Small molecule inhibitors of a human recombination-associated ATPase, RAD54

Homologous recombination (HR) is a principal support pathway for DNA replication and for recovery from DNA breaks and interstrand crosslinks, making it a rational target for inhibition in cancer therapy. The ATPase RAD54 functions in molecular events that promote DNA sequence-preservation during HR-mediated damage repair, including homology search, DNA strand exchange, and transition to DNA repair synthesis within a displacement loop intermediate. We developed a high-throughput biochemical screen to identify small-molecule inhibitors of human RAD54, using a phosphate detection assay to monitor RAD54 ATPase activity in the presence of double-stranded DNA (dsDNA). After filtering potential DNA intercalators and frequent hitters, we identified two chemotypes that reproducibly inhibited RAD54 ATPase in vitro. We evaluated these chemotypes for inhibition of RAD54-dsDNA binding and cancer cell survival. A halogenated carbazole/dihydroacridine scaffold inhibited a panel of SWI2/SNF2-related ATPases but not VCP/p97, an unrelated ATPase. Small molecules that interfere with key steps in HR-- such as inhibitors of RAD54--may expose DNA repair-dependent vulnerabilities in cancer cells.