Dissecting and Designing Inhibitor Selectivity Determinants at the S1 Site Using an Artificial Ala190 Protease (Ala190 uPA)

A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallo...

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Bibliographic Details
Published in:Journal of molecular biology 2004-11, Vol.344 (2), p.527-547
Main Authors: Katz, Bradley A., Luong, Christine, Ho, Joseph D., Somoza, John R., Gjerstad, Erik, Tang, Jie, Williams, Steven R., Verner, Erik, Mackman, Richard L., Young, Wendy B., Sprengeler, Paul A., Chan, Hedy, Mortara, Kyle, Janc, James W., McGrath, Mary E.
Format: Article
Language:English
Subjects:
Ala190 uPA
Alanine - chemistry
Alanine - metabolism
Amidines - chemistry
Benzimidazoles - pharmacology
Binding Sites
conserved water displacement
Crystallography, X-Ray
Drug Design
Guanidine - pharmacology
Humans
hydrogen bond deficit
Hydrogen Bonding
Indoles - chemistry
Molecular Structure
Mutagenesis, Site-Directed
Mutation
Protease Inhibitors - chemical synthesis
Protease Inhibitors - chemistry
Protease Inhibitors - pharmacology
Protein Binding
S1 site
selectivity
Serine - chemistry
Serine - metabolism
Structure-Activity Relationship
Substrate Specificity
Thermodynamics
Urokinase-Type Plasminogen Activator - antagonists & inhibitors
Urokinase-Type Plasminogen Activator - chemistry
Urokinase-Type Plasminogen Activator - genetics
Water - chemistry
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Description
Summary:A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallography and enzymology involving WT and Ala190 uPA were used to calculate free energy binding contributions of hydrogen bonds involving the Ser190 hydroxyl group (O γ Ser190) responsible for the remarkable selectivity of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors toward uPA and against natural Ala190 protease anti-targets. Crystal structures of uPA complexes of novel, active site-directed arylguanidine and 2-aminobenzimidazole inhibitors of WT uPA, together with associated K i values for WT and Ala190 uPA, also indicate a significant role of Ser190 in the binding of these classes of uPA inhibitors. Structures and associated K i values for a lead inhibitor (CA-11) bound to uPA and to five other proteases, as well as for other leads bound to multiple proteases, help reveal the features responsible for the potency ( K i=11 nM) and selectivity of the remarkably small inhibitor, CA-11. The 6-fluoro-5-amidinobenzimidzole, CA-11, is more than 1000-fold selective against natural Ala190 protease anti-targets, and more than 100-fold selective against other Ser190 anti-targets.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2004.09.032