Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human...

Area of Science:

• Biochemistry
• Structural Biology
• Medicinal Chemistry

Background:

• Human DNA polymerase theta (Polθ) is crucial for DNA double-strand break repair via microhomology.
• Polθ is a potential therapeutic target for cancers with deficient DNA repair pathways, including BRCA-deficient cancers.

Purpose of the Study:

• To describe the discovery and characterization of small molecule probes targeting Polθ.
• To elucidate the mechanism of inhibition and guide rational drug optimization.

Main Methods:

• Biochemical and biophysical characterization of Polθ inhibitors.
• Co-crystallization of Polθ with small molecule ligands.
• X-ray crystallography to determine enzyme-inhibitor structures.

Main Results:

• Identification and characterization of ART558 (in vitro probe) and ART812 (in vivo probe).
• Co-crystal structures reveal a unique inhibition mechanism involving stabilization of a closed enzyme conformation.
• Structural data enables rational optimization focused on reducing ligand conformational flexibility.

Conclusions:

• ART558 and ART812 are valuable chemical probes for studying Polθ function.
• The identified mechanism of inhibition provides a foundation for developing novel Polθ-targeted cancer therapeutics.
• Structural insights facilitate the design of optimized inhibitors with improved efficacy.