In Silico Studies of Inhibitors of Dihydrofolate Reductase And
Total Page：16
File Type：pdf, Size：1020Kb
Archana Moon*et al. /International Journal of Pharmacy & Technology ISSN: 0975-766X CODEN: IJPTFI Available Online through Research Article www.ijptonline.com IN SILICO STUDIES OF INHIBITORS OF DIHYDROFOLATE REDUCTASE AND DIHYDROPTEROATE SYNTHASE OF E.COLI Archana Moon1, Deeba Khan2, Pranjali Gajbhiye3 & Monali Jariya4 1,2,3&4 University Department of Biochemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur -440033. Email: [email protected] Received on: 10-02-2017 Accepted on: 22-03-2017 Abstract: In the past two decades, antibiotic resistance has become an increasingly severe health problem. Bacterial infections caused by resistant strains are problematic in numerous hospitals around the world, especially in patients compromised by age, illness, and treated with immune-suppressant drugs. Antibiotics have a distinct mechanism of action capable of modifying microbial metabolism and physiological processes such as translation, DNA replication and cell wall biosynthesis. In this context, it is essential to increase the understanding of resistance mechanisms in order to develop drugs with potential activity against these pathogens. Escherichia coli is the most common causative agent of urinary tract infection (UTI), and diagnosing this infection usually relies on bacteriologic methods. Urinary tract infection (UTI) is the second most common bacterial infection after respiratory tract infection. Approximately 75-95% of UTIs are caused by Escherichia coli. Sulfonamides are competitive inhibitors of dihydropteroate synthase, the bacterial enzyme responsible for the incorporation of para-aminobenzoic acid (PABA) into dihydropteroic acid, the immediate precursor of folic acid. Trimethoprim exerts a synergistic effect with sulfonamides. It is a potent and selective competitive inhibitor of microbial dihydrofolate reductase, the enzyme that reduces dihydrofolate to tetrahydrofolate. Docking studies are molecular modelling studies aimed at finding a proper fit between ligand and its binding site. The present paper deals with studies to corrobarate the microbial and molecular studies to treat MDR resistant bacterial infections. Autodock Suite, version 1.5 6rC2 for Docking has been utilized in this study. Keywords: Dihydrofolate reductase(DHFR), Dihydropteroate synthase (DHPS), Docking, E.coli, Multidrug resistant(MDR). IJPT| April-2017| Vol. 9 | Issue No.1 | 28816-28829 Page 28816 Archana Moon*et al. /International Journal of Pharmacy & Technology Introduction: UTI is a serious health problem affecting millions of people each year. Infections of the urinary tract are the second most common type of infection. These are one of the most common bacterial infections affecting humans throughout their lifespan. These infections are more common in females than in males (1). It is caused by Gram-negative bacteria such as Escherichia coli, Klebsiella species, Enterobacter species, Proteus species and Gram-positive bacteria like Enterococcus species, and Staphylococcus saprophyticus. Studies from various parts of India have shown occurrence of high rates of antimicrobial resistance among E coli. The resistance rates of uropathogenic E. coli to various antibiotics such as beta- lactams (57.4%), co-trimoxazole (48.5%), quinolones (74.5%), gentamicin (58.2%), amikacin (33.4%), cefuroxime (56%), nalidixic acid (77.7%) have been reported (2). UTI due to multi drug resistant (MDR) E. coli increases the cost of treatment, morbidity and mortality especially in developing countries like India (2). The infection caused by MDR bacteria leads to limited treatment options (3). Antibiotic resistance is a worldwide problem threatening the ability to treat infections (4). Despite advancements of higher generations of antibiotics, these antibacterial agents have not been up to the mark in eradicating these infectious diseases. One of the possible reasons responsible for this decline in therapeutic control of the disease is antibiotic resistance. The MDR bacteria due to continued mismanaged selective pressure have been credited for being responsible for genetic response to antibacterial therapy (5). Resistance in Gram-negative bacteria has been increasing, particularly over the last 6 years. Many of the isolates producing these enzymes are also resistant to trimethoprim, quinolones and aminoglycosides. Novel combinations of antibiotics are being used in the community and broad-spectrum agents such as carbapenems are being used increasingly as empirical treatment for severe infections (6). Microorganisms use various mechanisms to develop drug resistance, such as recombination of foreign DNA in bacterial chromosome, horizontal gene transfer and alteration in genetic material (7). The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a critical node, dihydrofolate reductase (DHFR), which is shared between prokaryotes and eukaryotes. The DHFR enzyme is currently targeted by methotrexate in anti-cancer therapies, by trimethoprim for antibacterial uses, and by pyrimethamine for anti-protozoal applications. An additional anti-folate target is dihyropteroate synthase (DHPS), which is unique to prokaryotes as they cannot acquire folate through dietary means. It has been demonstrated as a primary target for the longest standing antibiotic class, the sulfonamides, which act synergistically with DHFR inhibitors. IJPT| April-2017| Vol. 9 | Issue No.1 | 28816-28829 Page 28817 Archana Moon*et al. /International Journal of Pharmacy & Technology Investigations have revealed most DHPS enzymes possess the ability to utilize sulfa drugs metabolically, producing alternate products that presumably inhibit downstream enzymes requiring the produced dihydropteroate. These inhibitors are also likely to interact with the enzymatic neighbors in the folate pathway that bind products of the DHFR or DHPS enzymes and/or substrates of similar substructure (8) Fig (1). Commonly used sulfa drug is co-trimoxazole, an association of two antifolate agents, sulfamethoxazole and trimethoprim (9). Dihydrofolate reductase (DHFR) is a ubiquitous enzyme present in almost all eukaryotic and prokaryotic cells (11). Inhibition of DHFR results in a depletion of the reduced folate pool, inhibition of RNA and DNA synthesis, and cell death. For this reason, DHFR has been a critically important therapeutic drug target. DHFR inhibitors targeting the FH2 binding site have been used in the treatment of cancer, autoimmune diseases and bacterial and fungal infections (10). Most microorganisms must synthesize folate denovo since they lack the active transport system of higher vertebrate cells that allows these organisms to use dietary folates. DHPS (Gene folP) is the target of sulphonamides, which are substrate analogues that compete with para- aminobenzoic acid. Molecular docking has become an increasingly important tool for drug discovery (12). Detailed analysis was performed for a better understanding of the molecular interactions between the ligands and target DHFR & DHPS proteins of E. coli based on their efficiency to bind the active sites on the receptor utilizing Autodock (version 1.5 6rC2). The docking exhibits different binding poses out of which the one with minimum binding energy was selected. The favourable conformation was analysed with hydrogen bonding with the active site residues of DHFR & DHPS (13). Fig(1): Folate Pathway In Bacteria (20). IJPT| April-2017| Vol. 9 | Issue No.1 | 28816-28829 Page 28818 Archana Moon*et al. /International Journal of Pharmacy & Technology Materials & Methods: In silico studies Molecular docking is an important tool to study the interaction of ligands with active site residues of the receptor [14, 15]. The docking involves the use of sampling algorithm and a scoring function to evaluate the proper orientation and pose of ligand molecule in relation to the binding energy. The correct identification of this binding pose of one or more related ligands is important in establishing a structure-activity relationship in lead optimization. The second use of scoring functions is to rank different ligands to predict their relative experimental activity [15-17]. In silico studies were performed using Autodock 4 suite (version 1.5 6rC2). The ligands viz, Chlorogenic acid, Ellagic acid, Gallic Acid, Hippuric acid, Quercetin and Standard Antibiotics viz, Clavulanic acid, Cephalosporin, Cephalosporin C, Penicillin, Sulfamethoxazole and Trimethoprim were docked with DHFR & DHPS enzymes of E.coli. The structure of DHFR of E.coli was downloaded from PDB (protein data base) with PDB Id 2ANO (X ray diffraction structure, 2.6A0) & PDB Id 1AJ0 for DHPS of E.coli ( X ray diffraction structure, 2.0A0). Refer Fig (2). The PubSum database, utilizes 4 characters, as inputs, to get the ligands with their Ligplots. Ligplots indicate the interacting sites of the protein of interest i.e DHFR & DHPS Fig(3,4). Next a Ramachandran Plot Fig(5) to verify the protein structure was plotted. The structure of the ligands were downloaded from Pubchem (chemical structure data base) online portal and drawn in Marvin Sketch version 5.8.1. Fig(6,7). Docking was performed for DHFR & DHPS of E.coli with ligands (Chlorogenic acid, Ellagic acid, Gallic Acid, Hippuric acid and Quercetin and Standard Antibiotics (Clavulanic acid, Cephalosporin Cephalosporin C, Penicillin, Sulfamethoxazole and Trimethoprim). The docking results were analyzed on the basis of their binding energy and their interactions (13). Fig(2): PDB Structure of DHFR and DHPS Protein.