Solid Phase Combinatorial Library of Phosphinic Peptides for Discovery of Matrix Metalloproteinase Inhibitors

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.

Total Chemical Synthesis, Folding, and Assay of a Small Protein on a Water‐Compatible Solid Support

“…These highly pure proteins could be assayed for inhibitory activity towards a variety of enzymes by using strategies such as the one-bead-two-compound fluorescence-inhibitor assay. [19][20][21][22] We have demonstrated the synthesis and folding of a small trypsin inhibitor, EETI-II, in rapid fashion and high yield by solid-phase chemical ligation on a PEG-based solid support. We have also shown the ability to perform enzyme assays with the resin-immobilized inhibitor, as well as recovery of both the enzyme and inhibitor from the resin.…”

Automated Sorting of Beads from a “One‐Bead‐Two‐Compounds” Combinatorial Library of Metalloproteinase Inhibitors

“…The assay has previously been used to identify endo-proteinase inhibitors of Subtilisin Carlsberg, Cruzipain, Cathepsin B, Cathepsin L, Leishmania . Although the generation, incubation, and analysis of libraries have been optimized, the screening process has been problematic and far from quantitative in previous reports [14,15]. In addition, the excitation light of a fluorescence microscope is in the UV-region coinciding with the wavelength often used to release the inhibitor ladder part from a photo-labile linker for structure analysis thus resulting in premature cleavage.…”

“…In addition, the excitation light of a fluorescence microscope is in the UV-region coinciding with the wavelength often used to release the inhibitor ladder part from a photo-labile linker for structure analysis thus resulting in premature cleavage. This has limited the time window for reliable sorting and analysis [14].To overcome these problems, an instrument was developed for bead sorting based on the COPAS TM sorting technology for nematode handling. The performance of the custom engineered instrument in rapid sorting of spherical beads according to their fluorescent intensity was investigated.…”

“…Phosphinic peptide isosteres (Scheme 1) have been reported to be inhibitory transition state analogues of substrates [18]. Phosphinic peptides have previously been prepared by parallel synthesis [17][18][19] and in the combinatorial onebead-two-compounds format for identification of matrix metalloproteinase inhibitors [14].Using the fully automated high throughput bead sorting methodology, potent inhibitors of MMP-9 have been identified from a solid phase combinatorial library of phosphinic peptides based on the sequence, XX-GY{-PO 2 H-CH 2 }L-XX, in which X is an arbitrary amino acid and GY{PO 2 H-CH 2 }L is a Gly-Leu phosphinic dipeptide analogue. The preparation of the library, the incubation with enzyme and the structure determination were conducted in analogy with previously described procedures [14].…”

“…Phosphinic peptides have previously been prepared by parallel synthesis [17][18][19] and in the combinatorial onebead-two-compounds format for identification of matrix metalloproteinase inhibitors [14].…”