Structural basis for a Polθ helicase small-molecule inhibitor revealed by cryo-EM

Area of Science:

• Biochemistry
• Structural Biology
• Oncology

Background:

• DNA polymerase theta (Polθ) is a bifunctional enzyme with DNA helicase and polymerase activities.
• Polθ plays a critical role in DNA repair pathways, particularly in homology-directed repair (HDR).
• Polθ is synthetically lethal with HDR factors, making it a promising target for precision oncology in HDR-deficient cancers.

Purpose of the Study:

• To characterize the binding and mechanism of action of the Polθ helicase (Polθ-hel) inhibitor AB25583.
• To elucidate the structural basis for AB25583's potent and specific inhibition of Polθ-hel.
• To provide insights for the development of novel Polθ-targeting cancer therapeutics.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine complex structures.
• Biochemical assays to measure enzyme activity and inhibition (IC50).
• Cell-based assays to assess selective killing and drug synergy.

Main Results:

• AB25583 demonstrated a potent IC50 of 6 nM against Polθ-hel.
• Cryo-EM revealed predominantly dimeric Polθ-hel:AB25583 complex structures.
• AB25583 selectively kills BRCA1/2-deficient cells and synergizes with olaparib.
• Inhibition occurs via an allosteric mechanism targeting the helicase ATPase activity.

Conclusions:

• AB25583 is a highly specific and potent inhibitor of Polθ helicase.
• The structural insights into AB25583 binding and allosteric inhibition mechanism are crucial.
• These findings accelerate the development of Polθ-targeting drugs for HDR-deficient cancers.