Abstract
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that remodel the extracellular matrix environment and mitigate outside-in signaling. Loss of regulation of MMP activity plays a role in numerous pathological states. In particular, aberrant collagenolysis affects tumor invasion and metastasis, osteoarthritis, and cardiovascular and neurodegenerative diseases. To evaluate the collagen sequence preferences of MMPs, a positional scanning synthetic combinatorial library was synthesized herein and was used to investigate the P10' and P11' substrate subsites. The scaffold for the library was a triple-helical peptide mimic of the MMP cleavage site in types I-III collagen. A FRET-based enzyme activity assay was used to evaluate the sequence preferences of eight MMPs. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. On the basis of the screening results, several individual peptides were designed and evaluated. A triple-helical substrate incorporating Asp-Lys in the P10'-P11' subsites offered selectivity between MMP-14 and MMP-15, whereas Asp-Lys or Trp-Lys in these subsites discriminated between MMP-2 and MMP-9. Future screening of additional subsite positions will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery. Furthermore, selective substrates could serve as the basis for the design of specific triple-helical peptide inhibitors targeting only those MMPs that play a detrimental role in a disease of interest.
Highlights
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that remodel the extracellular matrix environment and mitigate outside-in signaling
The template FRET THP (fTHP) included subsites P10Ј and P11Ј, which are important based on collagen interactions with the HPX domains of MMPs [18, 19, 30, 53]
The resulting fTHP sublibrary with the defined P10Ј subsite is screened for relative activity by comparing initial velocities (RFU) of every sublibrary with MMPs
Summary
The template fTHP sequence (Fig. 1) contained consensus residues 769 –783 from the MMP hydrolysis sites in types I–III collagen and was sandwiched by five Gly–Pro–Hyp triplets [30, 53]. MMP-13 preferred Lys over Tyr in the P11Ј subsite mixtures (Fig. 5), whereas Ile–Lys fTHPb was a slightly worse substrate than the Ile–Tyr fTHPb for MMP-13 (Table 2). Unlike the P10Ј subsite Ile, the P10Ј subsite Asp mixture was the most favored by MMP-13 (Fig. 3), but when paired with Lys in the P11Ј subsite, the Asp–Lys fTHPb was not as preferred as either of the mixtures and yielded only 22.8% MMP-13 activity In the P11Ј sublibrary, four other mixtures reached a Tm above 40 °C: Val, Ala, Asp, and Leu. Four residues resulted in mixtures with Tm below 30 °C: Trp, Lys, Phe, and His. Pro, which occupies the P10Ј subsite in the reference peptide, yielded among the most stable P10Ј mixtures with a Tm ϭ 38.6 °C (Table 3). We can assume that differences between mixtures and individual peptides were not due to impurities found in the mixtures
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