[A method based on endogenous fluorescence determination for screening NAMPT inhibitors].

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Context:

• Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in NAD+ biosynthesis.
• Developing selective NAMPT inhibitors is crucial for therapeutic applications.
• Existing screening methods may not differentiate binding sites effectively.

Purpose:

• To establish a novel screening method for NAMPT inhibitors using endogenous fluorescence.
• To differentiate compounds that bind to the active site versus other regions of NAMPT.
• To evaluate the efficacy of known inhibitors and natural compounds.

Summary:

• A fluorescence-based assay was developed using wild-type and BMB-crosslinked NAMPT (nicotinamide phosphoribosyl transferase).
• FK866, a known inhibitor, decreased NAMPT fluorescence but not BMB-NAMPT fluorescence, indicating active site binding.
• Natural compounds like rosmarinic acid showed equivalent fluorescence changes in both proteins, suggesting non-active site binding and no enzymatic inhibition.

Impact:

• This method enables efficient screening and characterization of NAMPT inhibitors.
• It facilitates the identification of compounds with distinct binding mechanisms.
• The findings contribute to the development of targeted therapies involving NAMPT.