D. Mark Bickett

Substrate Specificity of Human Collagenase 3 Assessed Using a Phage-displayed Peptide Library

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.

N-hydroxyformamide peptidomimetics as TACE/matrix metalloprotease inhibitors: oral activity via P1' isobutyl substitution.

N-Hydroxyformamide-class metalloprotease inhibitors were designed and synthesized, which have potent broad-spectrum activity versus matrix metalloproteases and TNF-alpha converting enzyme (TACE). Compound 13c possesses good oral and intravenous pharmacokinetics in the rat and dog.

Rapid synthesis of novel dipeptide inhibitors of human collagenase and gelatinase using solid phase chemistry

Abstract Solid phase chemistry expedited the systematic modification of the C and N-terminal groups of cysteine derived lead compound 1 (collagenase IC 50 63nM), providing a series of matrix metalloproteinase inhibitors. Potent inhibitors of collagenase ( 1–2 , 4–6 , and 10–13 ) and gelatinase ( 4–8 ) were identified. Insights into the binding mode of selective inhibitors will be discussed.

A High Throughput Fluorogenic Substrate for Stromelysin (MMP‐3)

Stromelysin, a member of the matrix metalloproteinase family of enzymes, has been implicated in the pathogenesis of tumor metastasis and inflammatory diseases such as rheumatoid arthritis. To screen prospective inhibitors of this protease, we developed a fluorogenic substrate with excitation and emission spectra compatible with commercially available 96-well plate readers. The substrate is based on the addition of 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] hexanoic acid (NBD) (EX467/EM534) and 7-dimethylaminocoumarin-4-acetate (DMC) (EX368/EM459) to the previously reported peptide substrate for stromelysin, Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-NH2. The new substrate, NBD-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-Lys-(DMC)-NH2 is 95% quenched and the fluorescent product, Nva-Trp-Lys(DMC)-NH2 is easily detected (EX350/EM465). In competition assays the new fluorogenic substrate has a relative kcat/Km that is one half that of the parent peptide. The fluorophores NBD and DMC were chosen based on the high fluorescence yield of DMC and the overlap of the emission spectrum of DMC and excitation spectrum of NBD which results in an efficient energy transfer system in the intact substrate. These characteristics make this an excellent substrate for routine determination of in vitro activities of stromelysin inhibitors.

TNFα converting enzyme

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.

A high throughput assay for the TNF converting enzyme

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].