P. K. Mandal

Anti-Inflammatory Property of n-Hexadecanoic Acid: Structural Evidence and Kinetic Assessment

Ester bond hydrolysis of membrane phospholipids by Phospholipase A2 and consequent release of fatty acids are the initiating steps of inflammation. It is proposed in this study that the inhibition of phospholipase A2 is one of the ways to control inflammation. Investigations are carried out to identify the mode of inhibition of phospholipase A2 by the n‐hexadecanoic acid. It may help in designing of specific inhibitors of phospholipase A2 as anti‐inflammatory agents. The enzyme kinetics study proved that n‐hexadecanoic acid inhibits phospholipase A2 in a competitive manner. It was identified from the crystal structure at 2.5 Å resolution that the position of n‐hexadecanoic acid is in the active site of the phospholipase A2. The binding constant and binding energy have also been calculated using Isothermal Titration Calorimetry. Also, the binding energy of n‐hexadecanoic acid to phospholipase A2 was calculated by in silico method and compared with known inhibitors. It may be concluded from the structural and kinetics studies that the fatty acid, n‐hexadecanoic acid, is an inhibitor of phospholipase A2, hence, an anti‐inflammatory compound. The inferences from the present study validate the rigorous use of medicated oils rich in n‐hexadecanoic acid for the treatment of rheumatic symptoms in the traditional medical system of India, Ayurveda.

Binding to PLA2 May Contribute to the Anti-Inflammatory Activity of Catechol

Inhibiting PLA2 activity should, in theory, be an effective approach to control the inflammation. Several naturally occurring polyphenolic compounds have been reported as inhibitors of PLA2. Among the naturally occurring polyphenols, catechol (1,2‐dihydroxybenzene) possesses anti‐inflammatory activity. Catechol can inhibit cyclooxygenase and lipo‐oxygenase. By means of enzyme kinetic study, it was revealed that catechol can inhibit PLA2 also. Crystal structure showed that catechol binds to PLA2 at the opening of the active site cleft. This might stop the entry of substrate into the active site. Hence, catechol can be used as a lead compound for the development of novel anti‐inflammatory drugs with PLA2 as the target.

Structure of the tetradecanucleotide d(CCCCGGTACCGGGG)2 as an A‐DNA duplex

The crystal structure of the tetradecanucleotide sequence d(CCCCGGTACCGGGG)(2) has been determined at 2.5 Å resolution in the tetragonal space group P4(1). This sequence was designed with the expectation of a four-way junction. However, the sequence crystallized as an A-DNA duplex and represents more than one full turn of the A-helix. The crystallographic asymmetric unit consists of one tetradecanucleotide duplex. The structural parameters of the A-type DNA duplex structure and the crystal-packing arrangement are described. One Mn(2+) ion was identified with direct coordination to the N7 position of G(13) and a water molecule at the major-groove side of the C(2)·G(13) base pair.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of carbonyl reductase from Candida parapsilosis ATCC 7330.

The NAD(P)H-dependent carbonyl reductase from Candida parapsilosis ATCC 7330 catalyses the asymmetric reduction of ethyl 4-phenyl-2-oxobutanoate to ethyl (R)-4-phenyl-2-hydroxybutanoate, a precursor of angiotensin-converting enzyme inhibitors such as Cilazapril and Benazepril. The carbonyl reductase was expressed in Escherichia coli and purified by GST-affinity and size-exclusion chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.86 Å resolution. The asymmetric unit contained two molecules of carbonyl reductase, with a solvent content of 48%. The structure was solved by molecular replacement using cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae as a search model.

Interactions of Mn2+ with a non-self-complementary Z-type DNA duplex.

Crystal structures of the hexanucleotide d(CACGCG)·d(CGCGTG) were determined in two crystal lattices when different concentrations of the counterion Mn2+ were used in crystallization. The availability of Mn2+ during the crystallization process appears to play an important role in inducing different crystal packings that lead to crystals belonging to the two space groups P2(1) and P6(5). Analysis of the molecular interactions of Mn2+ with the Z-form duplexes shows direct coordination to the purine residues G and A.

Crystal structure of porcine pancreatic phospholipase A2 in complex with 2-methoxycyclohexa-2-5-diene-1,4-dione

Curcumin possess anti-inflammatory activity. In this study, the binding of curcumin with PLA2 was studied using X-ray crystallography. Since the electron density found at the active site did not match with curcumin, 2-methoxycyclohexa-2-5-diene-1,4-dione (MCW) (the photodegraded product of curcumin) was fitted in the unexplained electron density. To understand the binding mode of actual curcumin, molecular docking study was carried out. The crystallographic and docked structures were superimposed with respect to the ligand position and it was found that curcumin was binding in the hydrophobic cavity of PLA2 with a binding energy of−16.81 Kcal/mol. The binding mode was of such a nature that it prevented the entry of the substrate to the hydrophobic active site. This study indicates that curcumin can act as an inhibitor of PLA2. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:FLS:1.

Structure of d(CGGGTACCCG)4 as a four-way Holliday junction

The crystal structure of the decamer sequence d(CGGGTACCCG)(4) as a four-way Holliday junction has been determined at 2.35 Å resolution. The sequence was designed in order to understand the principles that govern the relationship between sequence and branching structure. It crystallized as a four-way junction structure with an overall geometry similar to those of previously determined Holliday junction structures.

The structure of d(CACACG).d(CGTGTG).

The crystal structure of d(CACACG).d(CGTGTG) was solved to a resolution of 2.05 A in space group P2(1). The duplex assumes the left-handed Z-DNA structure. The presence of two A.T base pairs in the hexamer does not greatly affect the conformation. The most significant changes compared with the regular structure of Z-DNA are in the values of twist in the central portion of the helix. This variation, as well as others in the values of roll, inclination etc., follow the pattern observed previously in the structure of d(CGCACG).d(CGTGCG).

Structure of d(CCCCGGTACCGGGG)2 at 1.65 Å resolution

The crystal structure of the tetradecanucleotide d(CCCCGGTACCGGGG)2 has previously been reported as an A-type double helix at a resolution of 2.5 Å in space group P41. Here, the structure of this sequence was determined at a significantly higher resolution of 1.65 Å in space group P4₁2₁2. The differences in crystal packing between the former and latter are described. The crystallographic asymmetric unit consists of one tetradecanucleotide duplex that spans more than one full turn of the A-helix. This structure allowed the unambiguous identification of solvent interactions.

Crystal structure of phospholipase A2 in complex with 1-naphthaleneacetic acid: PHOSPHOLIPASE A2 IN COMPLEX WITH 1-NAPHTHALENEACETIC ACID

Phospholipase A2 (PLA2) is one of the rate limiting enzymes involved in the production of arachidonic acid, a potent inflammatory mediator. PLA2 is widely distributed all over the animal kingdom. It is also seen in inflammatory exudation and venoms of different organisms. The studies demonstrated that PLA2 inhibitors have broad spectrum activities that they can either be used against inflammation or envenomation. In this study, the inhibitory activity of 1‐napthaleneacetic acid (NAA) against porcine pancreatic PLA2 has been explained through isothermal titration calorimetry and enzyme kinetics studies. The atomic level of interactions of NAA with PLA2 was also studied using X‐ray crystallography. Apart from these findings, the theoretical binding affinities and mode of interactions of two naphthalene‐based NSAIDs such as naproxen (NAP) and nabumetone (NAB) were studied through molecular modeling. The studies proved that the selected ligands are binding at the doorway of the active site cleft and hindering the substrate entry to the active site. The study brings out a potential scaffold for the designing of broad spectrum PLA2 inhibitors which can be used for inflammation or envenomation.