A protein scaffold, engineered SPINK2, for generation of inhibitors with high affinity and specificity against target proteases

Scientific Reports
Daisuke NishimiyaRyuji Hashimoto

Abstract

Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar KD and sub-nanomolar Ki values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that enginee...Continue Reading

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Citations

May 29, 2020·Science Translational Medicine·Nurit P AzouzMarc E Rothenberg
Aug 4, 2021·Acta Crystallographica. Section D, Structural Biology·Mi LiAlla Gustchina

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Methods Mentioned

BETA
phage display
protein folding
differential scanning calorimetry
size-exclusion chromatography
X
ray
PCR
phage-display
Cleavage
Chip

Software Mentioned

EpiMatrix
BIAcore
Phaser MR
BIAcore T200
MicroCal Origin
Refmac
GraphPad Prism
GraphPad
Protein Interaction Calculator
COOT

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