Synthesis and binding affinity of bidentate phenothiazines with two different photoactive groups

Area of Science:

• Biochemistry
• Medicinal Chemistry
• Molecular Biology

Background:

• Calmodulin (CaM) is a crucial calcium-binding protein regulating numerous enzymes.
• Understanding CaM-enzyme interactions is vital for drug discovery.
• Phenothiazines are known to interact with CaM, but targeted reagents are needed.

Purpose of the Study:

• To synthesize and evaluate bidentate photoaffinity reagents for mapping CaM-enzyme interaction domains.
• To develop novel phenothiazine derivatives with dual photoactive groups for cross-linking studies.

Main Methods:

• Synthesis of various phenothiazine derivatives incorporating 3-azidophenothiazine and benzophenone photoactive groups.
• Evaluation of binding affinity and inhibitory activity against calmodulin-mediated phosphodiesterase.
• Modification of phenothiazine structures with quaternary ammonium salts, amino acids, or carbohydrates to improve properties.

Main Results:

• Successfully synthesized phenothiazines with dual photoactive groups (3-azidophenothiazine and benzophenone).
• Identified structural modifications (quaternary ammonium salt) that enhanced solubility and inhibitory activity.
• Phenothiazines with quaternary N-methylammonium iodide moiety inhibited calmodulin-mediated phosphodiesterase activation comparably to chlorpromazine.

Conclusions:

• Novel bidentate photoaffinity reagents based on phenothiazines were successfully developed.
• Structural modifications are key to overcoming challenges in solubility and activity for CaM-targeting reagents.
• These reagents show potential for elucidating CaM-enzyme binding sites and guiding the development of new therapeutics.