Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases capable of degrading almost every component of the extracellular matrix (ECM). Dysregulated MMP activity leads to pathological conditions such as neurodegenerative diseases, cancer and other inflammation disorders. In the central nervous system, MMPs modulate the integrity of tight junction proteins, thus impacting BBB permeability and facilitating neuroinflammation, neuronal damage, and disease progression. MMPs are implicated in the regulation of various signaling pathways involved in cancer cell proliferation, survival, and migration, further underscoring their significance in tumor development and progression. Notably, MMP dysregulation is particularly pronounced in glioblastoma multiforme (GBM), a highly aggressive primary brain tumor. In GBM, MMPs, such as MMP-2/-9, are upregulated and associated with enhanced tumor invasion and therapeutic resistance. Their ability to degrade the ECM facilitates infiltration of tumor cells into surrounding brain tissue, contributing to the diffuse and infiltrative nature of GBM. The incomplete resection, high genetic heterogeneity, exclusive blood brain barrier (BBB), and immunosuppressive microenvironment of GBM pose ongoing treatment challenges which necessitating the prompt development of novel therapeutic approaches. Drug development and delivery for GBM requires understanding the challenges to transport the drug through BBB. Understanding the complex roles of MMPs in ECM and BBB is essential for developing targeted therapeutic strategies to inhibit MMP-mediated invasion and improve patient outcomes in this devastating disease. Tissue inhibitor of metalloproteinases (TIMPs) as the endogenous inhibitors of MMPs are excellent candidates for developing therapeutics targeting specific pathological MMPs driving these diseases. TIMPs have a high level of sequence and structure homology, with a broad range of binding and inhibition to the family of MMPs. It is important to identify the key motifs of TIMPs responsible for inhibition of MMPs to develop efficient therapeutics targeting specific MMPs. Protein and peptide variants based on TIMPs that drives binding and inhibition to specific MMPs has great potential as it provides more stability, tighter binding affinities, and higher selectivity compared to small molecules. The engineered TIMP variants also provide higher tissue penetration and cellular uptake due to their small molecular weight.
In this study, we studied effect of previously engineered minimal TIMP variants designed for MMPs inhibition on protecting BBB integrity in rat microvascular endothelial cells (RBMEC), and to prevent migration and invasion in GBM. For this purpose, effect of two minimal TIMPs variants, mTC1 and mTC3 were studied. Transwell Matrigel, MTT proliferation, and wound healing assays were used to evaluate the inhibitory effect of TIMPs and TIMPs variants on the migration and invasion in the two GBM cell lines, T98G and A172 and compared to wild-type TIMPs. The minimal TIMP variants significantly reduced cell migration to less than 10% and which is higher than wild-type TIMP proteins. The minimal TIMP variants also showed promising results in protecting BBB disrupted by MMP-9 in RBMEC cells by reducing permeability and increasing transendothelial electrical resistance (TEER), hallmarks of BBB integrity, significantly. These engineered protein variants were shown to be successfully delivered to the GBM cells or pass across BBB using microplate reader and fluorescence microscopy assays, further proving their potential for drug delivery across BBB to GBM cells. These engineered proteins could potentially lead to therapies for GBM, and neurodegenerative diseases. This research also shed light on our understanding of role of delivery across BBB and underscores the importance of delivery across BBB and protecting BBB integrity in developing effective treatments.
