Human Nonpancreatic Secretory Phospholipase A2 Research Articles

Green synthesis, density functional theory (DFT), molecular docking, molecular dynamics simulation and biological activity of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives as PLA2 and proteinase K inhibitors

In diseases like atherosclerosis, rheumatoid arthritis, and sepsis, phospholipase A2 (PLA2) and proteinase K play a role in inflammation by releasing arachidonic acid (AA). The crucial step in the inflammatory process is believed to be the release of prostaglandins after PLA2 mobilizes AA. Drugs obstructing the COX and LOX pathways in the arachidonic acid cascade, drugs that inhibit these enzymes can treat inflammatory processes. To combat against these inflammatory promoters,the authors herein report an effective method for the synthesis of a series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives (4a–h) under the effect of TiO2 as a photocatalyst in ethanolic medium, and their effect against phospholipase A2, as well as proteinase K, was evaluated. The results confirmed that using TiO2 (20 mg) as a photocatalyst exhibits excellent performance in terms of yield and reaction time. The yield of all the synthesized compounds is between 79 and 91 % in just 60 min. After closely evaluating the SAR of the examined compounds against both enzymes, it was discovered that compounds (4b, 4f) with electron-donating substituents have higher PLA2 inhibitory (%) activity than those with electron-removing substituents (4a, 4e). Compounds (4c, 4d) containing heterocyclic rings showed significantly higher proteinase inhibitory (%) activity when compared to other electron-donating or withdrawing compounds. It was further confirmed that studied compounds against both enzymes exhibited dose-dependent behavior. We studied the intermolecular interactions of the compounds by molecular docking with human non-pancreatic secretory phospholipase A2 and proteinase K. We evaluated the dynamical stability of the docked complexes using a 100 ns molecular dynamics simulation, which revealed stable interactions based on root mean square deviation/fluctuation (RMSD/RMSF), radius of gyration, Gibbs free energy landscapes, and principal component analyses (PCA). Moreover, the ADME properties of the compounds align with Lipinski’s rule of five, suggesting their potential as viable candidates for the development of therapies against inflammatory diseases. All compounds tested inhibited PLA2 more than proteinase K. However, compounds 4b and 4f better inhibited phospholipase A2, whereas compounds 4c and 4d showed better activity against proteinase K.

Synthesis and Biological Evaluation of Octahydroquinazolinones as Phospholipase A2, and Protease Inhibitors: Experimental and Theoretical Exploration

Phospholipase A2 (PLA2) promotes inflammation via lipid mediators and releases arachidonic acid (AA), and these enzymes have been found to be elevated in a variety of diseases, including rheumatoid arthritis, sepsis, and atherosclerosis. The mobilization of AA by PLA2 and subsequent synthesis of prostaglandins are regarded as critical events in inflammation. Inflammatory processes may be treated with drugs that inhibit PLA2, thereby blocking the COX and LOX pathways in the AA cascade. To address this issue, we report herein an efficient method for the synthesis of a series of octahydroquinazolinone compounds (4a-h) in the presence of the catalyst Pd-HPW/SiO2 and their phospholipase A2, as well as protease inhibitory activities. Among eight compounds, two of them exhibited overwhelming results against PLA2 and protease. By using FT-IR, Raman, NMR, and mass spectroscopy, two novel compounds were thoroughly studied. After carefully examining the SAR of the investigated compounds against these enzymes, it was found that compounds (4a, 4b) containing both electron-donating and electron-withdrawing groups on the phenyl ring exhibited higher activity than compounds with only one of these groups. DFT studies were employed to study the electronic nature and reactivity properties of the molecules by optimizing at the BLYP/cc-pVDZ. Natural bond orbitals helped to study the various electron delocalizations in the molecules, and the frontier molecular orbitals helped with the reactivity and stability parameters. The nature and extent of the expressed biological activity of the molecule were studied using molecular docking with human non-pancreatic secretory phospholipase A2 (hnps-PLA2) (PDB ID: 1DB4) and protease K (PDB ID: 2PWB). The drug-ability of the molecule has been tested using ADMET, and pharmacodynamics data have been extracted. Both the compounds qualify for ADME properties and follow Lipinski's rule of five.

Discovery of Novel Secretory Phospholipase A2 Inhibitors Using Virtual Screen

Human non-pancreatic secretory phospholipase A2 was reported to be associated with inflammatory diseases and considered as a potential drug target for inflammation and other related disease treatment. Although many human non-pancreatic secretory phospholipase A2 inhibitors were reported, few entered into the drug development stage due to various problems. In this study, we discovered seven novel human non-pancreatic secretory phospholipase A2 inhibitors using virtual screen. Of the 99 compounds tested by continuous fluorescence assay, seven are potent human non-pancreatic secretory phospholipase A2 inhibitors with micromolar IC50 values. Typical molecules include 9-fluorenylmethoxycarbonyl protected α-phenylalanine derivatives and azo compounds, which may serve as novel scaffold for developing potent human non-pancreatic secretory phospholipase A2 inhibitors. These compounds bind to human non-pancreatic secretory phospholipase A2 by interacting with the catalytic calcium ion and the hydrophobic regions in the substrate-binding pocket.

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) is a group IIA phospholipase A2 which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA2 is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA2 mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA2 and the two mutants. Both native hnpsPLA2 and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA2 is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.

Tailoring the pH Dependence of Human Non-pancreatic Secretory Phospholipase A2 by Engineering Surface Charges

Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) catalyzes the sn-2 acyl hydrolysis of phospholipids. It was reported that hnpsPLA2 is involved in various diseases like inflammation, cancer, and so on. This enzyme also exhibits anti-bacterial and anti-virus activities. It is active over a broad pH range, with higher activity at alkaline conditions. In order to make it suitable as a potential bactericide, high activity at neutral pH is preferable. We have tried to tailor the pH dependence of hnpsPLA2 activity by replacing its surface charged residues. Three surface charge replacements, Arg42Glu, Arg100Glu, and Glu89Lys, showed increased activities at neutral pH, which are 2.3, 2.8, and 2.3 times that of the wild-type enzyme at pH7. Both the positive-to-negative and negative-to-positive mutations lowered the optimum enzymatic reaction pH of hnpsPLA2, indicating that the enzyme pH profile depends on a delicate balance of charged residues. The activity changes are in good agreement with the recently proposed calcium-coordinated catalytic triad mechanism. This study also provides a general means of enhancing hnpsPLA2 activity at low pH.

Optimization of 5-hydroxytryptamines as dual function inhibitors targeting phospholipase A2 and leukotriene A4 hydrolase

Dual function inhibitors targeting phospholipase A2 (PLA2) and leukotriene A4 hydrolase (LTA4H) may balance the arachidonic acid (AA) metabolic network and be used as new anti-inflammatory drugs. In previous study, we discovered multi-target drugs towards the AA metabolic network, among which a dual-target inhibitor (JMC08-4) for human nonpancreatic secretory phospholipase A2 (hnps-PLA2) and human leukotriene A4 hydrolase (LTA4H-h) was found. Based on the structure of compound JMC08-4, new dual-target inhibitors were designed assisted by molecular docking. In this report, a series of 5-hydroxytryptamine compounds were synthesized; and most of these title compounds showed more potent inhibitory activity than compound JMC08-4 in the in vitro bioassay against these two enzymes. The best one inhibited hnps-PLA2 and LTA4H-h with IC50 values of 9.2 ± 0.5 μM and 2.4 ± 1.4 μM, respectively.

Discovery of Multitarget Inhibitors by Combining Molecular Docking with Common Pharmacophore Matching

Multitarget drugs have been to be found effective in controlling complex diseases. However, how to design multitarget drugs presents a great challenge. We have developed a computer-assisted strategy to screen for multitarget inhibitors using a combination of molecular docking and common pharmacophore matching. This strategy was successfully applied to screen for dual-target inhibitors against both the human leukotriene A(4) hydrolase (LTA4H-h) and the human nonpancreatic secretory phospholipase A2 (hnps-PLA2). Three compounds screened from the chemical database MDL Available Chemical Directory were found to inhibit these two enzymes at the 10 microM level. Moreover, one synthetic compound matching the common pharmacophores inhibits LTA4H-h and hnps-PLA2 with IC(50) values of 35 nM and 7.3 microM, respectively. The common pharmacophore model can also be used to search small molecule databases or collections of existing inhibitors, as well as to guide combinatorial library design to search for ideal multitarget inhibitors.

Chemically Induced Dimerization of Human Nonpancreatic Secretory Phospholipase A2 by Bis-indole Derivatives

A series of novel bis-indole compounds, 1,omega-bis(((3-acetamino-5-methoxy-2-methylindole)-2-methylene)phenoxy)alkane, have been designed and synthesized on the basis of the enzyme structure of human nonpancreatic secretory phospholipase A2 (hnps PLA2). Their inhibition activities against hnps PLA2 were improved compared to that of the monofunctional protocompound. These bivalent ligands not only inhibited hnps PLA2 but also drove the dimerization of hnps PLA2. Their dimerization ability correlated with the linker length and position. Further study on the potent compound 5 (1,5-bis(((3-acetamino-5-methoxy-2-methylindole)-2-methylene)phenoxy)pentane, IC50 = 24 nM) revealed that cooperative binding interactions between the two enzyme molecules also contributed to the stability of the ternary complex. The combination of bivalent ligands and hnps PLA2 can be used as a novel chemically induced dimerization (CID) system for designing regulatory inhibitors.

Indole-5-phenylcarbamate derivatives as human non-pancreatic secretory phospholipase A2 inhibitor

The synthesis of the human non-pancreatic secretory phospholipase A2 inhibitor (IC 50 = 1.81 ± 0.59 μM) is reported.

Carbocyclic[g]indole Inhibitors of Human Nonpancreatic s-PLA2

A vinyl azide cyclization method was used to synthesize three different carbocyclic[g]indole scaffolds as inhibitors of human nonpancreatic secretory phospholipase A2. Each scaffold demonstrated potent enzyme activity in a chromogenic assay system, with select examples also demonstrating potent activity in a secondary DOC/PC assay. Compound 11, representative of the cyclopent[g]indole series, gave an IC50 of 10 nM for the inhibition of hnps-PLA2 in the chromogenic assay.

Small Peptides Do Not Inhibit Human Non-Pancreatic Secretory Phospholipase-A2 (Type IIA)

Seven small peptides, that are among the most potent reported inhibitors of secreted mammalian phospholipases A2, were found not to inhibit processing of a small phospholipid substrate by human non-pancreatic secretory phospholipase A2 (type IIa), under conditions where certain non-peptides are potent inhibitors at nanomolar concentrations.

ChemInform Abstract: Indole Inhibitors of Human Nonpancreatic Secretory Phospholipase A2. Part 2. Indole‐3‐acetamides with Additional Functionality.

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Binding of Human Phospholipase A2 Type II to Proteoglycans: DIFFERENTIAL EFFECT OF GLYCOSAMINOGLYCANS ON ENZYME ACTIVITY

Phospholipase A2 acting on low density lipoproteins in the extracellular arterial intima may form proinflammatory lipid mediators. Human nonpancreatic secretory phospholipase A2 has three regions that may associate with sulfated glycosaminoglycans. The apoB-100 molecule in low density lipoproteins also has glycosaminoglycan binding regions that could mediate its retention in the arterial intima. Here we report that human nonpancreatic phospholipase A2 isolated from a transfected cell line binds to glycosaminoglycans secreted by cultured human arterial smooth muscle cells. A gel mobility shift assay showed that the affinity of phospholipase A2 for glycosaminoglycans from a heparan sulfate/chondroitin sulfate proteoglycan was higher than for chondroitin sulfate glycosaminoglycans from a larger versican-like proteoglycan. Affinity chromatography confirmed these results. All glycosaminoglycans tested, at concentrations up to 100 microM, increased the activity of phospholipase A2 toward phosphatidylcholine liposomes. Above this concentration, heparan sulfate and heparin inhibited the enzyme. Heparin and chondroitin 6-sulfate increased phospholipase A2 activity on low density lipoproteins up to 4-fold at 100 microM, whereas heparan sulfate had no effect. The results indicate that human nonpancreatic secretory phospholipase A2 interacts with proteoglycans via their glycosaminoglycan moiety and that the enzyme activity may be modulated by the association of the enzyme and its substrate to the sulfated polysaccharides.

Indole inhibitors of human nonpancreatic secretory phospholipase A2. 2. Indole-3-acetamides with additional functionality.

As reported in our previous paper, a series of indole-3-acetamides which possessed potency and selectivity as inhibitors of human nonpancreatic secretory phospholipase A2(hnps-PLA2) was developed. The design of these compounds was based on information derived from x-ray crystal structures determined for complexes between the enzyme and its inhibitors. We describe here the further implementation of this structure-based design strategy and continued SAR development to produce indole-3-acetamides with additional functionalities which provide increased interaction with important residues within the enzyme active site. These efforts led to inhibitors with substantially enhanced potency and selectivity.

Indole Inhibitors of Human Nonpancreatic Secretory Phospholipase A2. 1. Indole-3-acetamides

Phospholipases (PLAs) produce rate-limiting precursors in the biosynthesis of various types of biologically active lipids involved in inflammatory processes. Increased levels of human nonpancreatic secretory phospholipase A2 (hnps-PLA2) have been detected in several pathological conditions. An inhibitor of this enzyme could have therapeutic utility. A broad screening program was carried out to identify chemical structures which could inhibit hnps-PLA2. One of the lead compounds generated by the screening program was 5-methoxy-2-methyl-1-(phenylmethyl)-1H-indole-3-acetic acid (13a). We describe the syntheses, structure--activity relationships, and pharmacological activities of a series of indole-3-acetamides and related compounds derived from this lead. This SAR was undertaken with the aid of X-ray crystal structures of complexes between the inhibitors and hnps-PLA2 which were of great value in directing the SAR.

Structure-based design of the first potent and selective inhibitor of human non-pancreatic secretory phospholipase A2

A lead compound obtained from a high volume human non-pancreatic secretory phospholipase A2 (hnps-PLA2) screen has been developed into a potent inhibitor using detailed structural knowledge of inhibitor binding to the enzyme active site. Four crystal structures of hnps-PLA2 complexed with a series of increasingly potent indole inhibitors were determined and used as the structural basis for both understanding this binding and providing valuable insights for further development. The application of structure-based drug design has made possible improvements in the binding of this screening lead to the enzyme by nearly three orders of magnitude. Furthermore, the optimized structure (LY311727) displayed 1,500-fold selectivity when assayed against porcine pancreatic s-PLA2.

Structures of Free and Inhibited Human Secretory Phospholipase A 2 from Inflammatory Exudate

Phospholipase A2 (PLA2) participates in a wide range of cellular processes including inflammation and transmembrane signaling. A human nonpancreatic secretory PLA2 (hnps-PLA2) has been identified that is found in high concentrations in the synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. This enzyme is secreted from certain cell types in response to the proinflammatory cytokines, tumor necrosis factor or interleukin-1. The crystal structures of the calcium-bound form of this enzyme have been determined at physiological pH both in the presence [2.1 angstrom (A) resolution] and absence (2.2 A resolution) of a transition-state analogue. Although the critical features that suggest the chemistry of catalysis are identical to those inferred from the crystal structures of other extracellular PLA2s, the shape of the hydrophobic channel of hnps-PLA2 is uniquely modulated by substrate binding.