP.
This inhibiting mechanism is based on a simple thermodynamic principle: Fig. 20 depicts two parallel equilibria, one between the enzyme and the inhibitor and one between the enzyme and the substrate. Because they compete with the substrate for overlapping binding sites on the enzyme, competitive inhibitors frequently resemble the substrate in chemical structure, shape, and polarity pattern.
The inhibitory effect of various vanadium metal complexes on the PTP-1B enzyme was studied and found to be excellent. We also investigated the interactions of vanadium metal complexes with PTP-1B. To validate our findings, we used molecular modelling studies. For various vanadium complexes, the order of inhibition (Kinetics), the order of binding (binding constant), and the results of molecular modeling all agree.
The inhibitory activities of various vanadium metal complexes on PTP-1B enzyme are found to be in the following decreasing order:
i) Enzyme Kinetics studies: The decreasing order of their efficiency as inhibition found to be
[6-et-imi] > [6-me-imi] > [6-imi] > [6-B]
ii) PTP-1B binding order: Below is the decreasing order of binding (Kb) of metal complexes with PTP-1B.
[6-et-imi] > [6-me-imi] > [6-imi] > [6-B]
iii) Molecular Modeling studies: The binding constants for PTP-1B are listed in decreasing order [6-et-imi] > [6-me-imi] > [6-imi] > [6-B]
Final statement
According to the findings of the preceding experiments, vanadium metal complexes aid in the reduction of serum glucose levels in animals. They function as inhibitors of PTP-1B enzyme overexpression. The inhibition is competitive in nature. Among these complexes, [6-et-imi] was discovered to be the most promising for future research. However, more research is needed to determine its impact on humans. Pertinent to mention that vanadium salt was used as therapeutic agent by ancient people to treat DM in human beings. It is well established that vanadium has tremendous effect on reducing serum sugar level. The scientific way of explaining its effect is to be addressed.
Acknowledgments
The authors would like to thank the Head of the Chemistry Department at Osmania University in India for providing the resources required to conduct the current study, as well as DST in India for financial assistance. We are grateful to the University Grants Commission, India, for funding under the Basic Scientific Research Fellowship (BSR) scheme with file number F.4-1/2006(BSR)11-38/2008(BSR)/2013-2014/01.
Conflict of interest
There is no conflict of interest declared by the authors.
Author contributions
AyubShaik: Validation; Investigation Formal analysis; Writing Original Draft. Data curation. Vani Kondaparthy: Data curation; Formal analysis. Aliya Begum: Writing - Review & Editing.
Ameena Husain: Investigation; Methodology. Deva Das Manwal: Conceptualization; Methodology; Validation; Resources; Supervision.
Supplementary information
The data that supports the findings of this study can be found in the article's supplementary file.
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