Phage Display of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2)

Abstract

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34 -40 (L2) and 67-70 (L3), a library was generated containing 2 ؋ 10 10 variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys 1 -Gln 9 were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K i values, but TM8, containing Ser 2 to Asp and Ser 4 to Ala substitutions, was the most selective having a nanomolar K i value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 M. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.Normal biological processes, including embryo implantation, developmental remodeling of the extracellular matrix, and wound healing, require active MMPs.5 Excess active MMPs can have pathological effects and are tightly regulated at the levels of transcription, zymogen activation, and inhibition by endogenous high affinity protein inhibitors, the TIMPs. Disruption of the balance between active MMPs and their inhibitors results in diseases linked to unregulated matrix turnover, including arthritis, cancer, cardiovascular diseases, nephritis, neurological disorders, tissue ulceration, and fibrosis (1-4). The mammalian TIMPs are a family of four two-domain proteins (TIMP-1-4), with an N-terminal domain of ϳ125 amino acids and a C-terminal domain of ϳ65 amino acids; each domain is stabilized by three disulfide bonds. They show 41-52% identity in pairwise sequence comparisons.In general, the TIMPs show little discrimination in their inhibition of the 23 human MMPs. TIMP-1 inhibits most MMPs but is an exceptionally weak inhibitor of MT1-MMP, MT3-MMP, and MMP-19 (1, 2). In contrast, TIMP-2 forms high affinity complexes with all MMPs with K i values in the nanomolar range (3). TIMP-3 has a more extended inhibitory range that includes several disintegrin-metalloproteinases (ADAMs) such as ADAM-10, ADAM-12, ADAM-17, ADAM-28, and ADAM-33 together with various ADAMTS disintegrin metal...

Peptide-Based Selective Inhibitors of Matrix Metalloproteinase-Mediated Activities

“…In related work from the same laboratory, phage display was utilized with TIMP-2 to identify selective inhibitors of MMP-1 [99]. Three regions of TIMP-2 underwent randomization, residues 2–6 (excluding residue 3), 34–40, and 67–70.…”

Domain specific overexpression of TIMP-2 and TIMP-3 reveals MMP-independent functions of TIMPs duringXenopus laevisdevelopment

“…While many studies have demonstrated that TIMPs can signal through a number of important regulatory pathways, there is currently little information regarding how TIMP proteins actually function in vivo, particularly as their domains may act through both MMP-dependent and -independent mechanisms (Stetler-Stevenson 2008a). Accordingly, in this study we examined the individual domains of TIMP-2 and -3 in early stage X. laevis embryos to gain an understanding of how the individual N-and C-terminal domains may regulate key developmental events as there is evidence that TIMP N-and C-terminal domains may have unique properties and can function independently as individual domains (Bahudhanapati et al 2011;Brew and Nagase 2010;Chirco et al 2006) and maintain their native topology (Wu et al 2011).…”

“…This activity is thought to occur through the TIMP C-terminal domains and is independent of their MMP-inhibitory activity, which occurs exclusively at the N-terminus (DeClerck et al 1993;Nagase et al 2006). The 2 domains of TIMPs are thought to have evolved separately, and both domains have the ability to fold and function independently, as demonstrated with Nterminal domain expression in heterologous systems that still maintains a stable native structure, and can act as an MMP inhibitor (reviewed in Brew and Nagase 2010;Bahudhanapati et al 2011). …”

Matrix Metalloproteinase-10/TIMP-2 Structure and Analyses Define Conserved Core Interactions and Diverse Exosite Interactions in MMP/TIMP Complexes

“…Based on our structural analyses, we suggest that peripheral epitopes, presented not only by the AB loop but also by the C-terminal domain of the TIMP scaffold, might be more intensively targeted for optimization to enhance inhibitor selectivity toward individual MMPs. Such efforts could take advantage of diversity library screening and directed evolution, such as the phage display approach recently successful in identifying an MMP-1-selective variant of TIMP-2 [60]. Ultimately, continued efforts to understand and exploit the structural basis of molecular recognition of MMPs by TIMPs may facilitate development of new MMP-directed probes and therapeutics, taking advantage of emerging concepts for delivery of TIMP-based drugs and probes in vivo [61], [62], [63].…”