Engineering TIMP-Based Inhibitors with Minimal and Light-Controlled Designs to Modulate MMPs
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Authors
Hosseini, Seyed Arman
Issue Date
2025
Type
Dissertation
Language
en_US
Keywords
Directed Evolution , Matrix Metalloproteinases (MMPs) , Optogenetics , Protein Engineering , Tissue Inhibitors of Metalloproteinases (TIMPs) , Yeast Surface Display
Alternative Title
Abstract
Matrix metalloproteinases (MMPs) are key mediators of extracellular matrix remodeling and are implicated in a wide range of pathological conditions, including cancer, neurodegenerative diseases, and cardiovascular disorders. Tissue inhibitors of metalloproteinases (TIMPs), particularly TIMP-1, serve as endogenous regulators of MMP activity but face limitations in therapeutic applications due to their size, broad specificity, and lack of spatiotemporal control. This dissertation presents three complementary engineering strategies to overcome these challenges and advance the development of TIMP-based inhibitors with enhanced precision and functionality.In the first approach, minimal TIMP variants were engineered by recombining sequences from all four human TIMPs using DNA shuffling and screening the resulting libraries with yeast surface display and FACS. Several minimal inhibitors as short as 20 amino acids were identified with nanomolar binding affinities (Kd) and picomolar inhibition constants (Ki) toward MMP-3 and MMP-9. These compact variants retained critical inhibitory motifs, including the CXC motif, and demonstrated potency comparable to the full N-terminal domain of TIMP-1, highlighting their potential as modular therapeutic scaffolds.
The second strategy focused on reversible light-controlled inhibition by fusing the photoswitchable fluorescent protein Dronpa145N to TIMP-1. Two fusion orientations were constructed—N-TIMP-1-Dronpa145N and Dronpa145N-N-TIMP-1—and evaluated using structural modeling, flow cytometry, and inhibition assays. Only the N-terminal Dronpa145N fusion enabled light-dependent modulation of MMP-3 inhibition, with cyan light triggering dissociation of tetrameric Dronpa and restoring access to the TIMP domain. These findings emphasize the critical role of fusion orientation and steric architecture in designing optogenetic protein inhibitors.
In the third approach, irreversible light-triggered activation was achieved using PhoCl, a photocleavable fluorescent protein. TIMP-1 was fused to PhoCl through SpyTag/SpyCatcher domains to create a light-sensitive steric cage. Upon violet light exposure, PhoCl underwent photocleavage, releasing the inhibitory domain and restoring TIMP-1 activity. Functional assays and AlphaFold3 modeling confirmed that only the cleaved N-TIMP-1-SpyTag configuration adopted an accessible conformation for MMP-3 binding and inhibition, with a significant decrease in Ki post-cleavage. This system demonstrated precise optical control over protease inhibition and underscored the importance of domain accessibility in functional recovery.
Together, these studies establish a versatile platform for the design of minimal and optogenetically regulated TIMP-based inhibitors, enabling dynamic, light-responsive control of MMP activity. This work lays the foundation for next-generation therapeutics targeting protease-driven pathologies with improved specificity, modularity, and precision.