Phycocyanin of marine Oscillatoria sp. inhibits lipoxygenase by protein-protein interaction-induced change of active site entry apace: A model for non-specific biofunctions of phycocyanins.

An overview of the biological properties of phycocyanin (PC) amply illustrates that it may not have any specific functional feature towards any system at which it may elicit a specific function, but for the molecular interactions. Nevertheless, based on existing evidences, it is hypothesized that PC has more than one functional target with the interacting systems; therefore, it has diversity of effects. The mechanism of PC action remains elusive of a comprehensive idea. The present investigation focuses on the pro inflammatory enzyme, lipoxygenase (LOX) inhibiting property of PC purified from Oscillatoria sp. Enzyme kinetics studies show that the molecular composite of PC is required for its inhibition shown on LOX. Isothermal titration calorimetric study proves that one molecule of PC interacts with two molecules of LOX. Molecular dynamics simulation study pertaining to PC-LOX interactions shows it to be appropriate as a model to give molecular mechanistic insight into the varied biological properties of PC, demonstrated elsewhere in experimental studies including animal model studies. It explains that the PC-LOX interaction is of a function-freezing, protein-protein interaction in nature. The wide spectrum of properties of PC might be due to its function as a powerful protein hub showing non-specific protein-protein interactions.

A novel fibrinolytic enzyme from marine Pseudomonas aeruginosa KU1 and its rapid in vivo thrombolysis with little haemolysis.

A direct acting, extracellular, fibrinolytic enzyme, ~50 KDa from marine Pseudomonas aeruginosa KU1 (PEKU1), was purified. It was found to be a metalloprotease. 60% of the total activity of the purified PEKU1 was retained at 70 °C and the enzyme was practically denatured at 80 °C, 2 h. Metal ions, such as Na+, K+ and Co2+, were found to enhance slightly the fibrinolytic activity, while Fe2+, Mn2+ and Zn2+ were found to be inhibiting. The enzyme showed only less than 5% haemolysis, suggesting its thrombolytic administration safe. Tryptic digestion revealed its similarity to serralysin like alkaline protease of P. aeruginosa. In silico studies showed its binding of protease substrates and fibrin D-dimer in its active site. High affinity binding of bradykinin to the active site of PEKU1, confirmed by in vitro cleaving, suggested its future use as an analgesic. The purified enzyme with Na+, K+ and Co2+, and without Fe2+, Mn2+ and Zn2+ showed thrombolysis in vivo in carrageenan induced murine tail thrombolytic model. The enzyme PEKU1, a novel protease from marine isolate Pseudomonas aeruginosa KU1 has great potential to be developed as a therapeutic agent to combat cardiovascular diseases, as well as analgesic and anti-inflammatory drug in appropriate sites.

Crystal structure of 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. © 2018 IUBMB Life, 70(10):995-1001, 2018.

Derivatives form better lipoxygenase inhibitors than piperine: in vitro and in silico study.

Piperine is a secondary metabolite of black pepper. Its uses in medicine were already studied. However, its derivatives have not gained considerable attention. In the presented study, the Lipoxygenase (LOX) inhibitory activity of piperine and its derivatives, piperonylic acid, piperic acid, and piperonal have been assessed and compared by enzyme kinetics, ITC and molecular modeling experiments. The presented investigations expressed that all the studied compounds inhibited LOX by binding at its active site. The IC(50) values of these compounds were deduced from the kinetics data and found to be 85.79, 43.065, 45.17, and 50.78 µm for piperine, piperonylic acid, piperic acid, and piperonal, respectively. The binding free energies obtained from ITC experiments were -7.47, -8.33, -8.09, and -7.86 kcal/mol for piperine, piperonylic acid, piperic acid, and piperonal, respectively. Similarly, the glide scores obtained for piperine, piperonylic acid, piperic acid, and piperonal were -7.28, -10.32, -10.72, and -9.57 kcal/mol, respectively. The results of ITC and molecular modeling experiments suggested that piperonylic acid and piperonal exhibit stronger binding at the active site than piperine does. From the presented studies, it could be concluded that derivatives of piperine may be of higher significance than piperine for certain medicinal applications, implicating (Ayurvedic) fermented herbal drugs with piperine in them.

Interactions of selected indole derivatives with phospholipase A2: in silico and in vitro analysis.

Phospholipase A2 (PLA2) is one of the key enzymes involved in the formation of inflammatory mediators. Inhibition of PLA2 is considered to be one of the efficient methods to control inflammation. In silico docking studies of 160 selected indole derivatives performed against porcine pancreatic PLA2 (ppsPLA2) suggested that, CID2324681, CID8617 (indolebutyric acid or IBA), CID22097771 and CID802 (indoleacetic acid or IAA) exhibited highest binding energies. In silico analysis was carried out to predict some of the ADME properties. The binding potential of these compounds with human non pancreatic secretory PLA2 (hnpsPLA2) was determined using molecular docking studies. In order to corroborate the in silico results, enzyme kinetics and isothermal titration calorimetric analysis of the two selected compounds, IAA and IBA were performed against ppsPLA2. From the analysis, it was concluded that IAA and IBA can act as competitive inhibitors to the enzyme and may be used as anti inflammatory agents.

Anti-inflammatory property of n-hexadecanoic acid: structural evidence and kinetic assessment.

Ester bond hydrolysis of membrane phospholipids by Phospholipase A(2) and consequent release of fatty acids are the initiating steps of inflammation. It is proposed in this study that the inhibition of phospholipase A(2) is one of the ways to control inflammation. Investigations are carried out to identify the mode of inhibition of phospholipase A(2) by the n-hexadecanoic acid. It may help in designing of specific inhibitors of phospholipase A(2) as anti-inflammatory agents. The enzyme kinetics study proved that n-hexadecanoic acid inhibits phospholipase A(2) 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 A(2). The binding constant and binding energy have also been calculated using Isothermal Titration Calorimetry. Also, the binding energy of n-hexadecanoic acid to phospholipase A(2) 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 A(2), 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 PLA(2) activity should, in theory, be an effective approach to control the inflammation. Several naturally occurring polyphenolic compounds have been reported as inhibitors of PLA(2) . 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 PLA(2) also. Crystal structure showed that catechol binds to PLA(2) 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 PLA(2) as the target.