Institution: | 1. Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Israel;2. Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA;3. Paracelsus Medical University, Salzburg, Austria;1. Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA, F-31077 Toulouse, France;2. Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, UPS, F-31062 Toulouse, France;1. Agharkar Research Institute, Nanobioscience Group, G. G. Agarkar Road, Pune 411004, India;2. Savitribai Phule Pune University, Ganeshkhind, Pune 411 007, India;1. Monash University, Melbourne, Australia;2. Center for Inflammation and Tolerance; Cincinnati Children’s Hospital, Cincinnati, USA;3. U. Libre Brussels, Brussels, Belgium;4. Stanford University School of Medicine, Stanford, CA, USA;5. Southern Denmark University, Copenhagen, Denmark;6. Division of Experimental Feto-Maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg, Hamburg, Germany;7. Telethon Kids Institute, Perth, WA, Australia |
Abstract: | 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. |