Designed Loop Extension Followed by Combinatorial Screening Confers High Specificity to a Broad Matrix MetalloproteinaseInhibitor

• Published in: Journal of molecular biology Vol. 435; no. 13; p. 168095
• Main Authors: Bonadio, Alessandro, Wenig, Bernhard L., Hockla, Alexandra, Radisky, Evette S., Shifman, Julia M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 01.07.2023
• Subjects: matrix metalloproteinase 14; Matrix Metalloproteinase 14 - chemistry; Matrix Metalloproteinase 14 - genetics; Matrix Metalloproteinase 14 - metabolism; Matrix Metalloproteinase 3; Matrix Metalloproteinase Inhibitors - chemistry; Matrix Metalloproteinase Inhibitors - pharmacology; Mutagenesis; protein design; Protein Engineering; tissue inhibitor of metalloproteinases; MMP; protein engineering; tissue inhibitor of metalloproteinases; TIMP2; MSA; NGS; YSD; protein design; matrix metalloproteinase 14; FKIC
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2023.168095

[Display omitted] •Development of selective inhibitors of the MMP family members is challenging.•By computational design, YSD, and NGS, we extended a loop in the MMP inhibitor N-TIMP2.•The engineered variant exhibited exceptional specificity and high affinity.•Design of extended loops is an attractive strategy for enhancing binding specificity. 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.