Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The inhibitory N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP2), a natural broad MMP inhibitor, can provide a scaffold for protein engineering to create more selective MMP inhibitors. Here, we pursued a unique approach harnessing both computational design and combinatorial screening to confer high binding specificity toward a target MMP in preference to an anti-target MMP. We designed a loop extension of N-TIMP2 to allow new interactions with the non-conserved MMP surface and generated an efficient focused library for yeast surface display, which was then screened for high binding to the target MMP-14 and low binding to anti-target MMP-3. Deep sequencing analysis identified the most promising variants, which were expressed, purified, and tested for selectivity of inhibition. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, revealing 7500-fold greater specificity than WT N-TIMP2. High-confidence structural models were obtained by including NGS data in the AlphaFold multiple sequence alignment. The modeling together with experimental mutagenesis validated our design predictions, demonstrating that the loop extension packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates how introduction of loop extensions in a manner guided by target protein conservation data and loop design can offer an attractive strategy to achieve specificity in design of protein ligands.
Bibliographical noteFunding Information:
We thank Ora Furman-Schueler and Gideon Schreiber for their insightful discussions. This work was supported by the US-Israel Binational Science Foundation (BSF) 2017207 (J. M. S.) and NIH R01CA258274 (E.S.R. and J. M .S). In addition, J. M. S. acknowledges the support from ICRF and ISF 3486/20 and the U. of Toronto/HUJI research alliance in protein engineering and E.S.R. acknowledges NIH R01 GM132100.
We thank Ora Furman-Schueler and Gideon Schreiber for their insightful discussions. This work was supported by the US-Israel Binational Science Foundation (BSF) 2017207 (J. M. S.) and NIH R01CA258274 (E.S.R. and J. M .S). In addition, J. M. S. acknowledges the support from ICRF and ISF 3486/20 and the U. of Toronto/HUJI research alliance in protein engineering and E.S.R. acknowledges NIH R01 GM132100. AB conceived the project idea with inputs from JSM. AB performed most of the experimental studies and all the computational studies and wrote the software for the analysis of the NGS data. AB and JMS designed the research. BLW and AH performed the MMP-3 mutagenesis, MMP-3 and mutants expression and purification, enzyme inhibition studies of MMP-3 mutants, and the inhibition of cell invasion studies. JMS and ESR supervised the project, provided resources, and acquired funding. AB and JMS wrote the manuscript, with contributions from the other authors. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
© 2023 Elsevier Ltd
- matrix metalloproteinase 14
- protein design
- protein engineering
- tissue inhibitor of metalloproteinases