Current Topics in Medicinal Chemistry

ISSN: 1568-0266

Current Topics in Medicinal Chemistry
Volume 7, Number 8, 2007

Contents

Phospholipase Inhibition: Medicinal Chemistry and Therapeutic Potential of PLA₂ Inhibitors
Guest Editor: Dr. K.S. Rangappa


Editorial Pp. 741-742


Snake Venom Phospholipases A₂ Inhibitors: Medicinal Chemistry and Therapeutic Potential Pp. 743-756
Silvana Marcussi, Carolina D. Sant’Ana, Clayton Z. Oliveira, Aristides Q. Rueda, Danilo L. Menaldo, Rene O. Beleboni, Rodrigo G. Stabeli, José R. Giglio, Marcos R. M. Fontes and Andreimar M. Soares
[Abstract]


Structural Elements of Ligand Recognition Site in Secretory Phospholipase A₂ and Structure-Based Design of Specific Inhibitors Pp. 757-764
Nagendra Singh, Rishi K. Somvanshi, Sujata Sharma, Sharmistha Dey, Punit Kaur and Tej P. Singh
[Abstract]


PLA₂ Mediated Arachidonate Free Radicals: PLA₂ Inhibition and Neutralization of Free Radicals by Anti-Oxidants – A New Role as Anti-Inflammatory Molecule Pp. 765-777
B.L. Nanda, A. Nataraju, R. Rajesh, K.S. Rangappa, M.A. Shekar and B.S. Vishwanath
[Abstract]


Structural Biology of Recombinant Bovine Pancreatic Phospholipase A₂ and its Inhibitor Complexes Pp. 779-785
K. Sekar
[Abstract]


Chemistry and Structural Evaluation of Different Phospholipase A₂ Inhibitors in Arachidonic Acid Pathway Mediated Inflammation and Snake Venom Toxicity Pp. 787-800
J.N. Narendra Sharath Chandra, K.C. Ponnappa, C.T. Sadashiva, B.S. Priya, B.L. Nanda, T. Veerabasappa Gowda, B.S. Vishwanath and K.S. Rangappa
[Abstract]


Research Article


Group IIA Secretory PLA₂ Inhibition by Ursolic Acid: A Potent Anti Inflammatory Molecule Pp. 801-809
A. Nataraju, C.D. Raghavendra Gowda, R. Rajesh and B.S. Vishwanath
[Abstract]


Synthesis and Evaluation of Tricyclic Dipyrido Diazepinone Derivatives as Inhibitors of Secretory Phospholipase A₂ with Anti Inflammatory Activity Pp. 811-820
N.R. Thimmegowda, K.K. Dharmappa, C.S. Ananda kumar, M.P. Sadashiva, A.D. Sathish, B.L. Nanda, B.S. Vishwanath and K.S. Rangappa
[Abstract]


Molecule of Month




Abstracts
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Editorial

Phospholipase A₂ (PLA₂) enzymes are widely distributed in mammalian systems and in the venoms of snakes, bees, scorpions and spiders. Several isoforms of PLA₂ enzymes are reported in mammalian system and the secretory isoform (sPLA₂) exhibit strong homology with the snake venom PLA₂s, which are again secretory in nature. sPLA₂ enzymes from snake venom in spite of their strong homology are involved in wide variety of pharmacological activities.

Role of sPLA₂ enzyme in the manifestation of many inflammatory reactions in mammals is very well established. Apart from inflammatory reactions venom sPLA₂ enzymes exhibit highly deleterious toxicities such as neurotoxicity (pre/post synaptic), cardiotoxicity, cytotoxicity, myotoxicity, nephrotoxicity, local tissue necrosis and also affect hemostasis (anti/pro coagulant). In some snake venoms these toxic PLA₂s are the principal toxins in their venom composition. Since the catalytic sites are highly conserved in these sPLA₂ enzymes inhibitors of venom sPLA₂ enzymes also inhibit mammalian sPLA₂ isoforms and vice versa. Due to these reasons, inhibitors of sPLA₂ enzymes are immensely important as therapeutic agents against inflammation and venom toxicities.

During inflammatory reactions, release of arachidonic acid in the free form is very critical for the synthesis of proinflammatory lipid mediators such as prostaglandins, leukotrienes and lipoxins. The other lipid mediator is platelet activating factor. Glucocortico steroids and their derivatives exert their powerful anti-inflammatory property by decreasing the levels of free arachidonic acid and their subsequent pro-inflammatory lipid mediators. How steroids decrease the level of free arachidonic acid is still not clear? The key enzyme involved in the release of arachidonic acid is PLA₂ enzyme and for the subsequent synthesis of pro-inflammatory lipid mediators are cyclooxygenase and lipoxygenase enzymes. Clinical studies revealed that PLA₂ enzyme action is the rate limiting step and in mammals sPLA₂ isoforms are implicated as pro-inflammatory. Specific inhibitors of this isoforms could be used as anti-inflammatory drugs, which are as powerful as steroids.

Knowing the importance of PLA₂ inhibitors for their therapeutic applications many Pharmaceutical Industries and academic Institutions are involved in the search for novel specific PLA₂ inhibitors. In literature, wide varieties of PLA2 inhibitors are reported. These inhibitors are characterized from plant sources, marine sponges, and animal sera. Based on these natural inhibitors, several new molecules were synthesized chemically and tested for PLA₂ inhibition. By understanding the substrate nature and the active site of the molecules many substrates analogues were synthesized and tried for effective PLA₂ inhibition. For snake venom sPLA₂ enzymes, both polyclonal and monoclonal antibodies were raised. In spite of such a large variety of molecules only few molecules are there in the clinical trial and many of them are best suited for topical application only.

In this special issue, review articles are invited from experts in the field of PLA₂ enzymes searching for specific inhibitors. These reviews comprehensively cover the vast array of PLA₂ inhibitory molecules and summarize the actual state and scope in the field of inflammation and venom toxicity. Two of the review articles from the laboratory of Prof. T. P. Singh et al., and Dr. K. Sekar discusses the binding characteristics of various PLA₂ inhibitors (both natural and synthesized) at the molecular level in the co-crystallized enzyme-inhibitor complex. Prof. A.M. Soares and his group review the venom PLA₂ inhibitors from wide range of sources that includes plant extracts and compounds from marine animals, mammals and snakes serum/plasma. In addition to these poly or monoclonal and synthetic molecules are discussed for the neutralization of venom PLA₂ toxicity. The review article from the group of Prof. B.S. Vishwanath discusses the importance of PLA₂ mediated inflammation with respect to the generation of arachidonate free radicals. Several anti-oxidants are known to have anti-inflammatory properties and their molecular basis for such dual actions is discussed and has given new dimensions for the existing beneficial actions of antioxidants. The review article from the laboratory of Prof. K.S. Rangappa comprises the chemical derivatization of various PLA₂ inhibitor molecules whose parent molecule is a PLA₂ inhibitor from a natural source. These derivatizations help in understanding the importance of various functional groups necessary for effective enzyme inhibition. This review article is in collaboration with Prof. T. Veerabasappa Gowda’s group who has contributed significantly in the field of venom PLA₂. They jointly argue that sPLA₂ inhibitors can be beneficial to both inflammatory disorders and also for venom PLA₂ toxicity. Apart from these review articles two research articles from the group of Prof. K.S. Rangappa and Prof. B.S. Viswhanath are there on PLA₂ inhibitors. Though exhaustive literatures are available in this field a sincere attempt has been made to provide latest information on PLA₂ inhibitors as they are beneficial against inflammation and venom PLA₂ toxicity.

Since these specific PLA₂ inhibitors are viewed as replacement for steroid anti-inflammatory drugs, they have the potential to capture over a billion dollar pharma market world wide. The most rewarding will be the blessings from the aged patients suffering from chronic inflammatory diseases to all the scientists involved in the generation of such PLA₂ inhibitors.


Prof. K.S. Rangappa
Department of Studies in Chemistry,
University of Mysore, Mysore,
India


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Snake Venom Phospholipases A₂ Inhibitors: Medicinal Chemistry and Therapeutic Potential
Silvana Marcussi, Carolina D. Sant’Ana, Clayton Z. Oliveira, Aristides Q. Rueda, Danilo L. Menaldo, Rene O. Beleboni, Rodrigo G. Stabeli, José R. Giglio, Marcos R. M. Fontes and Andreimar M. Soares

Phospholipases A₂ (PLA₂s) are commonly found in snake venoms from Viperidae, Hydrophidae and Elaphidae families and have been extensively studied due to their pharmacological and physiopathological effects in living organisms. This article reports a review on natural and artificial inhibitors of enzymatic, toxic and pharmacological effects induced by snake venom PLA₂s. These inhibitors act on PLA₂s through different mechanisms, most of them still not completely understood, including binding to specific domains, denaturation, modification of specific amino acid residues and others. Several substances have been evaluated regarding their effects against snake venoms and isolated toxins, including plant extracts and compounds from marine animals, mammals and snakes serum plasma, in addition to poly or monoclonal antibodies and several synthetic molecules. Research involving these inhibitors may be useful to understand the mechanism of action of PLA₂s and their role in envenomations caused by snake bite. Furthermore, the biotechnological potential of PLA₂ inhibitors may provide therapeutic molecular models with antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes.


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Structural Elements of Ligand Recognition Site in Secretory Phospholipase A₂ and Structure-Based Design of Specific Inhibitors
Nagendra Singh, Rishi K. Somvanshi, Sujata Sharma, Sharmistha Dey, Punit Kaur and Tej P. Singh

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 α-helix, H2 (residues: 40-55), a short two stranded anti-parallel β-sheet referred to as β-wing (residues: 75-84), a third α-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.


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PLA₂ Mediated Arachidonate Free Radicals: PLA₂ Inhibition and Neutralization of Free Radicals by Anti-Oxidants – A New Role as Anti-Inflammatory Molecule
B.L. Nanda, A. Nataraju, R. Rajesh, K.S. Rangappa, M.A. Shekar and B.S. Vishwanath

PLA₂ enzyme catalyses the hydrolysis of cellular phospholipids at the sn-2 position to liberate arachidonic acid and lysophospholipid to generate a family of pro-inflammatory eicosanoids and platelet activating factor. The generation of pro-inflammatory eicosanoids involves a series of free radical intermediates with simultaneous release of reactive oxygen species (superoxide and hydroxyl radicals). Reactive oxygen species formed during arachidonic acid metabolism generates lipid peroxides and the cytotoxic products such as 4-hydroxy nonenal and acrolein, which induces cellular damage. Thus PLA₂ catalyzes the rate-limiting step in the production of pro-inflammatory eicosanoids and free radicals. These peroxides and reactive oxygen species in turn activates PLA₂ enzyme and further attenuates the inflammatory process. Therefore scavenging these free radicals and inhibition of PLA₂ enzyme simultaneously by a single molecule such as antioxidants is of great therapeutic relevance for the development of anti-inflammatory molecules. PLA₂ enzymes have been classified into calcium dependent cPLA₂ and sPLA₂ and calcium independent iPLA₂ forms. In several inflammatory diseases sPLA₂ group IIA is the most abundant isoform identified. This isoform is therefore targeted for the development of anti-inflammatory molecules. Many secondary metabolites from plants and marine sponges exhibit both anti-inflammatory and antioxidant properties. Some of them include flavonoids, terpenes and alkaloids. But in terms of PLA₂ inhibition and antioxidant activity, the structural aspects of flavonoids are well studied rather than terpenes and alkaloids. In this line, molecules having both anti-oxidant and PLA₂ inhibitions are reviewed. A single molecule with dual activities may prove to be a powerful anti-inflammatory drug.


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Structural Biology of Recombinant Bovine Pancreatic Phospholipase A₂ and its Inhibitor Complexes
K. Sekar

The enzyme phospholipase A₂ catalyzes the cleavage of the sn-2 acyl ester bond of phospholipids, leading to the production of free fatty acids and lysophospholipids, which leads to many inflammatory disorders. In view of its pharmaceutical interest, three phospholipase A₂ + inhibitor (namely, (i) L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine, Transition State Analogue, (ii) 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol, MJ33 and (iii) p-methoxybenzoic acid, anisic acid) complex structures have already been solved and analysed, using the data obtained from X-ray diffraction. These structures provide insight on the mode of binding of the inhibitor molecules at the active site of phospholipase A₂. The knowledge of the active site geometry in these inhibitor bound structures, yield valuable information in the design of more useful therapeutic agents. This report reviews only the inhibitor bound recombinant bovine pancreatic phospholipase A₂ structures solved using X-ray crystallography.


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Chemistry and Structural Evaluation of Different Phospholipase A₂ Inhibitors in Arachidonic Acid Pathway Mediated Inflammation and Snake Venom Toxicity
J.N. Narendra Sharath Chandra, K.C. Ponnappa, C.T. Sadashiva, B.S. Priya, B.L. Nanda, T. Veerabasappa Gowda, B.S. Vishwanath and K.S. Rangappa

PLA₂ inhibitors specific to Group I and II PLA₂ isoforms are therapeutically important as anti-inflammatory molecules and against venom toxicity. From various natural sources diversified molecules with PLA₂ inhibition and concomitant neutralization of inflammatory reactions and venom toxicity were characterized. Using these molecules, lead compounds are generated in several laboratories. Analogues of lead molecules were generated by substituting different types of functional groups in order to obtain a molecule with optimal PLA₂ inhibition. The lead molecules characterized as PLA₂ inhibitors are indoles, azetidinones, piperazines, isoxazolidines, isoxazolines, diazepinones, acenaphthenes and several substrate analogues. The lead optimization involves relative hydrophobicity and substitution of functional groups, such as electron withdrawing or donating. Many such groups are placed on hydrophobic moiety and their positional bioisosters are characterized. Among these analogue piperazine derivatives on optimization with respect to hydrophobicity and electronegativity showed inhibition at nanomolar levels. Structural analysis of many lead molecules indicated that a PLA₂ inhibitor should have both hydrophobic moiety and polar functional groups. Each lead molecule requires optimization in this regard for effective inhibition.


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Group IIA Secretory PLA₂ Inhibition by Ursolic Acid: A Potent Anti Inflammatory Molecule
A. Nataraju, C.D. Raghavendra Gowda, R. Rajesh and B.S. Vishwanath

Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) isolated from many medicinal plants has diverse pharmacologically important properties, including strong anti-inflammatory activity. However its interaction with pro-inflammatory PLA₂ is not known. Ursolic acid inhibited secretory PLA₂ (sPLA₂) enzymes purified from Vipera russelli, Naja naja venom and human pleural fluid and synovial fluid. IC₅₀ values determined for these enzymes ranged from 12 to 18 μM. Group II secretory PLA₂ from both venoms & human inflammatory source were found to be sensitive to inhibition in comparison with group I cobra venom sPLA₂. Variation in Ca²⁺ concentration from 2.5 -15 mM did not alter the level of inhibition. Similarly sPLA₂ inhibition by ursolic acid is independent of substrate concentration. Ursolic acid interacts with purified venom sPLA₂ enzymes and enhances relative fluorescence intensity in a dose dependent manner. In the presence of ursolic acid apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme and formation of enzyme-ursolic acid complex. This complex results in irreversible inhibition of sPLA₂ as evident by dialysis study. Inhibition of sPLA₂ induced mouse paw edema and indirect hemolytic activity confirmed its sPLA₂ inhibitory activity in vivo and in situ respectively. These studies revealed that the strong anti-inflammatory activity of ursolic acid is by inhibiting sPLA₂ enzymes.


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Synthesis and Evaluation of Tricyclic Dipyrido Diazepinone Derivatives as Inhibitors of Secretory Phospholipase A₂ with Anti Inflammatory Activity
N.R. Thimmegowda, K.K. Dharmappa, C.S. Ananda kumar, M.P. Sadashiva, A.D. Sathish, B.L. Nanda, B.S. Vishwanath and K.S. Rangappa

A series of tricyclic dipyrido diazepinone derivatives 6(a-f) bearing different substituents at the tenth position of diazepinone ring were designed and are characterized by ¹H NMR, FTIR and X-Ray crystallography studies. The synthesised derivatives are tested in-vitro phospholipase A₂ (PLA₂) enzyme inhibitory activity and in-vivo anti-inflammatory activity against purified group I and group II PLA₂ enzymes from the snake venom and human pleural fluid. Compounds bearing aromatic ring with different substituents at different positions shown varied specificity. The 6f derivative with strong electron withdrawing nitro (-NO₂) and trifluoromethyl (-CF₃) groups at ortho and para positions respectively shown greater inhibitory activity. Inhibitory effect of the compound appeared to be direct interaction with active site and likely competes with substrates as supported by substrate dependent and calcium independent assays. The IC₅₀ value of potent PLA₂ inhibitor 6f was 22.1 μM and showed similar potency in the neutralization of in vivo PLA₂ induced mouse paw edema and hemolytic activity.