Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.

A program to improve upon the in vitro, in vivo, and physicochemical properties of N-hydroxyformamide TACE inhibitor GW 3333 (1) is described. Using the primary structure of pro-TNF-alpha, along with a homology model of the catalytic domain of TACE based on the X-ray diffraction coordinates of adamalysin, we synthesized N-hydroxyformamide TACE inhibitors containing a P2' arginine side chain. Introduction of nitro and sulfonyl electron-withdrawing groups covalently bound to the P2' guanidine moiety rendered the inhibitors electronically neutral at cellular pH and led to potent inhibition of TNF-alpha release from stimulated macrophages. Inhibitors containing these arginine mimetics were found to have increased solubility in simulated gastric fluid (SGF) relative to 1, allowing for the incorporation of lipophilic P1' side chains which had the effect of retaining potent TACE inhibition, but reducing potency against matrix metalloproteases (MMPs) thus increasing overall selectivity against MMP1, MMP3, and MMP9. Selected compounds showed good to excellent in vivo TNF inhibition when administered via subcutaneous injection. One inhibitor, 28a, with roughly 10x selectivity over MMP1 and MMP3 and high solubility in SGF, was evaluated in the rat zymosan-induced pleuisy model of inflammation and found to inhibit zymosan-stimulated pleural TNF-alpha elevation by 30%.

Tumor necrosis factor-α (TNFα) has been implicated in cancer and inflammatory diseases since it was first characterized and eventually identified by researchers in several laboratories in the mid-1980s [1-5]. However, only recently has the patho-logical role of TNF in arthritis and Crohn’s disease been demonstrated in the clinic with the FDA’s approval of Enbrel and Remicade [6-8]. The recent success of these biological agents that neutralize TNF has led to intense efforts to find small molecule TNF antagonists that will mimic the consequences, if not the mechanism, of these agents. Because TNF interacts at multiple contact points with either of its two receptors, researchers have struggled to find small molecular weight inhibitors that antagonize this interaction. Therefore, most efforts have focused on targets upstream of TNF synthesis or secretion and downstream of TNF receptor engagement since these targets appear more amenable to modulation by small molecular weight inhibitors [9].

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide Library containing 2 x 10(8) independent recombinants, A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates, Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2,4-dinitrophenyl-GPLGMRGL-NH2 (CP), has a k(cat)/K-m value of 4.22 x 10(6) M-1 s(-1) for hydrolysis by collagenase 3, CP was synthesized as a consensus sequence deduced from the preferred sub sites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-l, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan, Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type N collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type Il and type IV collagens.

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 × 108 independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1′, P2′, and P3′ subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2,4-dinitrophenyl-GPLGMRGL-NH2 (CP), has a k cat/K m value of 4.22 × 106 m − 1s− 1 for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.

Tumor necrosis factor a (TNFα) is a pleiotropic cytokine that mediates inflammatory and apoptotic processes by binding to two different receptors and thereby initiating complex signaling transduction pathways [1]. While TNFa has been studied since the earlier part of this century, the purification and cloning of this protein in 1985 [2 – 5] precipitated a decade of intensive research. These efforts demonstrated the cross-disciplinary significance of this molecule and gave insight into the signaling pathways that initiated TNFα transcription, the unique control of its translation and the TNF receptors that bind the secreted, 17 kDa molecule. These receptors reside on cells of nearly every tissue and, in turn, transduce the signals that result in changes in cell behavior.

Nature 385, 733-736 (1997) The fourth author's name (D.M.B.) was accidentally omitted; he is in the Department of Molecular Biochemistry at Glaxo Wellcome Research and Development. Also, reference 8 was not included in the list. This was by G. M. McGeehan et al, "Regulation of tumour-necrosis factor-a processing by a metalloproteinase inhibitor", published in Nature 370, 558-561 (1994).

ADVERTISEMENT RETURN TO ISSUEPREVArticleMatrix metalloproteinase inhibitors containing a [(carboxyalkyl)amino] zinc ligand: Modification of the P1 and P2' residues. [Erratum to document cited in CA120:238898]Frank K. Brown, Peter J. Brown, D. Mark Bickett, C. Lynn Chambers, H. Geoff Davies, David N. Deaton, David Drewry, Michael Foley, Andrew B. McElroy, and Cite this: J. Med. Chem. 1994, 37, 10, 1546Publication Date (Print):May 1, 1994Publication History Published online1 May 2002Published inissue 1 May 1994https://pubs.acs.org/doi/10.1021/jm00036a021https://doi.org/10.1021/jm00036a021research-articleACS PublicationsRequest reuse permissionsArticle Views119Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

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