(715d) Engineering Interacting Protein Loops Targeting Metalloproteinases

My research group is interested in engineering and design of protein scaffolds as potential therapeutics for selective targeting of Metalloproteinases (MPs). MPs including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs) play a multifaceted role in several diseases, such as cancer, neurological disorders, cardiovascular diseases, and pre-term labor based on their critical role in remodeling the extracellular matrix (ECM) and other proteolytic activities in ECM. Protein-based MP inhibitors offer higher stability and selectivity which is critical for developing efficient therapeutics with low off-target effects. Tissue inhibitors of metalloproteinase (TIMPs), natural inhibitors of matrix metalloproteinases (MMPs), and monoclonal antibodies (mAbs), provide excellent protein scaffolds for engineering selective or multi-specific MP inhibitors. Our research group has engineered and designed TIMPs by a combination of directed evolution and computational approaches.

Through a blend of computational and experimental strategies, we design protein binders and inhibitors. Using directed evolution, rational design, and computational modeling we aim to uncover the key motifs and residues that facilitate selective inhibition of MPs. This work is vital for developing a new generation of highly selective protein drugs with reduced off-target effects. Advances in protein engineering and computational studies that predict protein structure and function provide robust tools for designing MP inhibitors as potential therapeutics for MP-related diseases. Here, we report our most recent progress in developing TIMPs with improved binding toward specific MPs by engineering their interacting loops targeting specific MPs such as MMP-3, -9, and ADAM-17. The four human TIMPs exhibit significant sequence and structural homology with each other, along with a broad range of inhibition selectivity and binding affinity to MMPs. We previously used DNA shuffling between the human TIMP family to generate a minimal TIMP hybrid library to identify the dominant minimal MMP inhibitory regions with higher flexibility and higher tissue penetration features. More recently, we engineered hybrid TIMPs using domain and loop swapping techniques for improving stability and binding selectivity to specific MPs such as MMP-3/-9, ADAM-17, and growth factors such as VEGFR2. For instance, we showed that the insertion of certain loops such as C-connector disrupts expression and binding, while MTL loop insertion improves and/or recovers this effect.

Further, we screened yeast-displayed TIMP-3 libraries with random mutations in the interacting loops using counter-selective strategies toward MMP-3, -9, and ADMA-17 using fluorescent-activated cell sorting (FACS). We used deep sequencing and bioinformatic tools to find key drivers of binding to specific MPs. A few interesting outcomes: 1) TIMP-3 could bind to MP targets including ADAM-17 even when no C-loop was present! 2) Position 1 and 2 of C-loop has great significance in dictating binding to MMP-9 vs. ADAM-17.

The protein engineering and protein design techniques developed in these studies could be used for the engineering and design of protein binders specifically enzyme inhibitors. The engineered highly selective MP inhibitors have great potential in developing highly efficient therapeutics with higher efficacy and lower side effects in MP-related diseases. The engineered TIMP scaffolds developed in these studies offer multi-inhibitory properties critical to addressing multi-dimensional biomarkers in complex diseases such as cancer which MPs play a key role.