No abstract

A solid phase combinatorial library of 165,000 phosphinic peptide inhibitors was prepared and screened for activity against MMP-12. The inhibitors of the library had the structure XXX-Gpsi(PO2H-CH2)L-XXX, in which X is an arbitrary amino acid and Gpsi(PO2H-CH2)L is a Gly-Leu phosphinic dipeptide analogue. The library was constructed as a one-bead-two-compounds library so that every bead contained a common quenched fluorogenic substrate and a different putative inhibitor. In addition, the inhibitor part was prepared by ladder synthesis. After incubation with MMP-12, beads containing active inhibitors were selected, and the inhibitor sequences were recorded using MALDI-TOF MS. Statistical analysis of the sequences obtained from 86 beads gave rise to a consensus sequence which was resynthesized along with 20 related sequences. Three truncated sequences and 16 sequences originally present on beads were also resynthesized. The inhibitors were investigated in an enzyme kinetic assay with MMP-12 showing that the compounds derived from the consensus sequence were strong inhibitors with Ki values down to 6 nM, whereas the sequences originally present on beads varied in potency with Ki values from micromolar to nanomolar. Truncated sequences derived from the consensus sequence were poor inhibitors of MMP-12.

No abstract

The solid-phase synthesis of an array of different pseudopeptides containing a phosphinic glycine ± leucine moiety (-GY{P(O)OH-CH 2 }L-) [1] is described. The resulting pseudopeptides were shown to act as matrix metalloproteinase-9 (MMP-9) inhibitors. Starting from available materials, the protected amino acid isosters benzyloxycarbonyl aminomethylphosphinic acid (glycine analogue) and ethyl a-isobutylacrylate (leucine analogue) were synthesized and coupled with the bis(trimethylsilyl)phosphonite. Protective group interchange yielded a protected phosphinic dipeptide building block 1 ready for use in solid-phase peptide synthesis. Solid-phase peptide synthesis was performed with 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry on a polyethylene glycol polyamide (PEGA) support and the coupling of 1 (1.5 equiv) was carried out with standard activation. The P 4 ± P 2 and P 2 ' ± P 4 ' positions of the pseudopeptides were designed by analogy of the cleavage sequences of different natural extracellular matrix protein substrates with a synthetic peptide substrate of MMP-9. The crude peptides were obtained in high yield and purity as determined by RP-HPLC, and were characterized by electrospray mass spectrometry and amino acid analysis after purification. Enzyme kinetic investigations with MMP-9 of the purified peptide inhibitors showed K i values in the range from mm to nm.

Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.