Matrix metalloproteinases (MMPs) play a multifaceted role in several diseases, such as cancer, neurological disorders, and cardiovascular diseases, based on their critical role in remodeling the extracellular matrix. The endogenous inhibitors of MMPs, tissue inhibitors of metalloproteinases (TIMPs), are a family of four in humans. TIMPs have a high level of sequence and structure homology, with a broad range of inhibition selectivity and binding affinity to the family of MMPs. We 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. Interestingly, several minimal TIMP variants selected after screening toward MMP-3cd or MMP-9cd, with lengths as short as 20 amino acids, maintained or improved binding to MMP-3cd and MMP-9cd. The TIMP-MMP binding dissociation constant (KD), in the nM range, and MMP inhibition constants (Ki), in the pM range, of these minimal TIMP variants were similar to the N-terminal domain of TIMP-1 on the yeast surface and in solution indicating the potency of these minimal variants as MMP inhibitors. We further used molecular modeling simulation, and molecular protein docking of the minimal TIMP variants in complex with MMP-3cd to understand the binding and inhibition mechanism of these variants.
We evaluated the therapeutic potential of these minimal TIMP variants using in vitro models of the blood-brain barrier (BBB). MMP-3 and MMP-9 were shown to disturb the BBB measured by permeability assays using FITC conjugated dextran (10kDa) across the rat brain endothelial cells (RBMECs) in a Transwell setting. TIMP minimal variants restored the permeability of BBB and increased transendothelial electrical resistance (TEER) comparable to wild-type TIMP-1 and TIMP-3 recombinant proteins. This research will shed light on the engineering and design of the next generation of enzyme inhibitors as potential protein therapeutics.