Enzyme PTP-1B Inhibition Studies by Vanadium Metal Complexes: a Kinetic Approach

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 overexpressed in patients with diabetes and obesity, suggesting that inhibiting PTP-1B may be a useful technique in their care. There are no clinically used PTP-1B inhibitors, despite the fact that numerous naturally occurring PTP-1B inhibitors have demonstrated great therapeutic promise. This is most likely due to their low activity or lack of selectivity. It is still important to look for more effective and focused PTP-1B inhibitors. A few organovanadium metal complexes were synthesized and 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.


Introduction
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 permeation, tissue uptake, and effectiveness, and reduce toxicity [1]. 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][4][5][6][7][8][9][10][11][12]. Various studies on diabetic patients revealed that PTP activity increases in type II diabetic (T2D) patients along with increased levels of the PTP-1B protein. Inhibition of the activity of the PTP-1B enzyme is thought to hold 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, liraglutide, etc.
PTP-1B is an active player in T2D patients. It is a unique enzyme that is included in the protein tyrosine phosphatase (PTP) family [13]. It is encoded by the PTPN1 gene in humans. It is a negative regulator of the insulin [14,15] signaling pathway, and it is considered to be an important therapeutic target for the treatment of T2D patients.
In view of the inhibitory action of vanadium metal complexes, many research groups are in the process of synthesizing novel vanadium complexes with the hope of getting an ideal metal complex to inhibit the PTP-1 [16][17][18], 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 [19,20], from vanadium metal and acetylacetone(4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione). Imidazole, 2-methyl-imidazole, and 1 3 2-ethyl-imidazole auxiliary ligands were employed. Using analytical techniques, metal complexes were characterized.
Before conducting inhibitory studies on the 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. The interaction of complexes with albumins can be investigated by a number of techniques like UV-visible, fluorescence spectral techniques, and viscometry, to name a few common ones. The binding constant (K b ) values from absorption studies and the Stern-Volmer constant (K SV ) values from fluorescence quenching studies are indicative of the binding strength of complexes. It has also been established that interactions between proteins and metal complexes are thought to be relatively strong if the K SV and K b between protein and drug are found to be in the order of 10 5 M −1 and 10 4 -10 5 M −1 , respectively [21][22][23]. The experimentally determined K b for the various vanadyl metal complexes under study is of the order of 10 5 M −1 , indicating that PTP-1B is a powerful binder of these vanadyl metal complexes in vivo [21]. Understanding the inhibition of PTP-1B overexpression by vanadium complexes will be aided by the K b 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 the PTP-1B enzyme with these vanadium metal complexes using spectrophotometer and fluorescence spectrometry [3,[24][25][26].

PTP-1B Binding Studies: Protocol Used
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 of 292-400 nm, with the 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.

Enzyme Kinetics
Experimental verification of the inhibitory activities of vanadium metal complexes was made for the purpose of 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 catalyzed by phosphatases (Fig. 2).
In this study, the synthetic substrate used is para-nitrophenyl phosphate (pNPP). The pNPP hydrolysis in the presence of PTP-1B (PTPase) will be studied, and the corresponding reaction is shown in Fig. 3.pNPP is a colorless substrate that hydrolyzes p-nitrophenol. The phenolate ion (pK a 7.2) is soluble and yellow in color. This color is conveniently measured at 405 nm on a spectrophotometer.
The purpose of this experiment is to evaluate the reaction rate. Since both compounds (reactants and products) are colorless, we cannot measure directly. The established procedure is to add NaOH. The benefits of adding NaOH are twofold, (i) it stops enzyme activity (reaction termination) and (ii) it generates a yellow color (due to p-nitrophenolate formation), which has maximum absorption at 405 nm. Measuring the A 405 of the product (p-nitrophenolate) is as good as measuring the quantity or concentration of p-nitrophenol produced in the reaction before the addition of NaOH.

Reaction Condition 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 λ max , 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 optical density (OD). The optimal conditions for hydrolysis of pNPP by PTP-1B enzyme are as follows: λ max =405 nm, pH = 7.5, T = 27°C, [E] = 40 nM, [S] = 15mM, [I] = 10 μm. (The detailed procedure for optimization is given in the Supplementary Information) 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 [12,27,28].  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 min at 27°C. The assays were terminated by the addition of 5 μl 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 1). The same soft data were generated twice more in the same manner. Soft data produced by the microplate reader include the absorbance or the optical density at various time intervals for various substrate concentrations.

The Procedure Used to Hydrolyze pNPP in the Presence of an Inhibitor and PTP-1B
While maintaining the other conditions mentioned in the previous section, this reaction was conducted in the presence of an inhibitor at a fixed concentration (5 μM) (Fig. 4).
After approximately 30 min of incubation at 27°C, the assays were terminated by the addition of 5 μl of 10 M NaOH. At 405 nm, absorbance was measured using a microplate reader.

PTP-1B Enzyme Molecular Docking Studies on Vanadium Metal Complexes
The crystal structure of human protein tyrosine phosphatase 1B (PDB id:4Y14) was downloaded from the Protein Data Bank (www. rcsb. org) and prepared using the Protein Preparation Wizard and the OPLS 2005 force field in the Schrodinger suite. ChemDraw was used to build and optimize metal complexes. Using Autogrid, a grid was created around the binding site. Autodock 4.2 was used to dock these into protein-binding sites. The DS 4.0 Visualizer was used to generate the molecular interaction diagrams. Table 2 shows the experimental data for [6-me-imi], with PTP-1B as a representative case. Tables S1, S2, and S3 (SI) show the remaining experimental data. Using the Origin software, a plot is drawn between the change in intensities (I) and the wavelengths (λ). For different concentrations of metal complexes, similar graphs are  The remaining figures are shown in Figs. S1, S3, and S7 (SI). The graph reveals that pure enzyme (represented by the red line) exhibits the highest intensity. When a metal complex is added, the intensity decreases (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 λ max (red shift/blue shift) corresponds to molecule changes.

Fluorescence Studies of PTP-1B Binding
A series of parameters (such as the Stern-Volmer quenching constant, quenching rate constant, binding constant, and a 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 Is Calculated as Follows
Using experimental data, a graph is plotted between [I o /I] vs. [Q]. Where I o is the enzyme fluorescence intensity, I is the fluorescence intensity of an enzyme in the presence of a metal complex, K SV is the quenching Stern-Volmer constant, [Q] is the quencher concentration (metal complexes), and K q is the enzyme bimolecular quenching rate constant. T o shows that in the absence of a quencher, the average lifetime of biomolecules is 10 −8 s [22].
A straight line is obtained by plotting data from Table 2 on a graph between I 0 /I vs. [C].
The slope of the graph gives the K SV . In this case, the slope is 26.12, the intercept is 1.18, and the K SV is 26.12 × 106 M −1 . The K SV values with 10 5 M −1 are considered to be indicative of relatively strong interaction between any protein and metal complexes. If the K SV values are divided by 10 −8 s, the obtained values are quenching rate constants. If the K q values obtained are in the order 10 15 M −1 s −1 , we can conclude that the complexation between PTP-1B and vanadium is through a static, single type of quenching mechanism.

Calculation of the Number of Binding Sites and the Binding Constants
The interactions between the [6-me-imi] complex and PTP-1B are depicted in Fig. 7  Using the data from Table 2, double logarithmic plots of log (complex) and log (I o -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 (K b ) As a result, the number of binding sites is ~1. This specifies the number of independent binding sites for complexes on PTP-1B [30].
Similar experimental results were obtained for different metal complexes, and Table 3 shows the associated K q , K b , and n with their interaction with PTP-1B.

Conclusion
These findings show that our vanadium complexes bind to the PTP-1B enzyme. However, their binding propensities differ in the following order.

Kinetics of Enzyme
A straight line was obtained by plotting absorbance vs. time using the Origin software. Reactions with various substrate concentrations also produced similar types of lines, which were all plotted on a single graph (Fig. 8). The rates of the reaction at various substrate concentrations were calculated from these plots. Table 4 lists the calculated rate constants. Figure 9 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 V max and K m . Figure 10 depicts the Lineweaver-Burk (L-B) plots (1/rate vs. 1/[S]) generated from Table S7 data. L-B plots, also known as double reciprocal plots, are the most commonly used linearization method and provide the most accurate values for V max and K m [31,32].   The Lineweaver-Burk (double reciprocal) plot, shown in Eq. (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 1 V vs. 1

[S]
(2)  Table S7, a pure enzyme Lineweaver-Burk plot was created    Table 6 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. 11). The M-M and L-B plots, which were also produced from Tables S8  and Table 6, are represented in Figs. 12 and 13, respectively. The same experiment was repeated with various inhibitor concentrations. ([6-me-imi] = 0.5 μM, 2.5 μM, and 5 μM) ( Table 7). Table 8 shows the OD values obtained over time at various inhibitor concentrations. The remaining tables are shown in Tables S4, S5, and S6 (SI). Figures 14 and 15 display the obtained M-M and L-B plots. The remaining figures are shown in Figs. S10, S11, S13, S14, S16, and S17 (SI).

pNPP Hydrolysis V max (app) and Km(app) Calculations in the presence of PTP-1B and inhibitor
In the presence of inhibitors, V max and K m are referred to as V max (apparent) and K m (apparent) . The steps for calculating V max (apparent) and K m (apparent) using M-M and L-B plots are the same as in the previous section. The measured V max (apparent) and K m (apparent) values are shown in Table 9. When the values in Table 10 are compared, it is clear that V max is unaffected while K m values are. As a result, because the vanadium

Calculation of IC 50
A substance's half-maximal inhibitory concentration (IC 50 ) 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 IC 50 , a series of dose-response data were generated. The OD and percentage inhibition were calculated using various inhibitor concentrations (Table 12). A graph is created by plotting Log [I] vs. % inhibition (on the x-axis) (on the y-axis). The graph is used to calculate 50% maximal inhibition (IC 50 ) using the following equation (Fig. 16). The remaining figures are shown in Figs. S12, S15, and S18 (SI).

Inhibition Constant Calculation
The inhibition constants using the formula, K i were calculated for each inhibitor [33].  Table 7   Where K m is the Michaelis-Menten constant without the inhibitor, [S] is the substrate concentration, K i is the inhibition constant (dissociation constant), and IC 50 is the half-maximal inhibitory concentration, We obtained K i = 4.37 × 10 −6 M or K i = 4.37 μM.

Discussion
In the presence of I, reaction rates are slower than in the absence of I. This finding (Table 13) indicates that the vanadium metal complex [6-me-imi] has an inhibitory effect. When the values of V max (apparent) and K m (apparent) are compared to V max and K m , it is discovered that V max and V max (app) are the same, but K m and K m (app) are not. This behavior suggests that binding is competitive [34]. It is well known that in the presence of a competitive inhibitor, the reaction can achieve a V max (app) value equal to the V max value, but this requires a higher substrate concentration. In other words, V max and V max (app) should be the same. K m (app) differs from K m in that it requires a greater concentration of substrate. As a result, as observed in our current investigation, K m (app) values will always be higher [35] than K m values. According to these theoretical equations, a competitive inhibitor influences only K m and not V max . In other words, whether I is present or not, V max values are the same. K m , on the other hand, is sensitive in the presence of I. The experimental values for pNPP hydrolysis  In the current studies, all of the inhibitors tested had a K i K m ratio of less than 1. All other inhibitors produced similar graphs, and their K i values are listed in Table 14.

Type of Inhibition Exerted on PTP-1B by Inhibitor
The following experiments were carried out to learn more about the type of inhibition [36][37][38]. Different inhibitor concentrations (0.5 M, 2.5 M, 5 M, 10 M, and 25 M) were prepared, as were various S concentrations (5 mM, 7.5 mM, 15 mM, 30 mM, and 50 mM). The concentration of the enzyme was held constant at 40 nM. A grid of solutions at each substrate concentration was created by varying inhibitor concentrations at a constant (40 nM) enzyme concentration. Absorbencies were measured after adding NaOH to reaction wells at 0 min, 10 min, 20 min, and 30 min intervals. A microplate reader was used to measure absorbance at 405 nm.
L-B plots were created and are depicted in Fig. 15. A family of lines intersected on the 1/V-axis in this plot, indicating competitive inhibition.

PTP-1B Molecular Modeling Studies
To determine the potential binding affinity and mode of interaction, all vanadium complexes were docked into the protein-binding site. The coordinates of the co-crystallized ligand present in the experimental crystal structure of PTP1B were used to select the active site of human protein tyrosine phosphatase 1B, which consists of amino acid residues Asp 181, Phe 182, Cys215, Ser 216, and Gln262 [39][40][41]. Autogrid was used to create a grid around this site. Using Autodock 4.2 [42,43], the metal complexes were docked into protein active sites; this enumerates and identifies suitable conformations of the molecule with minimum binding energy. The vanadium complexes exhibited appreciable hydrophobic π-π interaction and π-cation interaction with active site amino acids Try 46, Asp 48, and Phe 182. As a result, during docking, [6-et-imi] had a higher binding capacity with 2HNP than the other vanadium complexes. This may be attributed to the presence of a larger nonpolar alkyl group (ethyl group), which provides the best fit into the nonpolar binding pocket located on the receptor site. Results have been tabulated in The inhibitor preferential binding is listed in decreasing order.

Conclusion
In conclusion, one can say that the enzyme PTP-1B enhances the rate of hydrolysis of p-nitrophenyl phosphate. However, in the presence of inhibitors (vanadium metal complexes), the enzyme PTP-1B is not effective in increasing the rate of hydrolysis of p-nitrophenyl phosphate. So, we can say that the activity of the enzyme is diminished by the action of the inhibitor. Furthermore, the inhibition is competitive in nature, i.e., the inhibitor binds to the same active site where the substrate binds, as shown below. There is a competition between S and I for the active site of the enzyme. An earlier study on PTP-1B in the presence of different vanadium complexes also revealed the mechanism of competitive inhibition for the hydrolysis of pNPP. This inhibiting mechanism is based on a simple thermodynamic principle: Figure 19 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  19 Depicting competitive type of inhibition findings, we used molecular modeling 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 the PTP-1B enzyme are found to be in the following decreasing order: i) Enzyme kinetics studies: The decreasing order of their efficiency as inhibition was found to be [6-et-

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. It is pertinent to mention that vanadium salt was used as a therapeutic agent by ancient people to treat DM in humans. It is well established that vanadium has a tremendous effect on reducing serum sugar levels. The scientific way of explaining its effect is to be addressed.