Structure-guided inhibitor design for human FAAH by interspecies active site conversion

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes endocannabinoids like anandamide.
• Inhibition of FAAH offers therapeutic potential for analgesia, anxiolysis, and anti-inflammatory effects without direct cannabinoid receptor agonist side effects.
• Structure-based design of FAAH inhibitors is hindered by challenges in expressing the human enzyme.

Purpose of the Study:

• To overcome expression difficulties for human fatty acid amide hydrolase (FAAH).
• To generate a stable, high-yield FAAH enzyme suitable for structural studies and inhibitor design.
• To elucidate the structural basis for species selectivity in FAAH inhibition.

Main Methods:

• Site-directed mutagenesis was used to interconvert active sites of rat and human FAAH.
• A humanized rat FAAH (h/rFAAH) protein was engineered.
• Crystallography was employed to determine the structure of h/rFAAH complexed with the inhibitor PF-750.

Main Results:

• The engineered h/rFAAH protein demonstrated inhibitor sensitivity profiles similar to human FAAH while maintaining high expression yields.
• A 2.75-Å crystal structure of the h/rFAAH-PF-750 complex was obtained.
• The structure provided insights into the species selectivity of FAAH inhibitors, particularly the strong preference for human FAAH.

Conclusions:

• The humanized rat FAAH enzyme facilitates structure-based inhibitor design.
• The crystal structure reveals key interactions explaining inhibitor selectivity.
• This work guides the development of novel therapeutics targeting FAAH.