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Guru Gobind Singh Indraprastha University School of Biotechnology

The Seventh Dr Yellapragada SubbaRow Memorial Lectures 2008

12 January 2008

1. "Protein Structure and Structure Based Rating Drug Design" by Professor T P Singh, former HOD, Biophysics, All India Institute of Medical Sciences, New Delhi

Protein structure determination and Structure-based drug design


Prof T. P. Singh,

Department of Biophysics,

All India Institute of Medical Sciences, New Delhi - 110029

In the post-genomic era, there has been an enormous data explosion in terms of identification and characterization of proteins that cause disease in the human body. This phenomenal emergence of data, along with the need to reduce the time and cost of designing new drugs, has led to the development of rational approaches for ligand design and drug discovery.

The conceptual breakthrough developed in the Twentieth Century that a drug may have specific target at the molecular level led to the notion of receptor based drug design that has profoundly benefited the drug discovery process.

The strategy in the design of new agents has been to modify the structure of a lead compound by systematic chemistry in conjunction with binding studies and physiological evaluation to produce a compound of the required potency.

The most recent phase in the new drug discovery process has utilized the knowledge of the three-dimensional structures of target macromolecules or of related proteins. From the experimental observations at the atomic level of how inhibitors bind to the molecular targets, specific interactions that are important in the molecular recognition can be inferred. This knowledge can be applied to lead compounds and can be used for the de novo design as well. This approach also provides insights into mechanisms of action of existing drugs.

In this approach, the most important requirement is to identify the correct pathways that produce pro-disease compounds and find out the target proteins that are involved in these pathways. Using the rational method of structure-based approach, one designs the compounds that fit into the binding sites of target proteins. One of the several programmes that are currently in progress in my laboratory is the project on the development of potent anti-inflammatory agents using the structure-based approach. There are three main enzymes as potential targets in the pathway that produces pro-inflammatory compounds known as eicosanoids. The target enzymes are phospholipase A2s (PLA2), cyclo-oxygenases(COX) and lipoxygenases(LOX). Using PLA2 as a target protein, we have designed a number of molecules with high binding affinities as potential drugs against inflammation, rheumatism and arthritis. PLA2 catalyzes the hydrolysis of the sn-2 acyl ester linkage of phospholipids. One of the products of this reaction is arachidonic acid which works as a substrate for COX and LOX enzymes resulting in the production of pro-inflammatory eicosanoids. An increased concentration of these compounds causes various inflammatory disorders. Therefore, in order to restrict the level of concentrations of eicosanoids, inhibitors of PLA2 have been synthesized.

The development of tight inhibitors with high binding affinities requires the detailed knowledge of the stereochemistry of the binding site of PLA2. Therefore, crystal structures of a large number of isoforms of PLA2 have been determined. The three-dimensional structure of Phospholipase A2 reveals the details of the substrate binding site having a linear hydrophobic channel which has various linear and aromatic hydrophobic residues ending with an active site having residues such as His48, Asp49, Tyr52 and Asp99. The active site also contains a conserved water molecule that interacts with the active site residues, His48 and Asp49. This water molecule is an integral part of the active site of Phospholipase A2.

Six subsites within the phospholipase A2 molecule have been identified based on the detailed analysis of the structures of various complexes of phospholipase A2 ; Subsite 1 (residues 2-10), Subsite 2 (residues 17-23), Subsite 3 (residues 28-32), Subsite 4 (residues 48-52), Subsite 5 (residues 68-70) and Subsite 6 (residues 98-106).

The binding affinities of all known inhibitors for phospholipase A2 are in the range from 10-3 to 10-6M which make them poor candidates as drugs. After the detailed structural analyses, it has been concluded that the poor potency of these compounds can be attributed to the fact that these compounds are able to occupy only a few of the subsites within the overall binding space. Thus, there is an immense possibility to design new inhibitors, keeping in mind the range and scope of all the subsites. The goal is to design an inhibitor of phospholipase A2 which would occupy the maximum possible subsites and hence, would bind the molecule with an improved affinity. Keeping this goal in focus, the structures of complexes of phospholipase A2 with natural compounds, substrate analogues, indole derivatives, non-steroidal anti-inflammatory drugs (NSAIDs) and with designed peptides have been analyzed in detail.

The structure analyses of the complexes of PLA2 with natural compounds, aristolochic acid and Vitamin E are shown in Figs. 1a and 1b respectively.

The structures reveal that these compounds bind to the molecule at the substrate binding site of PLA2. The OH group of both these compounds is involved in the formations of two hydrogen bonds with the side chains of His48 and Asp49. These hydrogen bonds can be generated directly or through the conserved water molecule in the active site. All the natural compounds that bind to phospholipase A2 tend to occupy the same subsites of the molecule and hence display similar binding affinities.

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The structure analyses of the complexes of PLA2 with NSAIDS were carried out primarily to understand the mechanism of action of NSAIDs through PLA2 inhibition. While most of the NSAIDS bind to PLA2 complexes in the conventional manner as shown in Figs.

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2a and 2b while indomethacin, one of the most potent NSAIDS was found interacting with the molecule in a different mode so that one of the carboxylic group oxygen atoms forms a hydrogen bond with catalytic water molecule while the second oxygen atom interacts with Lys69 (Fig. 2c).


This finding gave a novel direction to the design of new inhibitors against phospholipase A2.

In order to harness the structural knowledge of phospholipase A2 ligand binding site for drug design, highly specific peptide inhibitors of phospholipase A2 which showed binding affinities at 10-9 M concentrations were designed, synthesized and co-crystallized with phospholipase A2.

A peptide with a sequence, Leu – Ala – Ile – Tyr – Ser (LAIYS) was designed with tyrosine and serine at the carboxyl terminus to impart interactions through OH group which can make bonds with His48 and Asp49 and Leu – Ala – Ile for hydrophobic interactions with the protein residues lining along the hydrophobic channel. The structural analysis of complex of LAIYS with phospholipase A2 revealed that the inhibitor occupied the substrate-binding site in a tight fit. As predicted, the hydroxyl group of tyrosine was found to be interacting with Asp49 and His48 while the hydrophobic residues of the inhibitor were found to be interacting with the residues of the hydrophobic channel


(Fig. 3a). The close fit of the peptide was substantiated with the high binding affinity of this peptide to PLA2 which is approximately 8.8 ´ 10-9 M. In an attempt to exploit the negative charge on Asp49 and positive charge on His48, a peptide Phe – Leu – Ser – Tyr – Lys (FLSYK) was designed. The structure of PLA2 complex with peptide FLSYK revealed that the Lys side chain was well placed in the active site and made a strong ionic interactions with the side chains of Asp49 and



His48 (Fig. 3b). Predictably, the peptide inhibitor displayed a high binding affinity of 1.1 ´ 10-9 M.




Hence, the detailed knowledge of stereochemistry of the active site, the identification of the subsites in the ligand recognition pocket and the interactions of the various inhibitors with phospholipase A2 can be exploited for the design of novel therapeutics against various inflammatory disorders.

2. "Clinical Cancer Proteomics: Discovery of Novel Drug Targets" by Professor Ranju Ralhan, Visiting Professor, Centre For Research in Mass Spectrometry, York University, Toronto, Canada and Professor, Department of Biochemistry, All India Institute of Medical Sciences, New Delhi :


Recent advances in genomic and proteomic technologies and mass spectrometry have paved the way for better understanding the human biology in health and disease. Diseases can now be investigated using a

systems biology approach, which includes epigenetics, genomics, transcriptomics and proteomics. Knowledge of the human proteome, structure and function of each protein and the complexities of protein-protein interactions, is critical for developing the most effective diagnostic techniques and disease treatments for the future. Functional proteomics combined with mass spectrometry promises the unveiling of the complex molecular events underlying the development of human diseases, particularly cancer as a prototype. My laboratory has been using genomics and tissue proteomics for unraveling the perturbed signaling pathways and biological networks in head and neck cancer and consequent discovery of potential cancer markers. The integration of knowledge harnessed from genomics and proteomics into systems biology is providing in depth understanding of the molecular basis of cancer. The application of high-throughput protein evaluation with a subset of predefined targets, identified through proteomics and pathway analysis in human tissues, is yielding novel biomarkers reflecting cancer development, establishing earlier detection strategies, and permitting monitoring of responses to therapy. My group is also using proteomic technologies for studies of dynamic protein expression, post-translational modifications and protein-protein interactions in head and neck cancer, that have culminated in the identification of many potential new drug targets for molecular therapeutics. Consequently, the prevailing dogma of use of generic anticancer drugs for management of cancer is being challenged by the new approach of targeted molecular therapeutics that in turn can translate into substantial strides toward improved patient care and outcomes. (Abstract by Prof Ralhan)