Cell experiment:

HEK293 cells are plated into 96-well tissue culture plates at a density of 300 cells per well in the presence or absence of 50 nM mitomycin C (MMC) for 24 hours at 37℃, 5% CO2. Media is subsequently replaced with fresh media containing 0.5% DMSO plus RI-2 for an additional 24 hours. RI-2 is then removed, and cultures are allowed to grow to a 50-70% confluence. Average survival from at least three replicates is measured using CellGlo reagentor. RI-2 is deemed successful in sensitizing cells to MMC if they generate significantly greater toxicity in the presence of MMC relative to the absence of MMC. Specifically, sensitization is scored as a “+” when non-overlapping standard errors are observed for at least two pairs of compound doses[1].

Description:

IC50: 44.17 μM

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. RI-2 is an optimized reversible RAD51 inhibitor.

In vitro: Although RI-2 did exhibit reduced activity relative to RI-1, it did retain enough activity to sensitize cells to MMC cell-based assays. RI-2 exhibited no reactivity toward glutathione after incubation for 24 hours. RAD51 protein that had been treated with RI-2 prior to washing exhibited no measurable loss of DNA binding activity, even though RI-2 is known to inhibit RAD51 with an IC50 of 44.17 μM. This indicates that RAD51 inhibition by RI-2 is fully reversible [1].

In vivo: RI-1, the analogue of RI-2, facilitated mapping of the binding pocket within known crystal structures of RAD51. However, this reactivity of RI-1 potentially may limit its development in pre-clinical animal models. RI-1 has been found to exhibit a relatively short half-life, due to reactivity in thiol-containing solutions [1].

Clinical trial: Up to now, RI-2 is still in the preclinical development stage.

Reference:
[1] Budke B, Kalin JH, Pawlowski M, Zelivianskaia AS, Wu M, Kozikowski AP, Connell PP.  An optimized RAD51 inhibitor that disrupts homologous recombination without requiring Michael acceptor reactivity. J Med Chem. 2013 Jan 10;56(1):254-63.