International Journal of Pharma and Bio Sciences

Transcription

1 International Journal of Pharma and Bio Sciences RESEARCH ARTICLE BIOINFORMATICS MOLECULAR SCREENING OF CITRATE AND STRUCTURALLY SIMILAR COMPOUNDS FOR PREVENTION OF LUNG TISSUE DESTRUCTION HEENA SHAH, PUSHPARANI MUDALIYAR AND CHANDRASHEKHAR KULKARNI MITCON Biopharma and Biotechnology Centre, Agriculture College Campus, Shivajinagar, Pune M.S. (India) HEENA SHAH MITCON Biopharma and Biotechnology Centre, Agriculture College Campus, Shivajinagar, Pune M.S. (India) ABSTRACT The major function of α1-antitrypsin is to protect the lung against the enzyme neutrophil elastase, which breaks down the connective tissue fibre elastin. However the aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis, and emphysema. One possible therapeutic strategy involves inhibiting the conformational changes involved in antitrypsin aggregation by using citrate ion, which has been shown to maintain the protein in an active conformation. In our study we identified compounds structurally similar to citrate and carried out docking of citrate ligand as well as the structurally similar ligands with alpha 1 antitrypsin. From the docking and the dynamics results we conclude that citrate and structurally similar compounds to that of citrate were potent inhibitors which can thus prevent aggregation and allow stable A1AT to work at its optimum concentration. B - 306

2 KEYWORDS alpha-1-antitrypsin, stabilising, citrate, docking, structurally similar. INTRODUCTION Alpha 1-Antitrypsin or α1-antitrypsin (A1AT) is a protease inhibitor belonging to the serpin superfamily. Serpins are a group of proteins with similar structures that are able to inhibit proteases 1. Alpha 1-antitrypsin protects the lungs from damage caused by protease enzymes, such as elastase and trypsin, that can be released as a result of an inflammatory response to tobacco smoke 2. Alpha-1-antitrypsin is mainly produced in the liver but its main function is to protect the lung against proteolytic damage from neutrophil elastase. Despite its name, alpha-1 antitrypsin reacts with neutrophil elastase much more readily than with trypsin and represents a major defence against the elastolytic burden in the lower airways posed by neutrophil elastase, which is produced by neutrophils in the lower respiratory tract 2. Serpins have been likened to mousetraps complete with bait, a loaded high-energy but unstable state, and a swinging arm. In the case of alpha-1 antitrypsin, the bait is a methionine amino acid side-chain in this active centre of the serpin. Docking of the neutrophil elastase on that residue cleaves the reactive centre, releases alpha-1 antitrypsin from its metastable high energy state, and allows the cleaved reactive loop to snap back, with the protease in tow, to the other pole of the molecule. Because that arm remains fairly short, it distorts and inactivates the elastase molecule by squeezing it on the other end of the alpha-1 antitrypsin molecule. While this process is mutually suicidal and ensures the destruction of both molecules, there is normally an excess of alpha-1 antitrypsin in the lung, thereby providing an adequate protective screen against the elastolytic burden of neutrophil elastase. Some forms of A1AT have a tendency to polymerise, being retained in the endoplasmic reticulum 3. This aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis, and emphysema. These aggregates characterized as an ordered polymeric structure, accumulate in the endoplasmic reticulum (ER) and lead to cell damage or death, which, in turn, lead to liver damage. Emphysema is a downstream result of AAT accumulation in the ER as less AAT is secreted into circulation, thus reducing the overall inhibitory capabilities of the plasma. Target proteinases of AAT, such as elastase, then operate unhindered leading to destruction of the connective tissue within the lungs and subsequently to emphysema. AAT aggregation involves a number of steps 4-6. An approach to prevent polymerisation of the protein is to use chemical chaperones to stabilize intermediates on the folding pathway. One possible therapeutic strategy, on the same lines, involves inhibiting the conformational changes involved in antitrypsin aggregation by using citrate ion. A single citrate molecule binds in a pocket between the A and B beta-sheets, a region known to be important in maintaining antitrypsin stability. Citrate binds to both the native and intermediate conformations of AAT, and in doing so can prevent unfolding and aggregation 7. We have identified compounds structurally similar to citrate which can bind in the active pocket of A1AT to prevent aggregation. Docking of citrate ligand as well as compounds structurally similar to citrate with alpha 1 antitrypsin was carried to confirm the action of newly identified ligands. B - 307

3 METHODS AND MATERIALS Identification and analysis of active site of the alpha-1-antitrypsine protein target The three dimensional structure of alpha 1 antitrypsin was identified from the Protein Data Bank (PDB). For finding out the active site of the Alpha-1-Antitrypsin protein (PDB ID: 3CWM) we used PDBsum. The human Alpha- 1-Antitrypsin receptor 3CWM was bound to the ligand citrate and their interactions were studied with the help up the PDBsum ligplot. The active site residues in this case were found to be the following: 1. ARG196 [Chain A] 2. MET226 [Chain A] 3. LYS243 [Chain A] 4. ARG281 [Chain A] Retrieval of ligands and similar structure Compounds structurally similar to stabilising agent sodium citrate were obtained via Tanimoto Similarity search predefining a threshold value of 0.7. The similar compounds obtained were docked with A1AT using ArgusLab. Docking of the ligand with the alpha-1- antitrypsin receptor Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using for example scoring functions 8. Furthermore, the relative orientation of the two interacting partners may affect the type of signal produced. Therefore docking is useful for predicting both the strength and type of signal produced. Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to predict the affinity and activity of the small molecule. Hence docking plays an important role in the rational design of drugs 9. RESULTS Table.1 The list of compounds obtained using Tanimoto Similarity search Tanimoto Similarity Score Name CAS Number Citric Acid Structure Score: Isocitric Acid Score: B - 308

4 Score: ISSN Vol 3/Issue 1/Jan Mar 2012 Alpha-Methylisocitric Acid 3-Isopropylmalic Acid Score: ,3-Dihydroxy-Valerianic Acid Score: Hydroxy-3-Methyl-Glutaric Acid Score: Tricarballylic Acid Score: (R)-Mevalonate Score: B - 309

5 2,4-Dihydroxy-3,3-Dimethyl-Butyrate Score: Malate Ion Score: D-tartaric acid Score: These compounds on docking with alpha 1 antitrypsin using ArgusLab tm gave the following values: Table.2 Binding energy of citrate and structurally similar compounds Name of Compound Binding energy in kcal/mol Citrate Malate Tartaric Acid B - 310

6 Figure.1 Citrate bound to active site of A1AT Figure.2 Malate bound to active site of A1AT. B - 311

7 Figure 3 Tartaric acid bound to A1AT active site. The other molecules viz. Isocitric Acid, Alpha- Methylisocitric Acid, 3-Isopropylmalic Acid, 2,3-Dihydroxy-Valerianic Acid, 3-Hydroxy-3- Methyl-Glutaric Acid, Tricarballylic Acid, Mevalonate and 2,4-Dihydroxy-3, 3-Dimethyl- Butyrate, did not generate any accepted poses. DISCUSSION From the results obtained, the observation made was that although compounds like isocitric acid, alpha-methylisocitric acid, 3- isopropylmalic acid, being very closely structurally and functionally similar to citrate, did not show any effective binding at the active site of A1AT, thus indicating an inefficiency in use as an alternative to citrate. However, positive results were obtained in case of malate and tartaric acid, which are far related to citrate. CONCLUSION From the observations made, the binding to the active site of A1AT is not dependent on the functional group similarity with citrate molecule, but is rather dependent on structural similarity with similar binding energy to that of citrate. From the binding energy studies it could be proposed that malate and tartaric acid can be used as better and more efficient alternative for citrate in enhancing stability of A1AT and thus preventing lung tissue destruction. The effectiveness of malate and tartaric acid to stabilize A1AT can be further investigated in vitro. B - 312

8 REFERENCES ISSN Vol 3/Issue 1/Jan Mar ) Gettins PG, Serpin structure, mechanism, and function, Chem Rev 102 (12): , (2002). 2) DeMeo DL, Silverman EK, α1- Antitrypsin deficiency 2: Genetic aspects of α1-antitrypsin deficiency: phenotypes and genetic modifiers of emphysema risk, Thorax 59 (3): , (2004). 3) Alkins SA, O'Malley P, Should healthcare systems pay for replacement therapy in patients with alpha(1)- antitrypsin deficiency? A critical review and cost-effectiveness analysis, Chest 117 (3): , (2000). 4) James, E.L. and Bottomley, S.P., The mechanism of a1-antitrypsin polymerization probed by fluorescence spectroscopy, Arch. Biochem. Biophys., 356: , (1998). 5) Dafforn, T.R., Mahadeva, R., Elliott, P.R., Sivasothy, P., and Lomas, D.A., A kinetic mechanism for the polymerization of a1-antitrypsin, J. Biol. Chem. 274: , (1999). 6) Devlin, G.L., Chow, M.K., Howlett, G.J., and Bottomley, S.P., Acid Denaturation of a1-antitrypsin: Characterization of a novel mechanism of serpin polymerization. J. Mol. Biol. 324: , (2002). 7) Pearce MC, Morton CJ, Feil SC, Hansen G, Adams JJ, Parker MW, Bottomley SP., Preventing serpin aggregation: the molecular mechanism of citrate action upon antitrypsin unfolding. Protein Sci., Dec;17(12): , (2008). 8) Lengauer T, Rarey M., Computational methods for biomolecular docking. Curr. Opin. Struct. Biol. 6 (3): 402 6, (1996). 9) Kitchen DB, Decornez H, Furr JR, Bajorath J., Docking and scoring in virtual screening for drug discovery: methods and applications. Nature reviews. Drug discovery 3(11): , (2004). B - 313