Mafosfamide

Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.

Cyclophosphamide (CP) is one of the most widely used anticancer drugs for various malignancies. However, its long-term use leads to ALDH1A1-mediated inactivation and subsequent resistance which necessitates the development of potential ALDH1A1 inhibitors. Currently, ALDH1A1 inhibitors from different chemical classes have been reported, but these failed to reach the market due to safety and efficacy problems. Developing a new treatment from the ground requires a huge amount of time, effort, and money, therefore it is worthwhile to improve CP efficacy by proposing better adjuvants as ALDH1A1 inhibitors. Herein, the database constituting the FDA-approved drugs with well-established safety and toxicity profiles was screened through already reported machine learning models by our research group. This model is validated for discriminating the ALDH1A1 inhibitors and non-inhibitors. Virtual screening protocol (VS) from this model identified four FDA-approved drugs, raloxifene, bazedoxifene, avanafil, and betrixaban as selective ALDH1A1 inhibitors. The molecular docking, dynamics, and water swap analysis also suggested these drugs to be promising ALDH1A1 inhibitors which were further validated for their CP resistance reversal potential by in-vitro analysis. The in-vitro enzymatic assay results indicated that raloxifene and bazedoxifene selectively inhibited the ALDH1A1 enzyme with IC₅₀ values of 2.35 and 4.41 μM respectively, whereas IC₅₀ values of both the drugs against ALDH2 and ALDH3A1 was >100 μM. Additional in-vitro studies with well-reported ALDH1A1 overexpressing A549 and MIA paCa-2 cell lines suggested that mafosfamide sensitivity was further ameliorated by the combination of both raloxifene and bazedoxifene. Collectively, in-silico and in-vitro studies indicate raloxifene and bazedoxifene act as promising adjuvants with CP that may improve the quality of treatment for cancer patients with minimal toxicities.