Current Topics in Medicinal Chemistry - Volume 7, Issue 8, 2007

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.....

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.

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.

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.

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.

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.

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 proinflammatory 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.

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 antiinflammatory 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.

A new “Alli” in weight loss. The FDA recently approved the country's first over-the-counter weight loss drug, appropriately named Alli (Orlistat), providing a readily available tool for the estimated 60 million Americans who are battling obesity [1]. Orlistat, first approved for prescription use in 1998, is a reversible inhibitor of gastric and pancreatic lipases, and causes a partial blockade of dietary fat absorption [2,3]. The compound, tetrahydrolipstatin, is derived from an endogenous lipostatin isolated from Streptomyces toxytricini [2]. Human studies showing that orlistat enhances weight loss in obese subjects were first reported in 1992 [4]. In conjunction with a low-calorie diet and moderate exercise, orlistat has been shown in doubleblind, placebo-controlled studies to reduce fat mass significantly more than diet and exercise alone [2,3]. Additional benefits of orlistat include reduced incidence of type 2 diabetes, lessening of risk factors for cardiovascular disease, and beneficial effects on blood pressure [2,3]. While Xenical, the prescription version of orlistat, is sold in 120 mg capsules, the nonprescription dose of Alli will be 60 mg. Studies performed by GlaxoSmithKline, which will manufacture Alli, indicate that the lower dose provides 85% of the weight loss benefit observed with the prescription dose [1]. While orlistat has a generally good safety record, many patients experience adverse gastrointestinal side effects due to an excess of excreted fat [2,3]. In addition, orlistat can reduce the absorption of fat-soluble vitamins and a handful of prescription drugs [2,3]. However, because very little drug is absorbed systemically, its negative drug interactions and potential for harmful systemic effects are minimal, making it relatively safe for nonprescription sale [1-3]. In addition to inhibiting gastrointestinal lipases, Orlistat is also an irreversible inhibitor of fatty acid synthase (FASN), an enzyme that synthesizes long-chain fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate (NADPH) [5]. Because FASN is minimally expressed in noncancerous cells, but is upregulated in many types of cancer cells, it has been identified as a possible therapeutic target for several types of cancer. Inhibitors of FASN, such as Orlistat and other β-lactones, have anti-proliferative and pro-apoptotic effects in cultured cancer cells expressing high levels of FASN, although the mechanisms underlying these effects are not well characterized. While Orlistat has no effect on non-cancerous cultured cells, cytotoxic effects have been observed against prostate, breast, colon, stomach, and ovarian cancer cells in culture [5]. Orlistat has also been shown to prevent tumor growth in a mouse xenograft model without obvious general toxicity, providing further evidence that it could be used to treat certain types of cancer [6]. Unfortunately, the low bioavailability of Orlistat would limit its application in human cancers to gastrointestinal tumors [5]. The synthesis of similar β-lactones with increased bioavailability and comparable levels of FASN inhibition could significantly enhance the therapeutic potential of Orlistat-like compounds. REFERENCES [1] Saul, Stephanie. ‘Over-the-counter weight loss drug is approved’ in the New York Times, Online edition, February 8, 2007. [2] Padwal, R.S.; Majumdar, S.R. ‘Drug treatments for obesity: orlistat, sibutramine, and rimonabant’ Lancet 2007, 369, 71-77. [3] Hennes, S.; Perry, C.M. ‘Orlistat: a review of its use in the management of obesity’ Drugs 2006, 66, 1625-1656. [4] Hauptman, J.B.; Jeunet, F.S.; Hartmann, D. ‘Initial studies in humans with the novel gastrointestinal lipase inhibitor Ro 18-0467’ Am J Clin Nutr. 1992, 55, 309S-313S. [5] Lupu, R.; Menendez, J.A. ‘Pharmacological inhibitors of Fatty Acid Synthase (FASN)-catalyzed endogenous fatty acid biogenesis: A new family of anti-cancer agents?’ Current Pharmaceutical Biotechnology 2006, 7, 483-494. [6] Kridel, S.J.; Axelrod, F.; Rozenkrantz, N.; Smith, J.W. ‘Orlistat is a novel inhibitor of fatty acid synthase with antitumor activity’ Cancer Res. 2004, 64, 2070-2075.