Immucillins in custom catalytic-site cavities
Andrew S Murkin1, Keith Clinch, Jennifer M Mason
1Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Bioorganic & Medicinal Chemistry Letters
|September 10, 2008
Summary
Purine nucleoside phosphorylase (PNP) inhibitors were developed using a His257Gly mutant. These Immucillin inhibitors achieve ultra-low dissociation constants, offering potent enzyme inhibition for therapeutic applications.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Medicinal chemistry
Background:
- Purine nucleoside phosphorylase (PNP) is a key enzyme in purine metabolism.
- Neighboring-group participation, involving His257, is crucial for PNP catalysis.
- The His257Gly mutation alters the enzyme's active site dynamics.
Purpose of the Study:
- To investigate the role of His257 in PNP-catalyzed reactions.
- To develop potent transition-state analogue inhibitors for the His257Gly mutant.
- To characterize the binding affinity of novel inhibitors.
Main Methods:
- Site-directed mutagenesis to create the His257Gly PNP mutant.
- Design and synthesis of hydrophobic 5'-substituted Immucillins.
- Enzyme inhibition assays to determine dissociation constants (Kd) and kinetic parameters (Km).
Main Results:
- The His257Gly mutation creates an accessible catalytic site.
- Hydrophobic 5'-substituted Immucillins act as potent transition-state analogue inhibitors.
- Achieved dissociation constants as low as 2 picomolar (pM).
- Observed K(m)/K(d) ratios up to 400,000,000, indicating high binding affinity.
Conclusions:
- His257 plays a critical role in facilitating neighboring-group participation in PNP.
- The His257Gly mutant provides a target for developing highly potent PNP inhibitors.
- Hydrophobic Immucillins represent a promising class of therapeutic agents targeting PNP.
