Design of a Targeted Covalent Probe to Interrogate the DNA Polymerase Activity of Polθ

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• Human DNA polymerase θ (Polθ) is crucial for microhomology-mediated end-joining (MMEJ).
• Polθ is a therapeutic target in homologous recombination (HR)-deficient cancers.
• Covalent inhibitors for Polθ have not been explored, unlike reversible ones.

Purpose of the Study:

• To design and synthesize a potent, selective covalent inhibitor of Polθ.
• To validate Cys2411 as a target site for covalent inhibition.
• To develop a chemical probe for studying Polθ biology.

Main Methods:

• Structure-based drug design utilizing X-ray crystallography.
• Synthesis of novel vinyl sulfone-containing compounds.
• Biochemical assays to determine inhibition kinetics (K_inact).
• High-resolution cocrystal structure determination.

Main Results:

• Identified Cys2411 as an accessible residue near the Polθ active site.
• Designed and synthesized RP-4029, a covalent inhibitor targeting Cys2411.
• RP-4029 demonstrated efficient covalent linkage to Cys2411 with K_inact = 11.6 s^-1.
• Cocrystal structure confirmed the covalent modification and validated the design.

Conclusions:

• Cys2411 is a privileged site for developing covalent Polθ inhibitors.
• RP-4029 is a highly potent and selective chemical probe for Polθ.
• This work opens new avenues for Polθ-targeted cancer therapies and biological research.