Using a site-directed fragment discovery method called tethering, we have identified a 60 nM small molecule antagonist of a cytokine/receptor interaction (IL-2/IL2Ralpha) with cell-based activity. Starting with a low micromolar hit, we employed a combination of tethering, structural biology, and computational analysis to design a focused set of 20 compounds. Eight of these compounds were at least 5-fold more active than the original hit. One of these compounds showed a 50-fold enhancement and represents the highest affinity inhibitor reported against this protein-protein target class. This method of coupling selected fragments with a low micromolar hit shows great potential for generating high-affinity lead compounds.

Fragment assembly has shown promise for discovering small-molecule antagonists for difficult targets, including protein-protein interactions. Here, we describe a process for identifying a 60 nM inhibitor of the interleukin-2 (IL-2)/IL-2 receptor (IL-2Ralpha) interaction. By use of fragment-based approaches, a compound with millimolar affinity was evolved to a hit series with low micromolar activity, and these compounds were optimized into a lead series with nanomolar affinity. Fragment assembly was useful not only for hit identification, but also for lead optimization. Throughout the discovery process, biophysical methods and structural biology demonstrated that compounds bound reversibly to IL-2 at the IL-2 receptor binding site.

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...

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PDB References: caspase-1 complexes with compound 1, 1rwk, r1rwksf; compound 4, 1rwm, r1rwmsf; compound 5, 1rwn, r1rwnsf; compound 6, 1rwo, r1rwosf; compound 8, 1rwp, r1rwpsf. Caspase-1 is a key endopeptidase responsible for the post-translational processing of the IL-1 and IL-18 cytokines and small-molecule inhibitors that modulate the activity of this enzyme are predicted to be important therapeutic treatments for many inflammatory diseases. A fragment-assembly approach, accompanied by structural analysis, was employed to generate caspase-1 inhibitors. With the aid of Tethering 1 with extenders (small molecules that bind to the active-site cysteine and contain a free thiol), two novel fragments that bound to the active site and made a disulfide bond with the extender were identified by mass spectrometry. Direct linking of each fragment to the extender generated submicromolar reversible inhibitors that significantly reduced secretion of IL-1 but not IL-6 from human peripheral blood mononuclear cells. Thus, Tethering with extenders facilitated rapid identification and synthesis of caspase-1 inhibitors with cell-based activity and subsequent structural analyses provided insights into the enzyme's ability to accommodate different inhibitor-binding modes in the active site.