Acylideneoxoindoles: A new class of reversible inhibitors of human transglutaminase 2

Abstract

Inhibitors of human transglutaminase 2 (TG2) are anticipated to be useful in the therapy of a variety of diseases including celiac sprue as well as certain CNS disorders and cancers. A class of 3-acylidene-2-oxoindoles was identified as potent reversible inhibitors of human TG2. Structureactivity relationship analysis of a lead compound led to the generation of several potent, competitive inhibitors. Analogues with significant non-competitive character were also identified, suggesting that the compounds bind at one or more allosteric regulatory sites on this multidomain enzyme. The most active compounds had Ki values below 1.0 µM in two different kinetic assays for human TG2, and may therefore be suitable for investigations into the role of TG2 in physiology and disease in animals.Keywords transglutaminase 2; oxoindole; celiac sprue; structure-activity relationships; allostery Transglutaminase 2 (TG2), a ubiquitous member of the mammalian transglutaminase enzyme family, is found in intracellular as well as extracellular environments of many organs. In the presence of Ca 2+ and the absence of guanine nucleotides, TG2 adopts an open, catalytically competent conformation, which activates γ-glutaminyl residues on proteins as acyl donors and cross-links these to ε-amino groups of lysyl residues. As a result, proteolytically resistant isopeptide bonds are formed between proteins. Hydrolysis of the γ-glutamyl acyl-enzyme intermediate results in deamidation of the substrate.1 , 2 TG2 is implicated in the pathogenesis of disorders including neurological diseases such as Huntington's, Alzheimer's and Parkinson's diseases, certain types of cancers and renal diseases, cystic fibrosis and celiac sprue,3 -8 and may therefore be a suitable therapeutic © 2010 Elsevier Ltd. All rights reserved. * Corresponding authors: khosla@stanford.edu (C.K.); john@numerate.com (J.H.G.).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Here, we present a structure-activity relationship (SAR) analysis for a new class of reversible inhibitors of human TG2, the acylidene oxoindoles. NIH Public AccessIsatin (indoline-2,3-dione) is an endogenous indole in mammals with a range of biological activities.19 , 20 Our motivation to screen this natural product as a candidate TG2 inhibitor was guided by the hypothesis that the cyclic α-keto amide structure of isatin may mimic the γ-carboxamide group of TG2 substrates. α-Keto amides, including isatin analogues, are widely utilized as reversible inhibitors of cysteine-dependent proteases.21 This led us to propose that isatin analogues may also be reversible inh...

Development of Potent and Selective Tissue Transglutaminase Inhibitors: Their Effect on TG2 Function and Application in Pathological Conditions

“…Sporadic examples have been disclosed so far (Duval et al, 2005;Klock et al, 2011;Ozaki et al, 2010Ozaki et al, , 2011Pardin et al, 2008aPardin et al, , 2008bPrime et al, 2012;Schaertl et al, 2010). Their pharmacological characterization in terms of potency, specificity, binding-site targeting, toxicity, and cellular permeability still remains to be revealed.…”

Activation of Extracellular Transglutaminase 2 by Thioredoxin

“…This cytokine is secreted by gluten-reactive, disease-specific T cells that reside in the small intestinal mucosa of celiac patients. Although the consequences of IFN-␥ release remain to be established, studies in model systems have shown that IFN-␥ can increase the trans-epithelial flux of antigen-sized peptides, thereby establishing a potentially autocrine process for gluteninduced toxicity (8). However, this self-potentiating mechanism depends upon the activity of TG2 in the extracellular matrix, because gluten peptides must be deamidated by the enzyme to transform into strong T cell antigens (31).…”

“…TG2 Activity Assay-TG2 activity was assayed using the glutamate dehydrogenase-coupled assay described previously (8). In brief, enzymatic deamidation of 20 mM ZQG was spectrophotometrically monitored at 340 nm in the presence of 1 M TG2 and a buffer containing 200 mM MOPS buffer (pH 7.2), 4 mM CaCl 2 , 0.35 mM NADH, glutamate dehydrogenase (36 units/ml), and 10 mM ␣-ketoglutarate.…”

A Colorful History: The Evolution of Indigoids

“…acetic acid and a catalytic amount of hydrochloric acid) (181,182). This method was then improved with the replacement of 2-methoxy-indol-3-one by isatin (27) and now has been applied widely (16,(183)(184)(185)(186)(187). It now represents the most well-known approach to the synthesis of isoindigo (11) (Scheme 45).…”