A KLK4 proteinase substrate capture approach to antagonize PAR1

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site.


Fig: Affinity predictions, purification, and characterization of KLK4 variants. (a) The computational binding landscape of KLK4/PAR1 interactions. KLK4 binding interface residues and their identity in the wild-type sequence are shown on the left, and the amino acids to which they were mutated are shown on the bottom. The change in binding free energies conferred by the mutations are color coded according to the following characteristics: blue—stabilizing, green—neutral, yellow—slightly destabilizing, and red—strongly destabilizing. The gray dots represent mutations that destabilize KLK4S207A. The calculations were performed using the in silico saturation mutagenesis protocol of Sharabi et al. (b) A representative size-exclusion chromatography (SEC) UV absorbance signal of pro-KLK4 proteins (shown here for KLK4WT) after the Ni–NTA elution purification step. (c) SDS-PAGE of KLK4WT and of KLK4S207A and KLK4S207A,L185Das selected purified pro-KLK4 variants. (d) CD spectra of KLK4WT, KLK4S207A and KLK4S207A,L185D (10 µM), as indicated with a solid black line, a dashed line and a solid gray line, respectively. (e) Catalytic activity of KLK4 WT and the KLK4 variants KLK4S207A and KLK4S207A,L185D. A BOC-VPR-AMC fluorescent substrate was added to KLK4WT (□), KLK4S207A (Δ), and KLK4S207A,L185D (○), all at final a concentration of 625 nM, or to buffer (control, ◊), and the fluorescent signal upon substrate cleavage was measured.

On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind ‘decoy’ PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This ‘substrate-capturing’ KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 ‘proteinase substrate capture’ approach to regulate proteinase-mediated PAR1 signaling.

Rabinovitch, E., Mihara, K., Sananes, A. et al. A KLK4 proteinase substrate capture approach to antagonize PAR1. Sci Rep 11, 16170 (2021). https://doi.org/10.1038/s41598-021-95666-4

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