Computational Insights on Inhibition of MSH3 Induced DNA Repair with Reserpine Analogs

 

G. Madhumathi, K. Anbarasu, S. Jayanthi*

Computational Drug Design Lab, Department of Biotechnology, School of Bio Sciences and Technology,

VIT University, Vellore - 632014, Tamil Nadu, India

 

ABSTRACT:

Colorectal cancer, like most cancers can be treated if detected at an early stage. Although it is common, no cure has been identified yet. Common treatment options like chemotherapy, radiation therapy and surgery have been used for pain management and prolonging and improving the quality of lives of patients. Targeting the DNA repair pathway is a fairly recent development in the treatment of cancers and can be used as a monotherapy option or in conjugation with conventional treatment options. This study aims at determining the efficiency of a certain class of drugs at inhibiting the DNA mismatch repair protein MSH3, which is an important protein involved in the onset of colorectal cancer. Rational drug design was performed on the experimental 3D structure of MSH3 by computational methods. Autodock was used to perform virtual screening of the initial set of drugs, after which the top leads, 4-Methoxybenzoyl and 3,4-Dimethoxybenzoyl were identified on the basis of their binding energies and weak interactions with the protein. Hence, MSH3 can be a potential drug target for inhibition of the onset of colorectal cancer.

 

 

 

INTRODUCTION:

Colorectal cancer (CRC), which is an adenocarcinoma of the colon and rectum, is the third most commonly occurring form of cancer and the fourth most common cause of death by cancer. Every year, approximately one million people are diagnosed with CRC, out of which 700,000 people die^1-4. A cascade of accumulation of mutated genes results in the sequential progression of an adenoma to carcinoma, resulting in a colorectal adenocarcinoma. The mutations occur in a specific order, which is, adenomatous polyposis coli (APC) gene mutations, then, global hypomethylation, K-ras mutations, deleted in colon cancer (DCC) gene mutations and lastly, p53 gene mutations.

 

 

Initiation of colorectal carcinogenesis can be triggered by APC mutations or mutations in the mismatch repair genes (MMR). Initiation by mutations in APC gene results in familial adenomatous polyposis coli (FAP), whereas initiation by mutations in MMR genes results in hereditary non polyposis colorectal cancer (HNPCC), which is a dominantly inherited syndrome^5-7. Mainly, three MMR genes are involved in HNPCC, which are MSH2, MLH1 and MSH6. The DNA mismatch repair (MMR) system is a DNA repair pathway for recognizing and eliminating errors in the DNA during replication and synthesis. It is evolutionarily conserved and helps in maintenance of genome stability. Errors in the DNA are identified as mismatches or insertion/deletion loops (IDLs) by a group of proteins called MutS homologues or MSH proteins^8,9. A single MutS protein is present in prokaryotes, while most eukaryotes contain two heterodimeric complexes MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSβ). The MSH2-MSH3 is involved in the repair of IDLs up to 17 nucleotides long and the MSH2-MSH6 is involved in the repair of mismatches and repairs of 1-2 nucleotides. The MSH complex binds to the region of mismatch or IDL on the DNA, after which it recruits MutL homologue (MLH) in the presence of ATP, forming a quaternary complex that activates the subsequent events in MMR. Following the binding of the quaternary complex, the DNA double strand unwinds, the faulty nucleotides are excised and the excised portion of DNA is synthesized again^10,11. Although the MMR pathway is more commonly involved in development of various cancers including CRC, it also allows the cancerous cells to survive DNA damage inflicted by chemotherapy and anti-cancer drugs, thus rendering the treatment ineffective or conferring drug resistance to the tumour cells. Thus, targeted inhibition of DNA MMR proteins is used in combinational therapy along with anti-cancer drugs or chemotherapy to increase the efficiency of the treatment. Inhibition of DNA MMR proteins can also be used exclusively as majority of developing tumour cells have altered DNA repair pathways, and hence depend on the remaining DNA repair pathways for proliferation and survival. This treatment method increases DNA damage in cancerous cells alone, and hence has the advantage of singularly targeting the cancerous cells and avoiding the loss of normal healthy cells^12,13. The MSH3 gene was first identified and described in 1989. It is located on chromosome 5q11-13¹⁴. Structural analyses have revealed the flexibility of the lesion sites containing the mismatched DNA. They can have different bending angles with mismatched bases flipped into either the major groove or the minor groove. It was also observed that the MSH3 protein dominates DNA binding in the MutSβ complex, as the DNA is biased towards MSH3^15,16. This model thus proves the efficiency of targeted inhibition of MSH3 as a potential anti-cancer combinational therapy or monotherapy.

 

MATERIALS AND METHODS:

Dataset:

The protein target used for screening inhibitors was Human MutSbeta complexed with an IDL of 3 bases and ADP. The structural model of MSH3 was retrieved from RCSB PDB (PDB ID - 3THX) in the PDB format. The model consists of 918 amino acids with N and C terminals. The target site chosen for the docking was the ATP binding cavity of MSH3 lined by the key residues Y1059, F1023, Y1048 and Y868¹⁵.

 

Docking:

The software AutoDock 4.2¹⁷ in PyMOL plugin was used in order to carry out docking on the chosen compounds and substantiate the binding conformation with the help of weak interactions. A default protocol was applied, with population size of 150 randomly placed individuals, a maximum number of 2.5 x 10⁵ energy evaluations, and a maximum number of 2.7 x 10⁴ generations, gene mutation rate of 0.02 and crossover rate of 0.8 were used. The final docking run was set as 100 in AutoDock for the possible binding conformations, i.e. 100 runs for each docking by using Genetic Algorithm (GA-LS) searches.

 

Various parameters like the binding energy (kcal/mol), hydrogen and hydrophobic interactions and the inhibition constant were used to determine the docking results. The results were visualized and subsequent analysis was done using the software LigPlot+ ¹⁸ which displays the hydrogen and hydrophobic interactions between the protein and the ligands.

 

Lipinski’ Rule of Five:

The Lipinski’s rule of five or Pfizer’s rule of five was proposed by Christopher Lipinski. According to the rule, a compound that follows the given criteria is more likely to be membrane permeable and in a form that can be easily absorbed by the body.

1.     It has a molecular weight less than 500 Daltons.

2.     Number of hydrogen bond donors (N-H and O-H bonds) should not exceed 5.

3.     Number of hydrogen bond acceptors (N and O atoms) should not exceed 10.

4.     Log P (logarithm of octanol-water partition coefficient) value should not exceed 5.

 

RESULTS AND DISCUSSION:

Targeting the DNA repair pathway is becoming a very popular treatment method to improve on the efficiency of chemotherapy or radiation. Its use as a monotherapy option is also becoming increasingly popular. The DNA or ATP binding pockets have been targeted for inhibition. In this study, a number of potent inhibitors were identified from a previous study conducted on inhibition of MSH2 and MSH6¹⁹. These compounds belong to a class of reserpine analogues and rational inhibitor design was used for identification of the lead compounds. The compounds were selected based on Lipinsky’s Rule of Five. A three-dimensional model of the structure of human MSH3 domain containing the ATP binding pocket was used and showed in Figure 1. MSH3 consists of an asymmetric ATP binding site along with ATPase activity which induces the binding of DNA, after which the ATPase activity is reduced to a great extent¹⁵. The structures of reserpine analogues were generated using the software Chemsketch and filtered using Lipinski’s rule of five. Eleven compounds were chosen in total (Table 1) and used as inhibitors for MSH3. The reserpine analogues consist of cyclohexyl and benzyl rings with the presence of several methoxy functional groups. The docking displayed promising results for inhibition of MSH3. The best binding affinity was expressed by the ligand 3,4- Dimethoxybenzoyl of -7.35 kcal/mol, closely followed by 4-Methoxybenzoyl of -7.34 kcal/mol, while the lowest binding affinity was expressed by the ligand Methyl Reserpate of -5.16 kcal/mol (Table 2). The top two lead candidates, 3,4-Dimethoxybenzoyl and 4-Methoxybenzoyl were chosen for visualization of their interaction using LigPlot+. The interactions are displayed in Figure 2. The hydrogen bond interactions of MSH3-3, 4-Dimethoxybenzoyl and MSH3-3-Methoxybenzoyl complexes were evaluated using PyMOL. In case of MSH3-3, 4-Dimethoxybenzoyl, the results displayed one hydrogen bond between the protein and the ligand. The residues involved in the interactions were Leu86, Leu862 and Val860. From the binding pose of 3,4-Dimethoxybenzoyl, it can be deduced that it effectively inhibits ATP binding, and hence MSH3. An interaction between the ‘N10’ and ‘O’ atom was observed. In case of 4-Methoxybenzoyl, one hydrogen bond was observed between the protein and the ligand. The residues involved in the interaction were Val860, Tyr837, Gly835, Leu862 and Asp859. The binding pose of 4-Methoxybenzoyl indicated effective MSH3 inhibition. An interaction between the ‘N9’ atom and ‘O’ atom was observed.

 

 

Figure 1: Experimental 3D structure of MSH3 from Homo sapiens.

 

Table 1: AutoDock binding energy results of ligand dataset. The top hits were highlighted in bold.

Compound	Binding energy
3-Methoxybenzoyl	-5.37
4-Methoxybenzoyl	-7.34
3,4-Dimethoxybenzoyl	-7.35
Benzoyl	-6.46
Cinnamoyl	-6.03
Deserpidine	-5.42
Evodiamine	-6.02
Methylenedioxy	-6.11
Methyl Reserpate	-5.16
Reserpine	-5.95
Syrosingopine	-5.48

 

Table 2: AutoDock energy terms results of hits.

Compound	Inhibitionconstant (um)	Finalintermolecularenergy (kcal/mol)	Electrostatic Energy (kcal/mol)	Vdw+ hbond+ desolv energy (kcal/mol)
3,4- Dimethoxybenzoyl	4.11 uM	-8.87	-0.90	-7.97
4-Methoxybenzoyl	4.15 uM	-8.78	-0.91	-7.86

 

 

a

b

Figure 2: AutoDock results of lead candidates visualizedinLigPlot⁺. Hydrogen bonds showed in green color dots and hydrophobic contacts in red color arc. a 3,4-Dimethoxybenzoyl complex b 4-Methoxybenzoylcomplex.

 

 

CONCLUSION:

The inhibition of the DNA mismatch repair protein MSH3 with the eleven drugs in the initial ligand dataset was computationally designed and results indicated the top lead compounds, which were 3-Methoxybenzoyl and 3, 4-Dimethoxybenzoyl. The lead drug compounds were selected on the basis of virtual screening using AutoDock. The lead compounds displayed formation of hydrogen bonds and hydrophobic interactions with the target that indicates stable protein-ligand interactions. They were also evaluated on the basis of Lipinski’s rule of five and results indicated that they are ideal ligands for the inhibition of MSH3. It can thus be concluded from this study that MSH3 can be a potential inhibition target using 3-Methoxybenzoyl and 3, 4-Dimethoxybenzoyl as a treatment method for colorectal cancer.

 

ACKNOWLEDGMENT:

The authors thank the management of VIT University for providing the facilities to carry out this work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 01.09.2017           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 3765-3768.