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REVEALING THE MECHANISM OF ACTION OF AN ANTI-DIABETIC DRUG VERAPAMILUSING THE MOLECULAR DOCKING APPROACH
Anukampa Pandey
Assistant Professor, Lakshmi Narain College of Professional Studies, Indore
Abstract- Diabetes Mellitus has become a major health issue worldwide presently;
confirmed diabetic patients in India are 67 million. Although some of the underlying causes and cornerstones of type 1 and type 2 diabetes are distinct (i.e. auto immunity and insulin resistance, respectively) but loss of functional- beta cell mass represents a crucial common feature in the pathogenesis of both diabetes types (1&2). Moreover, elevated TXNIP levels have emerged as a critical factor involved in cell dysfunction, cell death, and the resulting development of diabetes and its complications. On the other hand, Anath Shalev et.al in Aug 2014. Unraveled genetic deletion and pharmacological inhibition of cell TXNIP (Thioredoxin Interacting Protein) have been shown to protect against type 1 and type 2diabetes in Mouse. TXNIP is found to play a key role in Diabetes. Verapamil act as a potent drug in the inhibition of TXNIP expression (Figure 3.1). Experimentally, it was found that oral administration of Verapamil not only inhibits TXNIP expression but also reverts a diabetic Mouse into a normal Mouse. One of the possible mechanisms for the reversion of diabetic to non-diabetic mouse is through the inhibited TXNIP-Trx interaction system. This prompted us to hypothesize that Verapamil may directly be involve in regulating the TXNIP- Trx complex formation. Verapamil binding to the complex or the individual chains may regulate concentration of TXNIP. This strongly advocated the study the interaction of Verapamil to the interface of the TXNIP-Trx complex through docking studies. Verapamil was found to be involved in the interaction of the TXNIP-Trx complex. Other natural ligands were also studied and among them Devapamil was observed as a suitable analog of Verapamil.
Keywords: TXNIP, Trx, PPAR’S, VDUP1, CCB, miRNA.
1. INTRODUCTION
Diabetes mellitus (DM) commonly referred to as diabetes has become a major health issue worldwide. At present, confirmed diabetes patients in India are 62 million (IDF, 2015).
Disease is characterized by high blood sugar levels over a prolonged period. Acute complications include diabetic ketoacidosis and non-ketosis hyperosmolar coma. Serious long-term complications include cardiovascular disease, stroke, chronic kidney failure, foot ulcers, and damage to the eyes.
Diabetes is due to either the pancreas not producing enough insulin or the cells of the body not responding properly to the insulin produced. Pancreatic beta-cells are responsible for insulin production, and loss of functional beta-cell mass is now recognized as a critical step in the pathogenesis of both type 1 and type 2 diabetes (wikipedia.org).
1.1 Molecular Targets of Diabetes
The worldwide epidemic of Diabetes mellitus has been stimulating the search for new concepts and targets for the treatment of this incurable Disease. The list of potent targets of Diabetes mellitus includes PPAR’S, ChREBP, FOXO, IAPP, TXNIP (Kaadige, et.al 2010).
Recently, TXNIP is reported to be a potential target for the treatment of Diabetes mellitus (Cha-Molstad, Hyunjoo, et. al.2009).
Thioredoxin1 (Trx1) and Thioredoxin2 (Trx2) are ubiquitously expressed proteins in variety of cells. Trx are highly conserved in many organisms ranging from bacterial organisms to plants and mammals, indicating that the Trx system is essential for cellular survival and function. They control cellular reactive oxygen species by reducing the disulphides into thiol groups. Trx1 is mainly located in cytosol but also translocate to the nucleus and can be secreted from cells under certain circumstances, whereas Trx2 is located only in mitochondria (Eizirik et.al 2013).
The 12-kDa Trx1 proteins are characterized by the presence of three conserved prolines, with one located between the catalytic cysteine residues of the – Cys-Gly-Pro-Cys – motif. Two cysteine residues (Cys-32 and -35) of the active site – Cys-Gly-Pro-Cys – are responsible for this reducing activity. The crystal structures of Trx1 in both oxidized and reduced states have been resolved and revealed that Trx1 has a basic Trx-fold (consisting of
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four β2-strands surrounded by three α2-helices) with additional α2-helices and β2-strands at the N-terminus (Figure 1.1 A).
Trx1-knockout mice are lethal due to early development and morphogenesis failure of the mouse embryo, whereas Trx1-transgenic (Trx1 Tg) mice are more resistant to oxidative stress with longer life span compared with wild type mice. These results suggest that Trx1 is an essential molecule for cellular and organismal survival. (Yoshihara, et.al 2013)
Figure 1.1 A: Three dimensional structure of Thioredoxin consisting of four β2-strands surrounded by three α2-helices (PDB ID: 4LL1 chain B)
Figure 1.1 B: Three dimensional structure of TXNIP consisting of two terminals C- terminal and N terminal (PDB ID: 4LL1 A)
Recent studies have shown that an important Trx binding protein, Thioredoxin interacting protein [TXNIP/thioredoxin binding protein-2 (TBP-2)/vitamin D3 upregulated protein (VDUP1)] has the reciprocal function with Trx in the pathogenesis of disease such as autoimmune disease, cancer, and diabetes. TXNIP is a unique Trx binding protein that has a role as an endogenous inhibitor of Trx, since TXNIP binding to Trx inhibits their protein reducing activity and/or Trx expression( Reich, E, et al.2012) This finding has implicated that redox-sensitive proteins and related cellular processes such as metabolism, proliferation, and inflammation could be regulated by Trx/TXNIP signalling.
It has been known that the two TXNIP cysteines are important for Thioredoxin binding through a disulphide exchange reaction between oxidized TXNIP and reduced Trx.
Since TXNIP has a specific arrestin-like domain, which is responsible for protein–protein binding. The gene encoding TXNIP was first cloned in 1994 (20 years ago) from a 1, 25- dihydroxyvitamin D3-treated HL-60 human promyelocytic cell line and therefore was initially referred to as vitamin D3-up-regulated protein. TXNIP binds to and inhibits Thioredoxin and thereby can modulate the cellular redox state and induce oxidative stress.
(Jing, Gu, et al 2013), (Saitoh,et.al 2001).
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Figure 1.2: Schematic diagram of the cellular functions of TXNIP (taken from Anathshalev et al. 2014)
In the cytoplasm, TXNIP binds to and inhibits Thioredoxin 1 (Trx1) and thereby interferes with the ability of Trx1 to reduce oxidized proteins, resulting in oxidative stress and increased susceptibility to apoptosis. In addition, TXNIP can also enter the mitochondria where it interacts with mitochondrial Thioredoxin2 (Trx2), releasing ASK1 from its inhibition by Trx2 and allowing for phosphorylation and activation of ASK1.
This in turn leads to cytochrome c (Cyt C) release from the mitochondria, cleavage of caspase-3, and apoptosis. TXNIP has also been found to be localized in the nucleus and to modulate the expression of various microRNAs (e.g., miR-204). These microRNAs down regulate the expression of target genes including important beta-cell transcription factors such as MafA, which results in reduced insulin transcription and impaired beta-cell function. (A.shalev et.al.2008).
Figure 1.3: Mechanism of action of TXNIP and Trx (Yoshihara, Eiji, et al.2013)
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The TXNIP protein forms an intra-molecular disulphide bond conferred by 2 cysteines- Cys247 and Cys63, and Cys247 is also essential for the interaction of TXNIP with Cys32 of thioredoxin.
1.2 Structure of TXNIP
Human TXNIP is a 46-kDa ubiquitously expressed protein that contains 391 amino acid residues and is encoded on chromosome 1q21. TXNIP is highly conserved across species;
mouse protein contains 395amino acids, and is located in a region of mouse chromosome 3 that is syntenic to human 1q21. TXNIP belongs to the protein family of alpha-arrestins, but among these proteins only TXNIP is capable of interacting with Thioredoxin.
Figure 1.4: Representative structure of the Trx and TXNIP complex (Abais, et.al 2012) (a) Trx interacts exclusively with C-TXNIP in the complex structure. The β-strands shown in cyan. The cysteines in TXNIP are displayed as white carbon atoms. (Jungwon Hwang et al.) Beta-Cell TXNIP expression is strongly induced by glucose and is increased in diabetes as well as in response to ER stress, and this induction is conferred at the transcriptional level by ChREBP and at the posttranscriptional level by a decrease in miR-17 and is modulated by a number of additional factors. TXNIP in turn inhibits Thioredoxin function and promotes oxidative stress and Beta-cell death.
In addition, by modulating the expression of distinct microRNAs (miR-204 and miR- 124a) and with that the expression of their target genes (MafA and FoxA2) TXNIP inhibits insulin transcription while inducing IAPP transcription. These detrimental effects are further magnified by the fact that TXNIP promotes its own expression, again via ChREBP.
Although some of the underlying causes and cornerstones of type 1 and type 2 diabetes are distinct, i.e. autoimmunity and insulin resistance, respectively, loss of functional beta -cell mass represents a crucial common feature in the pathogenesis of both diabetes types.
Moreover, elevated TXNIP levels have emerged as a critical factor involved in beta- cell dysfunction, beta-cell death, and the resulting development of diabetes and its complications. On the other hand, genetic deletion and pharmacological inhibition of beta - cell TXNIP have been shown to protect against type 1 and type 2diabetes (Shalev, Anath, 2014).
1.3 Verapamil Clinical Trials and Diabetes
Verapamil, a calcium channel blocker (CCB), was recently reported to inhibit TXNIP expres- sion in INS-1 cells and human islets, and orally administered verapamil reduced TXNIP expression and β-cell apoptosis, enhanced endogenous insulin levels, and rescued mice from streptozotocin-induced diabetes. Verapamil also reportedly promoted β-cell survival and improved glucose homeostasis and insulin sensitivity in BTBR ob/ob mice.
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However, (Wang,et. al,2002) have shown that the magnitude of the protective effect may be too small to have an effect on type II diabetes in clinical settings, and trials in patients with type I diabetes may provide some insights. Further studies to scrutinize the effect of CCBs on glycaemic control in diabetic patients are also pending. (Noto, Hiroshi, et.
al.2013). Importantly, even when the oral verapamil administration was started after overt diabetes had fully developed, verapamil was able to rescue micefrom diabetes, normalize blood glucose levels, and restore insulin producing beta-cells very similar results have been observed in TXNIP-null mice fed a high-fat diet.
Furthermore, pharmacological inhibition of TXNIP again with verapamil has been shown to improve beta-cell survival and diabetes in BTRB ob/obmice .One obvious question is why such potentially beneficial effects of calcium channel blockers in terms of diabetes control and progression have not been recognized previously. Verapamil has been shown to have beneficial effects in diabetic cardiomyopathy (shalev, Anath 2014). Many transcriptional and posttranslational studies were performed and analysed to know the real mechanism of action of Verapamil but the mechanism by which Verapamil inhibits TXNIP expression remain elusive.(Hyunjoo, et al.2012).
1.4 Motivation behind the Work
The formation of a Redoxisome (TXNIP-Trx complex) results into Trx expression inhibition and TXNIP activation. A well-known molecular mechanism of disulphide bond switching between Cys247 in TXNIP and Cys32 and Cys35 in Trx is established (Patwari, Parth, et al., 2006). Further, studies revealed that reduction in TXNIP expression results in the prevention of Diabetes in mouse. Most importantly, the mice showed reversion from diabetic to normal condition. This strongly suggested that TXNIP can be used as an attractive therapeutic target for Diabetes.
Many wet laboratory experiments were performed to know the real mechanism behind the action of calcium channel blockers on TXNIP inhibition. CCB’s are found to regulate several transcriptional factors involved in TXNIP transcription. They are found to have a protective antioxidant effects which leads to TXNIP reduction in diabetic mouse.
Thus, reducing TXNIP expression by CCB’S may provide a novel approach to promote survival of Diabetic mouse (Junqin Chen, et al. 2009). Oral doses of Verapamil in Diabetic mouse revealed Verapamil as an important treatment tool in diabetes, the effects were found to be more pronounced if the treatment is initiated earlier in the disease progress (Guanlan Xu, et al, 2012).
Results of certain studies also revealed that Verapamil controls cardiac gene transcription by inhibiting the calcienurin pathway resulting in transcriptional repression of genes such as TXNIP (Cha-Molstad, Hyunjoo, et al. 2012). But, still the mechanism of action of CCB’s remain elusive as although CCB’s might inhibit proapoptotic beta-cell TXNIP expression thereby enhancing beta-cell survival and function, certain experiments revealed that use of CCB’s not significantly associated with incident Diabetes compared to other hypertensive agents.
Further studies on the complex interaction between CCB’s and TXNIP are warranted before CCB’s can be advocated as a measure for Diabetes prevention or treatment (Noto, Hiroshi, et al., 2013). This lead to the idea of In-silico analysis of mechanism of action of CCB’s (particularly Verapamil) and Protein TXNIP-Trx complex to reveal the underling mechanism of action.
The queries regarding the concentration of TXNIP that has already been transcribed by the patient suffering from Diabetes, the expression of Trx that has been blocked by TXNIP overexpression as well as key role of TXNIP in certain metabolic pathways resulted to think in the direction of inhibition of Trx-TXNIP complex by the ligand instead of its post translational and transcriptional inhibition.
2. MATERIALS AND METHODS
2.1 Structural analysis of the target (TXNIP-Trx) of Verapamil
Verapamil structure was searched from many servers PubChem (Wang, et.al,2009), KEGG (Kanehisa et.al,2000) to obtain its mol2 and pdb formats and then the structure was analysed in Chimera(Chen et al,2014) and prepared for Docking and Inverse docking.
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Figure 2.1 Visualization of the Interacting residues involved in the TXNIP-Trx complex formation.
2.2 Molecular Docking of Verapamil to the Protein
The structure file of ligands in pdb format (Westbrook, John D et.al 2003) were taken and prepared well for docking by adding charges and hydrogen bonds. Similarly the proteins of interest (TXNIP, Trx, TXNIP-Trx complex) were prepared by addition of Kollman charges and polar hydrogen bonds. Docking was performed by using Auto Dock (v 4.2) (William E. Hart, et al) tool and final results were run on the Cygwin terminal, The compatibility of ligands was checked based on the values of minimum binding energies information of Auto Dock tool.
Figure 2.2|Region of complex used for griding
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Table 2.1 |Verapamil and list of receptors used for docking Ligand Receptor
Verapamil Trx Verapamil TXNIP
Verapamil TXNIP-Trx complex 2.3 Comparison of TXNIP sequences from Human and Mouse
For comparing the mouse and human TXNIP sequence similarity , the sequences of both the species for TXNIP protein was retrieved from UniProt (UniProt Consortium, 2008) .Both the sequences were downloaded in fasta format and then compared by using BLAST tool (Johnson, Mark, et al.) at NCBI. Then the results were observed and analysed.
2.4 Structural comparison of TXNIP of Human and Mouse
For analysing TXNIP structure, first went through the literature and found that the structure consists of beta strands having N-terminal and C-terminal and the crystalline structure was obtained in the form of a complex having Trx attached to its N-terminal on visualisation of the structure is performed in the Chimera 1.10(Chen et al, 2014) and by using Matchmaker tool of Chimera 1.10(Chen et al, 2014) superimposition of the both the structures were performed. Then by the visualization of the interacting partners of Trx it was analysed that, what all residues of TXNIP interacts with Trx which may act as the catalytic site for ligands.
Figure 2.3. TXNIP structure, the TXNIP (shown by Tan colour) represents the Human TXNIP and the TXNIP (shown in deep sky blue colour) represents the Mouse TXNIP.
3. RESULTS AND DISCUSSION
TXNIP is found to play a key role in Diabetes. Verapamil act as a potent drug in the inhibition of TXNIP expression (Figure 3.1). Experimentally, it was found that oral administration of Verapamil not only inhibits TXNIP expression but also reverts a diabetic Mouse into a normal Mouse. One of the possible mechanisms for the reversion of diabetic to non-diabetic mouse is through the inhibited TXNIP-Trx interaction system.
This prompted us to hypothesize that verapamil may directly be involve in regulating the TXNIP-Trx complex formation. Verapamil binding to the complex or the individual chains may regulate concentration of TXNIP. This strongly advocated the study the interaction of Verapamil to the interface of the TXNIP-Trx complex through docking studies.
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3.1 Docking of Verapamil onto the TXNIP and Trx molecules
In order to understand the molecular mechanism of action of Verapamil, we performed docking experiments. Here, Verapamil was docked onto the TXNIP-Trx complex and individual chains of Trx and TXNIP molecules. These three sets mimic the binding of the drug molecule in complex and free form of protein molecule (Figure 3.1). The affinity of the verapamil for the individual chains Trx, TXNIP and TXNIP-Trx complex was checked (table 3.1). The lowest binding energy for the ligand and complex interaction indicates the preference of the ligand for the TXNIP-Trx protein complex.
Figure 3.1|Docking of Verapamil onto TXNIP and Trx molecule.
Verapamil bound to Trx (shown in green), TXNIP (shown in yellow) and TXNIP-Trx complex (shown in yellow and green complex). Verapamil (calcium channel blocker), a drug molecule involved in various therapies including cardiomapathy, retinal dysfunctions and recently found to be as a potent drug for the prevention of Diabetes in Mouse.
Table 3.1 Binding affinity of Verapamil Ligand Receptor Minimum binding
energy(Kcal/joule)
Verapamil Trx -5
Verapamil TXNIP -5.58 Verapamil TXNIP-Trx -6.78 3.2 Verapamil TXNIP-TRX interaction
Based on the ligand-protein interaction analysis, we suggest two models for the action of verapamil.
a. Binding of Verapamil to Trx and
b. Binding of Verapamil to TXNIP-Trx complex.
Model A: Binding of Verapamil to Trx
The blind docking experiment of Verapamil to Trx revealed that Trx has a binding site away from the TXNIP-Trx interface (Figure 3.2). The Cys32 of Trx stabilizes the interaction with TXNIP, causing the β-cell apoptosis and reduction in the insulin production. Verapamil interacts with Cys32 of Trx through its backbone and does not hamper its interaction with TXNIP.
Direct binding of verapamil to Cys32 may interfere with the Trx-TXNIP complex formation. However, this binding may prevent the function of reduction of oxidative stress by Trx. This suggests that we should have a ligand bound to the Trx that indirectly hampers the binding of Trx-TXNIP, without affecting Trx function. Moreover, Verapamil showed a weak binding with the Trx surface, suggesting its easy release from the protein.
Verapamil
CID:2520
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Figure 3.2 Catalytic pocket of Trx.
Trx docked with Verapamil (shown in hot pink), H-bond (shown in black color), andVerapamil–Trx interaction (shown in red). Verapamil interacting with the Cys32 backbone.
B) Model of Verapamil and TXNIP-Trx complex
As shown in figure 3.3, docking experiment revealed that Verapamil binds to TXNIP-Trx complex interface. Our results suggest that verapamil interaction with the Cys 247 of TXNIP may destabilize the TXNIP-Trx complex. The cys247 of TXNIP and cys32 of Trx stabilize the interface of the TXNIP-Trx complex through disulphide bridges. Moreover, a highly constrained conformation of Verapamil (figure 3.3) as shown in docking complex, may hamper the energetics of the interface.
Also, The Cys247of TXNIP may interact with the verapamil and interfere the binding of incoming Trx, thereby reducing the oxidative stress, a favourable situation for the cell.
The model suggest that due to weak attachment of verapamil to the surface, the Verapamil- TXNIP will be transiently formed, justifying the proposed reversal mechanism. It will be interesting to visualize and study the effect of such a constraint conformation of ligand on the stability of the molecule through molecular simulation dynamics study in future.
Figure 3.3 |Overview of the catalytic pocket of TXNIP-Trx complex: (a) TXNIP (shown in yellow) and Trx (shown in green).Verapamil (shown in hot pink). Residues showing
interaction with Verapamil are depicted in red.
C ys
3 2 C ys
3 2
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Figure 3.4| Surface view ofthe catalytic pocket showing the constraint ligand. TXNIP- Trx complex (shown in yellow and green surface) with the docked ligand (shown in hot
pink).
The interactions between Verapamil in the centre (shown in hot pink) and the cysteine residues involved in the interaction is labelled Cys.
Figure 3.5|Ribbon structure showing the residues of strands and helix involved in interaction: (a) The Ribbon structure denotes the protein complex(TXNIP -Trx) and the docked ligand is shown at the center (in hot pink) ,(b) The ligand (Verapamil ) is interacting
with many amino acid residues labelled and shown in red,(c) Verapamil interaction with surrounding residues.
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Figure 3.6 | Schematic diagram of the model depicting the mechanism of action of interaction between TXNIP-Trx complex
3.3 Sequences and structure analysis of TXNIP in Mouse and human
The reversal of diabetes is seen in mouse model system. It will be interesting to check that the same system can be reverted back in Humans as well. The comparative sequence analysis (see methods), as shown in figure 3.8 reveals that Human and Mouse TXNIP are 96% identical (Figure 3.7 A). Moreover the Cys 247 residues along with other residues of TXNIP-Trx interface are conserved. The substituted amino acid residues were located in the regions away from the interaction site and disulphide bond formation site of complex.
Structural superposition revealed that a low 0.75 value of RMSD, indication high structural conservation (Figure 3.8 7). This suggests that Verapamil may target TXNIP-Trx systems of Human.
Figure 3.7|A) Superimposition of TXNIP: the superimposition of the TXNIP structure by (matchmaker tool in chimera)
4. CONCLUSION
Our study revealed the probable mechanism of action of Verapamil. Based on the ligand- protein interaction analysis, we suggest two models for the action of verapamil. Verapamil interacts with Cys32 of Trx through its backbone and does not hamper its interaction with TXNIP. Direct binding of verapamil to Cys32 may interfere with the Trx-TXNIP complex formation. However, this binding may prevent the function of reduction of oxidative stress by Trx. This suggests that we should have a ligand bound to the Trx that indirectly (a)
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hampers the binding of Trx-TXNIP, without affecting Trx function. Our study also suggests that Verapamil interaction with the Cys 247 of TXNIP may destabilize the TXNIP-Trx complex.
The cys247 of TXNIP and cys32 of Trx stabilize the interface of the TXNIP-Trx complex through disulphide bridges. Moreover, a highly constrained conformation of Verapamil (figure 3.3) as shown in docking complex, may hamper the energetics of the interface. Also, The Cys247of TXNIP may interact with the verapamil and interfere the binding of incoming Trx, thereby reducing the oxidative stress, a favourable situation for the cell. The study also revealed that Verapamil have 1207 possible targets.
This may lead to certain side effects afterwards due to this reason further search for thenatural analog of Verapamil which may also act as a potent drug for the prevention of Diabetes as instead of Verapamil is also under investigations. Finally, Verapamil was found to exhibit the properties and interactions that make them a probable and suitable potent drug that can be used as anti-diabetic agents.
4.1 Future Perspective
Diabetic therapies have significantly improved over the last several decades, but curative approaches are still missing, even though loss of functional beta cell mass by apoptosis has now been well established as a critical step in pathogenesis of type1 and type 2 diabetes, TXNIP has emerged as an attractive novel therapeutic target for diabetes and its ability not only prevent but also reverse diabetes in mice.
In future it will be interesting to visualize and study the effect of such a constraint conformation of Verapamil on the stability of the molecule through molecular simulation dynamics study. However, based on its binding affinity, we may utilize the Mandelate scaffold to generate potent drugs against diabetes in future. These studies provide insight into the mechanism of potent inhibitors of TXNIP (i.e. Verapamil and Devapamil) in mouse as well as in human.This may help us to introduce a curative approach for diabetes.
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