Rishi K. Somvanshi

Specific binding of non-steroidal anti-inflammatory drugs (NSAIDs) to phospholipase A2: structure of the complex formed between phospholipase A2 and diclofenac at 2.7 A resolution.

Type IIA secretory phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids to release fatty acids and lysophospholipids. In order to elucidate the role of PLA2 in inflammatory disorders and to determine the mode of binding of non-steroidal anti-inflammatory drugs (NSAIDs) to PLA2, the detailed three-dimensional structure of a complex formed between a group IIA PLA2 from Daboia russelli pulchella and 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid (diclofenac) has been determined. The preformed complex was crystallized by equilibrating the protein solution against a mixture of 0.20 M ammonium sulfate and 30% PEG 4000. The crystals belong to space group P4(3), with unit-cell parameters a = b = 53.0, c = 48.4 A. The structure was solved by the molecular-replacement method and refined to R(cryst) and R(free) factors of 0.192 and 0.211, respectively, using reflections to 2.7 A resolution. The structure showed that diclofenac occupies a very favourable position in the centre of the substrate-binding hydrophobic channel that allows a number of intermolecular interactions. The binding mode of diclofenac involved crucial interactions with important residues for substrate recognition such as Asp49, His48 and Gly30. In addition, it included three new interactions involving its Cl atoms with Phe5, Ala18 and Tyr22. It also showed an extensive network of hydrophobic interactions involving almost all of the residues of the substrate-binding hydrophobic channel. The binding affinity of diclofenac was determined using surface plasmon resonance, which gave an equilibrium constant of 4.8 +/- 0.2 x 10(-8) M.

Non-steroidal anti-inflammatory drugs as potent inhibitors of phospholipase A2: structure of the complex of phospholipase A2 with niflumic acid at 2.5 Å resolution

Phospholipase A(2) (PLA(2); EC 3.1.3.4) catalyzes the first step of the production of proinflammatory compounds collectively known as eicosanoids. The binding of phospholipid substrates to PLA(2) occurs through a well formed hydrophobic channel. Surface plasmon resonance studies have shown that niflumic acid binds to Naja naja sagittifera PLA(2) with an affinity that corresponds to a dissociation constant (K(d)) of 4.3 x 10(-5) M. Binding studies of PLA(2) with niflumic acid were also carried out using a standard PLA(2) kit that gave an approximate binding constant, K(i), of 1.26 +/- 0.05 x 10(-6) M. Therefore, in order to establish the viability of PLA(2) as a potential target molecule for drug design against inflammation, arthritis and rheumatism, the three-dimensional structure of the complex of PLA(2) with the known anti-inflammatory agent niflumic acid [2-[3-(trifluoromethyl)anilino]nicotinic acid] has been determined at 2.5 Angstroms resolution. The structure of the complex has been refined to an R factor of 0.187. The structure determination reveals the presence of one niflumic acid molecule at the substrate-binding site of PLA(2). It shows that niflumic acid interacts with the important active-site residues His48 and Asp49 through two water molecules. It is observed that the niflumic acid molecule is completely buried in the substrate-binding hydrophobic channel. The conformations of the binding site in PLA(2) as well as that of niflumic acid are not altered upon binding. However, the orientation of the side chain of Trp19, which is located at the entry of the substrate-binding site, has changed from that found in the native PLA(2), indicating its familiar role.

Structural Elements of Ligand Recognition Site in Secretory Phospholipase A2 and Structure-Based Design of Specific Inhibitors

Phospholipases A2 (phosphotide 2-acylhydrolases, PLA2s, EC 3.1.1.4) are widely distributed enzymes in the animal world. They catalyze the hydrolysis of the sn-2 acyl ester linkage of phospholipids, producing fatty acids and lysophospholipids. The mammalian type II secreted phospholipase A2 (PLA2-II) is one of the most extensively studied member of low molecular weight (13-18 kDa) PLA2s. PLA2-II contains 120-125 amino acid residues and seven disulphide bridges. The important features of overall structure of PLA2-II contain an N-terminal helix, H1 (residues: 2-12), an external loop (residues: 14-23), a calcium binding loop (Ca2+-loop, residues: 25-35), a second alpha-helix, H2 (residues: 40-55), a short two stranded anti-parallel beta-sheet referred to as beta-wing (residues: 75-84), a third alpha-helix, H3 (residues: 90-108) which is antiparallel to H2 and two single helical turns, SH4 (residues: 114-117) and SH5 (residues: 121-125). The three-dimensional structure of PLA2-II has defined a conserved active site within a hydrophobic channel lined by invariant hydrophobic residues. The active site residues His48, Asp49, Tyr52 and Asp99 are directly connected to the channel. An important water molecule that bridges His48 and Asp49 through hydrogen bonds is a part of catalytic network. Based on the structures of various complexes of group II PLA2, the ligand-recognition site has been divided into six subsites consisting of residues 2-10 (subsite 1), residues 17-23 (subsite 2), residues 28-32 (subsite 3), residues 48-52 (subsite 4), residues 68-70 (subsite 5) and residues 98-106 (subsite 6). It is observed that most of the currently available ligands saturate only part of the ligand-recognition site leaving a wide scope to improve the ligand complementarity. Naturally, the ligands that interact with the largest number of subsites would also correspond to the maximum affinity. Therefore, for the design of potent inhibitors of PLA2, the stereochemical knowledge of the binding site as well as their potential to interact with ligands must be known so as to make the structure-based ligand design successful.

Development of novel peptide inhibitor of Lipoxygenase based on biochemical and BIAcore evidences

Lipoxygenase (LOX) are enzymes implicated in a broad range of inflammatory diseases, cancer, asthma and atherosclerosis. These diverse biological properties lead to the interesting target for the inhibition of this metabolic pathway of LOX. The drugs available in the market against LOX reported to have various side effects. To develop potent and selective therapeutic agents against LOX, it is essential to have the knowledge of its active site. Due to the lack of structural data of human LOX, researchers are using soybean LOX (sLOX) because of their availability and similarities in the active site structure. Based on the crystal structure of sLOX-3 and its complex with known inhibitors, we have designed a tripeptide, FWY which strongly inhibits sLOX-3 activity. The inhibition by peptide has been tested with purified sLOX-3 and with LOX present in blood serum of breast cancer patients in the presence of substrate linoleic acid and arachidonic acid respectively. The dissociation constant (K(D)) of the peptide with sLOX-3 as determined by Surface Plasmon Resonance (SPR) was 3.59x10(-9) M. The kinetic constant (K(i)) and IC(50), as determined biochemical methods were 7.41x10(-8) M and 0.15x10(-6) M respectively.

Detection of native peptides as potent inhibitors of enzymes

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in α‐chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7u2003±u20030.3u2003×u200310−11u2003m and thus works as a highly potent competitive inhibitor. The commercially available α‐chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP‐treated α‐chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295u2003K. Twenty milligrams of lyophilized protein were dissolved in 1u2003mL of 25u2003mm sodium acetate buffer, pHu20034.8. It was equilibrated against the same buffer containing 1.2u2003m ammonium sulfate. The rectangular crystals of small dimensions of 0.24u2003×u20030.15u2003×u20030.10u2003mm3 were obtained. The X‐ray intensity data were collected at 2.2u2003Å resolution and the structure was refined to an R‐factor of 0.192. An extra electron density was observed at the binding site of α‐chymotrypsin, which was readily interpreted as a 14 residue fragment of α‐chymotrypsin corresponding to Ile‐Val‐Asn‐Gly‐Glu‐Glu‐Ala‐Val‐Pro‐Gly‐Ser‐Trp‐Pro‐Trp(16–29). The electron density for the eight residues of the C‐terminus, i.e. Ala22–Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N‐terminus, Ile16–Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0u2003σ between the active site Ser195 Oγ and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35u2003Å between Ser195 Oγ and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16–Trp29. In addition to the O–C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of α‐chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of α‐chymotrypsin.

Detection of native peptides as potent inhibitors of enzymes. Crystal structure of the complex formed between treated bovine alpha-chymotrypsin and an autocatalytically produced fragment, IIe-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp, at 2.2 angstroms resolution.

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in α‐chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7u2003±u20030.3u2003×u200310−11u2003m and thus works as a highly potent competitive inhibitor. The commercially available α‐chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP‐treated α‐chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295u2003K. Twenty milligrams of lyophilized protein were dissolved in 1u2003mL of 25u2003mm sodium acetate buffer, pHu20034.8. It was equilibrated against the same buffer containing 1.2u2003m ammonium sulfate. The rectangular crystals of small dimensions of 0.24u2003×u20030.15u2003×u20030.10u2003mm3 were obtained. The X‐ray intensity data were collected at 2.2u2003Å resolution and the structure was refined to an R‐factor of 0.192. An extra electron density was observed at the binding site of α‐chymotrypsin, which was readily interpreted as a 14 residue fragment of α‐chymotrypsin corresponding to Ile‐Val‐Asn‐Gly‐Glu‐Glu‐Ala‐Val‐Pro‐Gly‐Ser‐Trp‐Pro‐Trp(16–29). The electron density for the eight residues of the C‐terminus, i.e. Ala22–Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N‐terminus, Ile16–Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0u2003σ between the active site Ser195 Oγ and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35u2003Å between Ser195 Oγ and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16–Trp29. In addition to the O–C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of α‐chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of α‐chymotrypsin.

SD-8, a novel therapeutic agent active against multidrug-resistant Gram positive cocci

Anti-bacterial drug resistance is one of the most critical concerns among the scientist worldwide. The novel antimicrobial decapeptide SD-8 is designed and its minimal inhibitory concentration and therapeutic index (TI) was found in the range of 1–8xa0μg/ml and 45–360, respectively, against major group of Gram positive pathogens (GPP). The peptide was also found to be least hemolytic at a concentration of 180xa0μg/ml, i.e., nearly 77 times higher than its average effective concentration. The kinetics assay showed that the killing time is 120xa0min for methicillin-sensitive Staphylococcus aureus (MSSA) and 90xa0min for methicillin-resistant S. aureus (MRSA). Membrane permeabilization is the cause of peptide antimicrobial activity as shown by the transmission electron microscopy studies. The peptide showed the anti-inflammatory property by inhibiting COX-2 with a KD and Ki values of 2.36xa0×xa010−9 and 4.8xa0×xa010−8 M, respectively. The peptide was also found to be effective in vivo as derived from histopathological observations in a Staphylococcal skin infection rat model with MRSA as causative organism.

Detection of native peptides as potent inhibitors of enzymes: Crystal structure of the complex formed between treated bovine α-chymotrypsin and an autocatalytically produced fragment, Ile-Val-Asn-Gly-Glu-Glu-Ala-

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in α‐chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7u2003±u20030.3u2003×u200310−11u2003m and thus works as a highly potent competitive inhibitor. The commercially available α‐chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP‐treated α‐chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295u2003K. Twenty milligrams of lyophilized protein were dissolved in 1u2003mL of 25u2003mm sodium acetate buffer, pHu20034.8. It was equilibrated against the same buffer containing 1.2u2003m ammonium sulfate. The rectangular crystals of small dimensions of 0.24u2003×u20030.15u2003×u20030.10u2003mm3 were obtained. The X‐ray intensity data were collected at 2.2u2003Å resolution and the structure was refined to an R‐factor of 0.192. An extra electron density was observed at the binding site of α‐chymotrypsin, which was readily interpreted as a 14 residue fragment of α‐chymotrypsin corresponding to Ile‐Val‐Asn‐Gly‐Glu‐Glu‐Ala‐Val‐Pro‐Gly‐Ser‐Trp‐Pro‐Trp(16–29). The electron density for the eight residues of the C‐terminus, i.e. Ala22–Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N‐terminus, Ile16–Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0u2003σ between the active site Ser195 Oγ and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35u2003Å between Ser195 Oγ and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16–Trp29. In addition to the O–C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of α‐chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of α‐chymotrypsin.