A protein–small molecule conjugate as a bispecific inhibitor of proteases implicated in cancer metastasis

Matrix metalloproteinase-9 (MMP-9) and kallikrein 6 (KLK6) are key extracellular proteases implicated in metastatic progression. In colorectal and pancreatic cancers, their overexpression correlates with aggressive disease and poor prognosis, making them attractive therapeutic targets. While monospecific inhibition of either protease shows limited efficacy, simultaneous dual inhibition has not yet been explored as a strategy to block metastasis. To address the lack of an efficient inhibitor of the above metastatic cancers, we prepared a bispecific inhibitor by the site-specific conjugation, via click chemistry, of a selective inhibitor of MMP-9 to a small molecule specifically inhibiting KLK6; the inhibitor comprised the N-terminal domain of tissue inhibitor of metalloproteinases 2 (N-TIMP2), bearing the non-canonical amino acid propargyl lysine (PrK), conjugated to a small molecule known as DKFZ-938. The bispecific inhibitor, thus designated N-TIMP2 PrK –DKFZ-938, exhibited inhibitory activity against purified KLK6 and both purified and cellular MMP9 in the absence and presence of KLK6. Functional assays of the bispecific inhibitor in HCT-116, PANC-1, and CAPAN-2 cancer cell lines expressing both MMP-9 and KLK6 demonstrated dose-dependent inhibition of migration and invasion that outperformed the respective monospecific inhibitors, alone or in admixture, without affecting cell viability. These results thus show N-TIMP2 PrK –DKFZ-938 to be a promising dual-targeting therapeutic prototype, providing simultaneous inhibition of two proteases that cooperatively drive metastasis.