RATIONAL AND COMBINATORIAL APPROACHES TO DEVELOP THE N-TERMINAL DOMAIN OF TIMP2 WITH HIGH SPECIFICITY TOWARDS MMP14 AND MMP9 FOR CANCER THERAPY

Matrix Metalloproteases (MMPs), in particular MMP14 and MMP9, play a major role in cancer progression, angiogenesis and metastasis due to their ability to degrade the extra cellular matrix components. Though many attempts have been made to develop inhibitors for MMPs there are no clinically approved agent that possess selective MMP inhibition. Lack of specificity of broad-spectrum inhibitors causes severe side effects because several MMPs promote either essential biological processes or anti-cancer activity, or both. Tissue inhibitor of matrix metalloproteases 2 (TIMP2) is an endogenous non specific regulator of all MMPs. To possess its inhibitory effect, the N- terminal domain of TIMP2 (N-TIMP2) interacts with the catalytic site of all MMPs. Our aim is to engineer N-TIMP2 variants with high affinity and specificity towards MMP14 and MMP9. To achieve this goal, five rounds of MMP-14 screening of a focused yeast displayed N-TIMP2 library with seven randomly mutated N-TIMP/MMP14 interface positions resulted in identification of four unique N-TIMP2 variants. Most importantly, these soluble N-TIMP variants showed significantly enhanced K_i values (pico-molar range) towards MMP14. These proteins are currently being tested for MMP14 binding and inhibition in cell assays. Simultaneous screening of the focused library against MMP14 and MMP9 yielded several highly selective clones towards both targets. These protein variants are currently produced and tested for MMP14 and MMP9 binding and inhibition in in-vitro and cell-based assays. Our approach enables us to identify specific N-TIMP2 based inhibitors that can be further utilized in cancer diagnostics, prognosis assessment and for personalized treatment. Furthermore, this approach can be extended to other MMPs increasing the variability and availability of cancer therapeutics.