A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility

Area of Science:

• Biochemistry
• Structural Biology
• Drug Discovery

Background:

• Peptides are increasingly recognized for their potential as protease inhibitors.
• The serine protease murine urokinase-type plasminogen activator (uPA) is a key target in various physiological and pathological processes.

Purpose of the Study:

• To elucidate the inhibitory mechanism and exosite interactions of mupain-1, a phage-displayed peptide inhibitor of murine urokinase-type plasminogen activator (uPA).
• To investigate strategies for enhancing the affinity and specificity of peptidic inhibitors.

Main Methods:

• X-ray crystal structure analysis
• Site-directed mutagenesis
• Liquid state NMR
• Surface plasmon resonance
• Isothermal titration calorimetry
• Analysis of wild-type and engineered murine and human uPA variants

Main Results:

• Mupain-1's Arg6 residue inserts into the uPA S1 pocket, with improper carbonyl alignment explaining its inhibitory rather than substrate role.
• Novel unnatural Arg analogues at the P1 position enhanced affinity through altered S1 and exosite interactions.
• Peptide modifications, like Ala-substitution of Asp9, improved binding affinity despite entropic penalties, suggesting increased flexibility enhances exosite interactions.

Conclusions:

• The study demonstrates a new inhibitory mechanism for peptidic inhibitors targeting uPA, involving specific P1 residue interactions and unique exosite engagement.
• Increased peptide flexibility can facilitate favorable exosite interactions, offering a novel approach to enhance inhibitor affinity and specificity.
• This work provides a conceptual framework for designing potent and specific peptidic inhibitors by manipulating peptide flexibility and incorporating unnatural amino acids.