An Optimized RAD51 Inhibitor That Disrupts Homologous Recombination without Requiring Michael Acceptor Reactivity

Abstract

Homologous recombination (HR) is an essential process in cells that provides repair of DNA double-strand breaks and lesions that block DNA replication. RAD51 is an evolutionarily conserved protein that is central to HR. Overexpression of RAD51 protein is common in cancer cells and represents a potential therapeutic target in oncology. We previously described a chemical inhibitor of RAD51, called RI-1 (referred to as compound 1 in this report). The chloromaleimide group of this compound is thought to act as a Michael acceptor and react with the thiol group on C319 of RAD51, using a conjugate addition-elimination mechanism. In order to reduce the likelihood of off-target effects and to improve compound stability in biological systems, we developed an analog of compound 1 that lacks maleimide-based reactivity but retains RAD51 inhibitory activity. This compound, 1-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)-4-morpholino-1H-pyrrole-2,5-dione, named RI-2 (referred to as compound 7a in this report), appears to bind reversibly to the same site on the RAD51 protein as does compound 1. Like compound 1, compound 7a specifically inhibits HR repair in human cells.

MUC1-Mediated Metabolic Alterations Regulate Response to Radiotherapy in Pancreatic Cancer

“…In our irradiation study, we observed that MUC1-expressing cells stained less positively for γH2AX twelve hours post-irradiation in comparison to either control or MUC1-knock down cells (Figure 2a). Furthermore, we evaluated the role of non-homologous end joining repair (NHEJ) and homologous repair (HR) in DNA damage repair of MUC1-expressing cells using NHEJ inhibitor Nu-7026 and HR inhibitor RI-1, respectively (30,31). Both the treatments abrogated MUC1-induced differences in γH2AX staining as the level of γH2AX staining increased by combination treatments with DNA damage repair inhibitors and radiation (Figure 2a).…”

Development of Small Molecules that Specifically Inhibit the D-loop Activity of RAD51

“…27,28 Similarly, we previously demonstrated that both RI-1 and RI-2 induce elevated levels of SSA activity. 19 By contrast, we found that 9h does not stimulate SSA, using a relevant concentration range that inhibits HR (Figure 5b). This suggests that 9h stabilizes nucleoprotein filaments in a nonfunctional state, which are incapable of D-loop activity and simultaneously shielded from related (e.g., RAD52-mediated) pathways that promote SSA.…”

“…Other characterized RAD51 inhibitors including our previously described inhibitors RI-1 and RI-2 act by preventing RAD51 from loading onto damaged DNA. 13,19 Compound 1 and its analogues represent potential cancer therapeutics aimed at sensitizing tumors to DNA-damaging therapies. Considering its high potency for inhibiting both D-loop formation and HR, compound 9h provides an important candidate for further investigation.…”

“…[14][15][16] Although several small molecule RAD51 inhibitors have been developed, most of these inhibitors act by preventing the formation of RAD51-ssDNA nucleoprotein filaments. 13,14,[16][17][18][19] One possible exception is halenaquinone, a chemical purported to specifically inhibit D-loop formation by RAD51. 20 However, halenaquinone also reduces the subnuclear appearance of RAD51 foci at DSB sites in cells, which raises questions as to its mechanistic interaction with RAD51 in cells.…”

Quinazolinone derivatives as inhibitors of homologous recombinase RAD51

“…RAD51 has been recognized as a potential oncotarget due to its critical role in HR, and contributes to an aggressive cellular phenotype and resistance to therapeutics. Several small molecule RAD51 inhibitors have been discovered by high-throughput screening of compound libraries, notably B02 [5][6][7] , the RI series [8][9][10] and the IBR2 series 11,12 ( Figure 1). Alternatively, a fragmentbased screening approach at Cambridge identified another series of compounds 13,14 ( Figure 1).…”