Utilizing Bioorthogonal Chemistry and Site-Specific PEGylation for Improving the Pharmacokinetic Properties of N-TIMP2

Matrix metalloproteinases (MMPs) are a family of enzymes that regulate the degradation of the extracellular matrix protein components, in biological processes such as angiogenesis and wound healing. Imbalance in their activity might lead to a variety of diseases, including different types of cancer and cardiovascular diseases. MMPs activity is regulated by natural Tissue Inhibitors of Metalloproteinases (TIMPs), a family of four members that can inhibit MMP`s activity, but some can also mediate activation of some pro-MMPs. TIMP-2, for example, can both inhibit MMPs by binding to their catalytic zinc-binding site via its N-terminal, but can also lead to pro-MMP-2 activation via its C-terminal.

Engineering approach showed that the N-terminal domain of TIMP-2 (residues 1-127) is sufficient to inhibit MMPs activation, without activating pro-MMP-2. However, the short circulation half-life of this inhibitor makes a serious limitation for using it as a therapeutic drug. Therefore, we decided to PEGylate N-TIMP2 in order to increase its size and to improve its pharmacokinetics properties.

The common approaches for protein PEGylation include the conjugation of PEG molecules to the canonical amino acids in a way that targets all amino acids of the same type randomly, which can be undesired. We have decided to use bioorthogonal chemistry and to incorporate a non-canonical amino acid into N-TIMP2, allowing a site-specific PEGylation and a better control for this modification.

We have managed to incorporate the non-canonical amino acid propargyl lysine into N-TIMP2 in different positions. These residues are aimed to be conjugated with PEG-azide (5K and 20K) in a click chemistry reaction, followed by examination of the inhibition activity towards MMP-14 as well as the pharmacokinetic properties of the modified variants in in vitro and in vivo studies.