Utilizing bioorthogonal chemistry for improving the pharmacokinetic properties and inhibitory potency of N-TIMP2

Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that regulate the degradation of the extracellular matrix components. Imbalance in their activity might lead to a variety of malignancies, including cancer and cardiovascular diseases. MMPs activity is regulated by natural Tissue Inhibitors of Metalloproteinases (TIMPs) that can inhibit MMP`s activity. Synthetic MMP inhibitors exhibit their inhibitory effect by chelating the MMP catalytic Zn²⁺, but have some drawbacks such as poor pharmacokinetic properties and severe adverse effects, which limit their use as therapeutic drugs.

The N-terminal domain of TIMP-2 (N-TIMP2) is sufficient to inhibit MMPs activity, without leading to pro-MMP-2 activation, like FL-TIMP-2 does. Yet, its short circulation half-life limits its use as a therapeutic drug. PEGylation of N-TIMP2 might be a useful method in order to increase its size and to improve its pharmacokinetics properties. However, the common approaches for protein PEGylation are not site-specific and target randomly amino acids on the target protein, which can be undesired. Thus, in this study we are incorporating a non-canonical amino acid (NCAA) into N-TIMP2, allowing a site-specific PEGylation. In addition, we are incorporating an N-TIMP2 with another NCAA with a zinc-binding residue in order to improve its inhibitory effect towards MMP-14. For that, we incorporated propargyl lysine (PrK) into N-TIMP2 at different positions. PrK incorporation was validated by fluorescence labeling and mass spectrometry. PEGylation of N-TIMP2-31TAG with PEG-azide by a click chemistry reaction resulted in 66% labeling. We are currently evaluating the inhibition potency of the PEGylated N-TIMP2 towards MMP-14 as well as the pharmacokinetic properties of the modified N-TIMP2 variants in preclinical cancer models.