Inhibition of the complete set of mammalian secreted phospholipases A2 by indole analogues: a structure-guided study

doi:10.1016/j.bmc.2004.01.022

Bioorganic & Medicinal Chemistry

Volume 12, Issue 7, 1 April 2004, Pages 1737-1749

https://doi.org/10.1016/j.bmc.2004.01.022 Get rights and content

Abstract

Structure-guided design was employed in a search for potent and selective inhibitors of mammalian secreted phospholipases A₂ (sPLA₂s). Using the X-ray structures of human groups IIA and X sPLA₂s (hGIIA and hGX) as templates, homology structural models were made for the other human and mouse sPLA₂s (hGIB, mGIB, mGIIA, mGIIC, hGIID, mGIID, hGIIE, mGIIE, hGIIF, mGIIF, hGV, mGV, and mGX). Me-Indoxam is a previously discovered indole analogue that binds tightly to many sPLA₂s, and the X-ray structure of the hGX-Me-Indoxam complex was determined at a resolution of 2.0 Å. Modeling suggests that the residues near the N₁-substituent of Me-Indoxam vary significantly among the mammalian sPLA₂s, and therefore a library of 83 N₁-variants was prepared by parallel synthesis. Several Me-Indoxam analogues bearing a 4-(2-oxy-ethanoic acid) side chain were potent inhibitors (IC₅₀ <0.05 μM) of hGIIA, mGIIA, mGIIC, hGIIE, mGIIE, hGV, and mGV, while they displayed intermediate potency (0.05–5 μM) against hGIB, mGIB, hGX, and mGX, and poorly inhibited (>5 μM) hGIID, mGIID, hGIIF, and mGIIF. Me-Indoxam analogues bearing a 5-(4-oxy-butanoic acid) side chain were generally less potent inhibitors. Although no compounds were found to be highly specific for a single human or mouse sPLA₂, combinations of Me-Indoxam analogues were discovered that could be used to distinguish the action of various sPLA₂s in cellular events. For example, Me-Indoxam and compound 5 are approximately 5-fold more potent on hGIIA than on hGV, and compound 21 is 10-fold more potent on hGV versus hGIIA.

Graphic

Introduction

The phospholipase A₂ class of enzymes catalyze the hydrolysis of the sn-2 ester of glycero-phospholipids to release fatty acids and lysophospholipids.¹ One or more of these enzymes is thought to initiate pro-inflammatory cascades by liberating arachidonic acid from membrane phospholipids for the biosynthesis of the eicosanoids (prostaglandins, leukotrienes, and others). The current thinking is that arachidonic acid liberation from the membranes of mammalian cells is catalyzed by the 87-kDa cytosolic phospholipase A₂ (group IVA) and by one or more low molecular weight, secreted phospholipases A₂ (sPLA₂s).2, 3 Understanding the role of sPLA₂s in this process is complicated by the fact that mammals contain a large number of these enzymes. Humans contain the groups IB, IIA, IID, IIE, IIF, III, V, X, and XIIA sPLA₂s (designated hGIB, hGIIA, etc.), whereas the mouse contains all of these sPLA₂s (designated mGIB, mGIIA, etc.) plus the group IIC enzyme (which is present in the human genome as a pseudogene).4, 5 Deciphering the role played by sPLA₂s in arachidonic acid release as well as in other physiological events would be greatly aided by the availability of potent and specific inhibitors of each mammalian homologue.

Of the numerous known sPLA₂ inhibitors, many are non-specific agents that act by perturbing the physical structure of the membrane substrate.⁶ sPLA₂s are water-soluble enzymes that must bind to the membrane surface to access their water-insoluble substrates, (interfacial enzymes).⁷ Methods have been developed for the proper quantitative analysis of inhibitors of interfacial enzymes that allow ‘true’, active site-directed, competitive inhibitors to be investigated.6, 8, 9 Inhibitors that have been convincingly established to be potent and active site-directed competitive inhibitors of sPLA₂s either by proper kinetic analysis or by determination of the enzyme-inhibitor X-ray structure include phospholipid analogues (i.e., sn-2 amides and phosphonates)10, 11, 12, 13, 14 as well as non-phospholipid analogues including the plant-derived alkaloid aristolochic acid,¹⁵ 1,3-dioxane-4,6-dione-5-carboxamides,16, 17 fatty acid amides,¹⁸ aromatic sulfonamides (i.e., SB-203347),¹⁹ plant-derived sterols,²⁰ bis-carboxylates,21, 22 complex poly-p-hydroxy benzoate natural products,23, 24, 25 highly substituted pyrazoles,²⁶ short peptides,27, 28 vitamin E analogues,²⁹ and indole analogues.30, 31, 32, 33 The X-ray structure of some of these compounds bound to hGIIA, porcine and bovine pancreatic sPLA₂s, and venom sPLA₂s have been determined.15, 17, 27, 28, 29, 30, 34, 35, 36, 37, 38, 39, 40, 41

Of these sPLA₂ inhibitors, the indole analogues developed by workers at Lilly and Shionogi laboratories are particularly appealing for further study as they appear to have properties most suitable for use in studies with cultured mammalian cells and are the most generally potent among the full set of human and mouse sPLA₂s.⁴² We have tested the indole analogue Me-Indoxam (Fig. 1) on all of the human and mouse sPLA₂s except the human and mouse group III and mouse group XIIA enzymes, and found it to be a potent inhibitor of mGIIA, hGIIA, mGIIC, mGIIE, hGIIE, mGV, and hGV (IC₅₀∼0.01–0.02 μM), and a modestly potent inhibitor of mGIB, hGIB, mGX, and hGX (IC₅₀ ∼0.1–1 μM).⁴² On the other hand Me-Indoxam is a poor inhibitor of mGIID, hGIID, mGIIF, hGIIF, and hGXIIA (IC₅₀>10 μM).⁴²

The groups V and X sPLA₂s are particularly appealing for further study because these enzymes display high enzymatic activity when added exogenously to a variety of mammalian cells,43, 44, 45, 46, 47, 48 whereas the other mammalian sPLA₂s are poorly active in this assay⁴² (the human and mouse group III enzymes remain to be analyzed on mammalian cells). These two sPLA₂s display the unique ability to bind to phosphatidylcholine-rich vesicles in vitro, which accounts for their ability to act on the phosphatidylcholine-rich outer leaflet of the mammalian cell plasma membrane.42, 43, 44, 45 Thus, among the sPLA₂ family members, group V and X enzymes seem to be the strongest candidates for playing a role in arachidonic acid liberation for eicosanoid production. For example, a role for mGV in lipopolysaccharide-induced arachidonic acid release from a murine macrophage-like cell line has been reported.⁴⁹ The group V and X sPLA₂s also show relatively high activity on serum lipoproteins, and may play a role in the development of atherosclerosis.50, 51

In the present study, we extend our structural studies of hGX⁵² by reporting the 2.0 Å X-ray crystal structure of this sPLA₂ complexed with Me-Indoxam. This structure was then used as a guide for preparing analogues of the lead compound Me-Indoxam with the hope of identifying compounds with improved potency and specificity for one or more of the various human and mouse sPLA₂s. We report the preparation, by small-scale, parallel organic synthesis, of a library of 83 Me-Indoxam analogues, and provide inhibition values of many of these compounds toward the full set of human and mouse sPLA₂s.

Section snippets

Crystal structure of the complex of hGX with Me-Indoxam

The X-ray crystal structure of hGX-Me-Indoxam was solved in a protein crystal form with two independent subunits in the asymmetric unit. The overall structure is well refined with good geometry with an R_working of 0.219 and an R_free of 0.267. These statistics are similar to the converged R_factors for the ligand free form of hGX that was previously reported from a protein crystal also with symmetry of the C222₁ space group and similar unit cell lengths.⁵² Furthermore, there are parallels between

Inhibition patterns among sPLA₂ enzyme species

The large amount of inhibition data obtained with the 4-(2-oxy-ethanoic acid) analogues and the human sPLA₂s (potency of 48 compounds on eight group I, II, V, and X enzymes) is conveniently summarized by the gray scale array shown in Figure 5A. Figure 5B shows the array for the same compounds as inhibitors of the eight mouse sPLA₂s. Figure 5C shows a comparison of the results obtained with human and mouse sPLA₂s (mGIIC is removed since there is no hGIIC and mGXIIA is removed since inhibition

Conclusion

In an attempt to find potent and selective inhibitors of mammalian secretory phospholipases A₂, a library of 83 indole analogues was synthesized and tested against the full set of mouse and human sPLA₂s. Homology models of the human and mouse sPLA₂s were made based on the X-ray structures of the hGIIA and hGX and inhibitors were designed with structure-guided techniques. The X-ray structure was determined of Me-Indoxam, an indole analogue that is a competitive tight binding inhibitor of several

Crystallization and X-ray data collection

The purified recombinant hGX sPLA₂ was prepared as described elsewhere.⁵² The hGX-Me-Indoxam complex was co-crystallized at ambient temperature in 2 μL hanging drops of a mixture of 1 μL of a pre-equilibrated solution containing 15 mg/mL protein, 0.56 mM Me-Indoxam, 10 mM CaCl₂, 10% (v/v) MPD, 0.25% (v/v) DMSO, and 1 μL of a crystallization reservoir solution of 10 mM CaCl₂, 12% (w/v) PEG 3500, 10% (v/v) MPD, 2% (v/v) ethylene glycol and 0.2 M Hepes buffer, pH 7.4. Crystals of rectangular shape

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^☆: Supplementary data associated with this article can be found, in the online version at, doi:10.1016/j.bmc.2004.01.022

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Inhibition of the complete set of mammalian secreted phospholipases A2 by indole analogues: a structure-guided study☆

Abstract

Introduction

Section snippets

Crystal structure of the complex of hGX with Me-Indoxam

Inhibition patterns among sPLA2 enzyme species

Conclusion

Crystallization and X-ray data collection

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Bioorg. Med. Chem. Lett.

Phytochemistry

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Adv. Protein Chem.

Methods Enzymol.

Methods Enzymol.

Tetrahedron

The Phospholipases

J. Biochem.

Am. J. Respir. Crit. Care Med.

Biochemistry

Ann. Rev. Biochem.

Faseb J.

Interfacial Enzyme Kinetics

Inhibition of the complete set of mammalian secreted phospholipases A₂ by indole analogues: a structure-guided study☆

Inhibition patterns among sPLA₂ enzyme species