Application of inhibitor of targeted FIGNL1-FIRRM-MACIR complex in preparation of tumor drugs and composition of inhibitor of targeted FIGNL1-FIRRM-MACIR complex

Abstract

The invention provides an application of an inhibitor of a targeted FIGNL1-FIRRM-MACIR complex in preparation of a tumor drug and a composition of the inhibitor of the targeted FIGNL1-FIRRM-MACIR complex. According to the research, the MACIR is identified as a bridge-like adaptive subunit of a FIGNL1-FIRRMAAA + ATP (Adenosine Triphosphate) enzyme complex. The MACIR is directly combined with the FIRRM and the DNA to promote the FIGNL1 mediated RAD51 filamentous structure to be unfolded, so that the damage repair of cross-linking (ICLs) between DNA chains induced by aldehydes or platinum drugs is promoted, and the genome stability is maintained. Pharmacological inhibition is carried out on the FIGNL1-FIRRM-MACIR complex by using two inhibitors, namely, 4-{2-[(5-chloro-2-methoxyanilino) carbonyl] anilino}-4-oxobutyric acid and 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1, 6-dihydro-2-pyrimidinyl] sulfenyl}-N-(3-methoxyphenyl) acetamide, which are used for the FIGNL1-FIRRM-MACIR complex, so that the FIGNL1-FIRRM-MACIR complex can be prepared by using the two inhibitors. The cytotoxic effect of platinum drugs in ovarian cancer can be obviously enhanced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of inhibitors targeting the FIGNL1-FIRRM-MACIR complex in the preparation of oncology drugs and their compositions. Background Technology

[0002] Interstrand crosslinks (ICLs) are a serious form of genomic DNA damage that can be induced by intracellular metabolic byproducts or exogenous harmful chemicals, including aldehydes and platinum-based drugs. ICLs simultaneously block DNA replication and RNA transcription, leading to DNA strand breaks and genomic instability. To address this damage, cells have evolved a specific repair mechanism—the Fanconi Anemia (FA) pathway. This pathway coordinates ICL repair through a multi-step process, including activation of the FA core complex, recruitment of structure-specific endonucleases to unlink crosslinked structures, and repair of resulting DNA double-strand breaks (DSBs) via homologous recombination (HR). Defects in the FA pathway severely impair ICL repair efficiency and can lead to Fanconi anemia, developmental abnormalities, immune system disorders, or tumorigenesis. On the other hand, platinum-based chemotherapy drugs treat various malignancies, including ovarian cancer, by inducing the accumulation of ICL damage. In the FA pathway, the recombinase RAD51 plays a central role in HR repair by mediating homology search, DNA pairing, and strand invasion through the formation of nucleoprotein filaments on single-stranded DNA (ssDNA). Therefore, elucidating the molecular mechanisms regulating the assembly and disassembly of RAD51 nucleoprotein filaments is of great significance for understanding ICL repair, maintenance of genomic stability, tumorigenesis, and chemotherapy response.

[0003] The reported regulatory mechanisms of RAD51 can be broadly categorized into positive and negative types. For example, classic tumor suppressor factors BRCA2 and BRCA1 promote the assembly of RAD51 filaments and enhance homologous DNA pairing. After RAD51 completes its function, helicases such as RTEL1, BLM, and RECQ5 can act as anti-recombinases to promptly dissociate the RAD51 filament structure. Notably, we and other research teams recently discovered a novel type of anti-recombinase—the FIGNL1-FIRRM (also known as C1orf112) complex—whose mechanism of action differs from all previously known RAD51 antagonists. The catalytic subunit FIGNL1 of this complex belongs to the AAA+ ATPase family and can form a hexameric ring structure that binds to the N-terminal tail of RAD51. Through conformational remodeling, it ultimately achieves the dissociation of the RAD51 filament structure via protein unfolding. Given that AAA+ATPase-type unfolding enzymes (such as the classic p97) typically rely on multiple adaptor proteins to recognize and remodel different substrates, exploring whether there are bridging-like adaptor proteins that specifically enhance the effect of FIGNL1-FIRRM on RAD51 filaments is of significant research value and can help develop novel therapeutics that target this regulatory axis to sensitize tumor chemotherapy. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides the application of an inhibitor targeting the FIGNL1-FIRRM-MACIR complex in the preparation of tumor drugs.

[0005] In one embodiment, the tumor is a malignant tumor that develops platinum-based drug resistance due to ICL damage mechanisms.

[0006] In one embodiment, the tumor is ovarian cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, or bladder cancer.

[0007] In one embodiment, the inhibitor is 4-{2-[(5-chloro-2-methoxyaniline)carbonyl]aniline}-4-oxobutyric acid or 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamide.

[0008] In one embodiment, the present invention provides the use of an inhibitor that targets and inhibits the FIGNL1-FIRRM-MACIR complex in the preparation of a combination of platinum-based chemotherapy drugs for tumors, the combination comprising the inhibitor and a platinum-based chemotherapy drug.

[0009] In one embodiment, the platinum-based chemotherapy drug is one or more of cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin.

[0010] In one embodiment, the present invention provides a combination drug composition for sensitizing platinum-based chemotherapy in tumors, comprising an inhibitor of the FIGNL1-FIRRM-MACIR complex and a platinum-based chemotherapy drug, and further comprising a pharmaceutically acceptable carrier, diluent or excipient.

[0011] In one embodiment, the present invention provides the use of 4-{2-[(5-chloro-2-methoxyaniline)carbonyl]aniline}-4-oxobutyric acid or 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamide in the preparation of tumor drugs.

[0012] To identify potential adaptor proteins for FIGNL1-FIRRM, we conducted this study and successfully identified C5orf30 as a candidate molecule. C5orf30, also known as macrophage immune metabolic regulator (MACIR, Human Gene ID: 285148; aliases: FLJ25291, C5orf30, corresponding protein of 206 amino acids), has previously been reported to regulate macrophage polarization, promote the expression of anti-inflammatory cytokines, and inhibit the production of pro-inflammatory factors. Mutations in the MACIR gene increase susceptibility to rheumatoid arthritis (RA) and exacerbate inflammatory damage in joint tissues. Notably, significantly elevated levels of aldehydes such as acetaldehyde and malondialdehyde were detected in the synovial fluid of RA patients, suggesting a potential link between DNA crosslinking-related DNA damage and the immune-inflammatory response in RA. Furthermore, CCNH-MACIR fusion genes have been reported in various tumors, which may promote immune escape and lead to poor prognosis by upregulating MACIR expression. However, it remains unclear whether and how MACIR participates in the repair of DNA interstrand crosslinking damage.

[0013] In this study, we combined proteomics analysis and genetic interaction database screening to identify MACIR as a potential adaptor protein for the FIGNL1-FIRRM unfolding enzyme. Further research showed that MACIR can directly interact with FIRRM and form a ternary complex with FIGNL1. Functionally, MACIR enhances the anti-recombinase activity of the FIGNL1-FIRRM complex and promotes the dissociation of RAD51 from DNA, thereby helping cells cope with aldehyde or platinum-based drug-induced ICL damage and maintain genome stability. In vitro biochemical experiments further confirmed that MACIR, through high-affinity binding to DNA, acts as an adaptor protein for the FIGNL1-FIRRM unfolding enzyme, assisting in the dissociation of RAD51 nucleoprotein filaments.

[0014] In the later stages of HR repair in the FA pathway, the ternary complex formed by MACIR and FIGNL1-FIRRM efficiently removes the RAD51 filamentous structure, thereby promoting the repair of ICL damage induced by aldehydes or platinum drugs. Conversely, the absence or pharmacological targeting of the FIGNL1-FIRRM-MACIR complex can significantly enhance the cytotoxicity of platinum drugs to ovarian cancer cells.

[0015] The significance of the FIGNL1-FIRRM-MACIR complex in tumors was also investigated. High expression of this complex was closely associated with poor prognosis in ovarian cancer patients, and pharmacological targeting of this complex enhanced the cytotoxic effects of platinum-based drugs on ovarian cancer. In summary, this study identified MACIR as a novel adaptor protein of the FIGNL1-FIRRM unfolding enzyme, elucidated its role in the dissociation of the RAD51 filament structure, and proposed a potential therapeutic strategy to improve the sensitivity of ovarian cancer to platinum-based chemosensitive agents by targeting the FIGNL1-FIRRM-MACIR complex.

[0016] Besides aldehydes, platinum-based drugs are also common inducing factors of ICL damage. These drugs induce tumor cell death by forming cross-links with DNA, blocking replication and transcription. In high-grade gynecological malignancies such as ovarian cancer, platinum-based chemotherapy remains the first-line standard treatment. However, the development of platinum resistance poses a significant clinical challenge. This study reveals the molecular mechanism by which the FIGNL1-FIRRM-MACIR complex participates in ICL repair, providing a theoretical basis for targeting this complex to overcome platinum resistance in ovarian cancer. Knockdown of the FIGNL1-FIRRM-MACIR complex using siRNA or inhibition of the FIGNL1-FIRRM-MACIR complex with small molecule compounds significantly enhances the sensitivity of ovarian cancer cells to platinum-based drugs.

[0017] In summary, this study identified MACIR as a bridging-like aptamer subunit of the FIGNL1-FIRRMAAA+ATPase complex. MACIR promotes FIGNL1-mediated RAD51 filament unfolding by directly binding to FIRRM and DNA, thereby facilitating aldehyde- or platinum-based drug-induced ICL damage repair and maintaining genomic stability. Pharmacological inhibition of this complex significantly enhances the cytotoxic effects of platinum-based drugs in ovarian cancer. This study deepened our understanding of the RAD51 unfolding mechanism and proposed a novel strategy to target and inhibit the FIGNL1-FIRRM-MACIR complex as a means to enhance the efficacy of platinum-based chemotherapy. Two inhibitors targeting the FIGNL1-FIRRM-MACIR complex, 4-{2-[(5-chloro-2-methoxyaniline)carbonyl]aniline}-4-oxobutyric acid and 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamide, were identified and their role in enhancing the efficacy of platinum-based chemotherapy was confirmed. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a diagram showing the results of a co-immunoprecipitation experiment performed in HEK293T cells to analyze the interaction between exogenously overexpressed MACIR and endogenous FIRRM or FIGNL1. Figure 2 This is a diagram showing the results of Co-IP experiments analyzing the interaction between exogenously overexpressed FIRRM or FIGNL1 and endogenous MACIR. Figure 3 The results of the Co-IP experiment were analyzed after co-overexpression of MACIR and FIRRM or FIGNL1. Figure 4 The diagram shows the results of the Co-IP experiment analyzing the interaction between endogenous FIRRM, FIGNL1 and MACIR. Figure 5 This is a graph showing the results of Western blot analysis of endogenous FIRRM and FIGNL1 protein levels after siRNA knockdown of MACIR. Figure 6The results of the analysis of MACIR, FIRRM, FIGNL1 and RAD51 proteins expressed in Escherichia coli or Saccharomyces cerevisiae, and the purified proteins by SDS-PAGE and Coomassie Brilliant Blue staining are shown in the figure. Figure 7 The results of in vitro incubation of MACIR and FIRRM proteins and pull-down experiments using Flag magnetic beads are shown in the image. The supernatant containing unbound proteins and the eluent from the magnetic beads are analyzed by immunoblotting. Figure 8 This is a graph showing the results of detecting the direct interaction between MACIR and FIGNL1 using an in vitro pull-down experiment; Figure 9 This is a graph showing the results of detecting the direct interaction between MACIR and RAD51 using an in vitro pull-down experiment. Figure 10 The image shows the results of a two-step pull-down experiment performed in vitro on MACIR, FIRRM, and FIGNL1 proteins using GST magnetic beads and Flag magnetic beads. Figure 11 The results of a two-step Co-IP experiment using Flag magnetic beads and S magnetic beads were obtained by co-expressing MACIR and FIRRM with different affinity tags in HEK293T cells. Figure 12 The sensitivity of MACIR or FIRRM knocked-down U2OS cells to cisplatin and mitomycin C (MMC) was evaluated using the CCK-8 assay. Knockdown efficiency was validated by Western blot. The results are presented as mean ± SD (n=3; *p<0.05, **p<0.01, ***p<0.001). Figure 13 The graph shows the results of the CCK-8 assay to evaluate the sensitivity of MACIR or FIRRM knockdown U2OS cells to formaldehyde, acetaldehyde, and malondialdehyde. Data are expressed as mean ± SD (n=3; **p<0.01, ***p<0.001). Figure 14 The figure shows the results of evaluating the viability of MACIR-knockdown U2OS cells after treatment with the indicated drugs using a clonogenic assay. Data are expressed as mean ± SD (n=3; **p<0.01, ***p<0.001). Figure 15 The results of immunofluorescence analysis of γH2AX sites in HeLa cells that were knocked down by MACIR or FIRRM were obtained after treatment with 3.3mM cisplatin for 2 hours and cultured for 12 hours or 48 hours. Figure 16The results of immunofluorescence analysis of the γH2AX site were obtained after HeLa cells knocked down by FIRRM and MACIR alone or simultaneously were treated with cisplatin and released for 48 h. Data are expressed as mean ± SD (n>50; **p<0.01, ****p<0.0001, ns). Figure 17 The image shows the DNA damage results of MACIR knocked-down HeLa cells after MMC treatment and release for 12h or 48h, detected by the neutral comet assay. The comet tail length is expressed as mean ± SD (n>50; ****p<0.0001, ns). Figure 18 The results of the neutral comet assay were obtained at 48h and 72h after MACIR knockdown. The length of the comet tail is expressed as mean ± SD (n>50; p<0.0001). Figure 19 The results are shown in the figure, which are the levels of ssDNA and dsDNA in the cytoplasm detected by immunofluorescence 72 h after MACIR, FIRRM or MRE11 knockdown. The data are expressed as mean ± SD (n>6; *p<0.05, **p<0.01, ***p<0.001). Figure 20 The expression levels of the indicated inflammatory factors were detected after knocking down MACIR, FIRRM, or MRE11 in MCF-7 cells (see figure below). The knockdown efficiency of siRNA was verified by qPCR (see figure above). Data are expressed as mean ± SD (n=3; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, no significant difference). Figure 21 The MACIR nuclear / cytoplasmic fluorescence intensity ratio was analyzed by immunofluorescence 12 h after MMC treatment; the total MACIR protein level was detected by Western blot. The data are expressed as mean ± SD (n>100; p<0.0001). Figure 22 This is a graph showing the results of laser micro-irradiation experiments to observe the recruitment of GFP-MACIR at DNA damage sites. Data are expressed as mean ± SD (n=3; ***p<0.001). Figure 23 The results of PLA assay analysis of the interaction between MACIR and RAD51 were obtained after FIRRM knockdown cells were treated with MMC and released for 12 h. Data are expressed as mean ± SD (n>50; ***p<0.001, ****p<0.0001). Figure 24The results of RAD51 focus plots were analyzed by immunofluorescence after HeLa cells knocked down by MACIR or FIRRM were treated with MMC and released to the indicated time points. Data are expressed as mean ± SD (n>50; ***p<0.001, ns). Figure 25 The results of PLA assay analysis of the interaction between FIRRM and RAD51 were obtained after MACIR knockdown cells were treated with MMC and released for 12 h. Data are expressed as mean ± SD (n>50; p<0.0001). Figure 26 The image shows the results of Western blot analysis of whole cell lysates, soluble components, and chromatin components after MACIR knockdown cells were treated with 1 μM MMC for 24 h. Figure 27 The effect of MACIR protein on the depolymerization of RAD51 filaments was evaluated by biochemical experiments. The levels of RAD51 on the supernatant and magnetic beads were analyzed by immunoblotting, and the results were plotted using ImageJ. Data are expressed as mean ± SD (n=3; ns, no significant difference). Figure 28 The image shows the analysis results after FIRRM-FIGNL1 and MACIR were applied to the RAD51 filamentary structure. The data are expressed as mean ± SD (n=3; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Figure 29 This is a graph showing the results of in vitro DNA binding assays to analyze the binding ability of MACIR, FIGNL1, and RAD51 proteins to ssDNA and dsDNA. Figure 30 The results of the analysis of FIRRM-FIGNL1 and MACIR on RAD51 filamentous structures without the addition of ssDNA capture agents are shown in the figure. Data are expressed as mean ± SD (n=3; **p<0.01, ***p<0.001, ****p<0.0001). Figure 31 This is a graph showing the results of detecting the ssDNA binding activity of truncated MACIR protein using an in vitro DNA binding assay. Figure 32 The direct interaction between truncated MACIR protein and FIRRM was analyzed using an in vitro pull-down assay. The results of the supernatant (S) and eluent (E) were analyzed by immunoblotting. Figure 33 This is a diagram showing the results of the Co-IP experiment in cells to detect the interaction between the truncated MACIR mutant and FIRRM. Figure 34 The image shows the results of constructing truncated mutants (ΔM1 and ΔM2) of MACIR as shown, and analyzing the purified proteins using Coomassie brilliant blue staining. Figure 35 This is a graph showing the results of detecting the ssDNA binding activity of truncated MACIR protein using an in vitro DNA binding assay. Figure 36 The direct interaction between truncated MACIR protein and FIRRM was detected by in vitro pull-down assay. The results of immunoblotting analysis were obtained from the supernatant (S) containing unbound protein and the magnetic bead eluent (E). Figure 37 This is a diagram showing the results of the Co-IP experiment in cells to detect the interaction between the truncated MACIR mutant and FIRRM. Figure 38 Wild-type MACIR, ΔMiddle, or ΔN-terminal mutants were overexpressed in MACIR-knockdown HeLa cells. After treatment with 3.3 μM cisplatin for 2 h and release for 48 h, the γH2AX site was detected by immunofluorescence and quantitative results were obtained. Data are expressed as mean ± SD (n>80; p<0.0001, ns, no significant difference). Figure 39 This is a diagram showing the results of the Co-IP experiment to detect the interaction between the FIRRM truncated mutant and MACIR in cells; Figure 40 The graph shows the results of overexpressing wild-type FIRRM or ΔN mutant in FIRRM knockdown HeLa cells, respectively. Data are expressed as mean ± SD (n>50; ****p<0.0001, ns). Figure 41 This is a graph showing the results of analyzing the expression levels of MACIR in various tumors using the Kaplan–Meier Plotter database; Figure 42 This is a graph showing the results of a survival analysis (including overall survival and progression-free survival) of ovarian cancer patients who received platinum-based chemotherapy; Figure 43 The sensitivity of SKOV3 cells knocked down by MACIR, FIRRM, or FIGNL1 to cisplatin and carboplatin was evaluated using the CCK-8 assay. Knockdown efficiency was validated by Western blot. The results are presented as mean ± SD (n=3; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Figure 44The sensitivity of OVCAR8 cells knocked down by MACIR, FIRRM, or FIGNL1 to cisplatin and carboplatin was evaluated using the CCK-8 assay. Knockdown efficiency was verified by Western blot. The results are presented as mean ± SD (n=3; **p<0.01, ***p<0.001). Figure 45 The results of the colony formation assay to evaluate the viability of MACIR or FIRRM knockdown SKOV3 cells after treatment with the indicated drugs are presented in a figure. Data are expressed as mean ± SD (n=3; *p<0.05, **p<0.01, ****p<0.0001). Figure 46 The results of the colony formation assay to evaluate the viability of MACIR or FIRRM knockdown OVCAR8 cells after treatment with the indicated drugs are presented as mean ± SD (n=3; **p<0.01, ***p<0.001, ****p<0.0001). Figure 47 The figure shows the results of the ATPase activity assay to detect the effect of the small molecule compound on the activity of FIGNL1 ATPase. The data are expressed as mean ± SD (n=3; **p<0.01, ***p<0.001, ****p<0.0001). Figure 48 This is a graph showing the results of in vitro interaction analysis between FIGNL1 protein and candidate compounds using micro-thermophoresis (MST). Figure 49 HeLa cells were pretreated with 500 μM inhibitor for 12 h, then treated with 3.3 mM cisplatin for 2 h, and cultured in inhibitor-containing medium for 48 h. The results of immunofluorescence detection of γH2AX sites were presented as mean ± SD (n>100; p<0.0001). Figure 50 After pretreating FIGNL1 knockdown HeLa cells with 500 μM inhibitor (compound 8) for 12 h, they were treated with 3.3 mM cisplatin for 2 h and then cultured in medium containing the inhibitor for 48 h. The γH2AX focal plane results were detected by immunofluorescence. Data are expressed as mean ± SD (n>100; p<0.0001, ns, no significant difference). Figure 51 HeLa cells with FIGNL1 knockdown were pretreated with 125 μM inhibitor (compound 10) for 12 h, then treated with 3.3 mM cisplatin for 2 h, and cultured in medium containing the inhibitor for another 48 h. The γH2AX focal plane results were detected by immunofluorescence. Data are expressed as mean ± SD (n>50; p<0.0001, ns). Figure 52 The figure shows the results of the CCK-8 assay used to evaluate the effects of compound 8 (250 μM) and compound 10 (125 μM) on the sensitivity of SKOV3 and OVCAR8 cells to cisplatin and carboplatin. Data are expressed as mean ± SD (n=3; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Figure 53 The figure shows the results of evaluating the effects of compounds 8 and 10 on the colony-forming ability of SKOV3 and OVCAR8 cells under platinum-based drug treatment using a colony-forming assay. Data are expressed as mean ± SD (n=3; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application. The present invention will be further described below with reference to the accompanying drawings and embodiments. I. Materials and Methods

[0021] 1. Cell Culture Human HEK293T, HeLa, U2OS, MCF-7, SKOV3, and OVCAR8 cells were cultured in a humidified incubator at 37°C with 5% CO2. The cell culture medium was DMEM (Gibco) supplemented with 10% fetal bovine serum (TransGen) and 1% penicillin-streptomycin (MACGene).

[0022] 2. Construction of plasmids and siRNA The coding sequences for MACIR / C5orf30, FIRRM / C1orf112, and FIGNL1 were cloned into the pCW57, pHBLV, and pMAL vector backbones, respectively, and their corresponding tags were fused. All constructs were validated by sequencing. Plasmid transfection was performed using TurboFect (ThermoScientific) or GenJet (SignaGen) transfection reagents; siRNA transfection was performed using LipofectamineRNAiMAX (ThermoScientific). All siRNAs were synthesized by GenePharma.

[0023] 3. Immunoprecipitation and Western blot After cell collection, cells were lysed for 15 min on ice using CLB-100 lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with a protease inhibitor mixture (Sigma-Aldrich). The cells were then centrifuged at 14,000 rpm for 10 min at 4°C, and the soluble fraction was collected. The supernatant was incubated with anti-FlagM2 affinity magnetic beads (Sigma-Aldrich), S-protein agarose beads (Sigma-Aldrich), or GST beads (GE Healthcare) at 4°C for 4 h. After washing the magnetic beads five times with CLB-100 buffer, the cells were boiled at 95°C with 2×SDS loading buffer for 5 min. Finally, the eluted proteins were separated by SDS-PAGE and analyzed by Western blotting using specific antibodies.

[0024] 4. Expression and purification of MACIR To construct the pMAL-c2PS-Flag-MACIR-10×His plasmid, the coding sequence of MACIR was cloned into the pMAL-c2PS-Flag-10×His vector. This plasmid was transformed into *Escherichia coli* cells. When the OD600 of the cell culture medium reached 0.8, expression was induced for 16 h at 16°C with 0.1 mM IPTG. Subsequently, the cells were collected and lysed by sonication in a T buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 1 mM MDTT, 0.05% Igepal) containing 300 mM KCl and protease inhibitors (aprotinin, chymostatin, leupeptin, and pepstatin A (5 μg / mL each) and 1 mM PMSF).

[0025] After centrifugation at 13,000 rpm for 30 min, the supernatant was collected and incubated with starch affinity magnetic beads (amylose beads, New England Biolabs) at 4°C for 3 h. The beads were then washed four times with T buffer containing 300 mM KCl. The protein was eluted with T buffer containing 10 mM maltose and 300 mM KCl. The eluted protein was then incubated with Ni-NTA resin (QIAGEN) at 4°C for 3 h with 15 mM imidazole. After washing three times with T buffer containing 15 mM imidazole and 300 mM KCl, the bound protein was eluted with T buffer containing 200 mM imidazole and 150 mM KCl. Finally, the purified protein was concentrated, aliquoted, and stored at −80°C.

[0026] 5. Expression and purification of FIRRM To construct the pGEX-MBP-FIRRM-10×His plasmid, the FIRRM coding sequence with a C-terminal 10×His tag was cloned into the pGEX-6P1 vector, and the original GST tag was replaced with an MBP tag. The constructed plasmid was transformed into E. coli cells and induced for expression at 16°C with 0.1 mM IPTG for 16 h.

[0027] After bacterial cell collection, the cells were lysed by sonication in T buffer containing 400 mM KCl and a protease inhibitor. After centrifugation at 13,000 rpm for 30 min, the supernatant was collected and incubated with starch affinity magnetic beads at 4°C for 3 h. The magnetic beads were washed four times with T buffer containing 300 mM KCl, and proteins were eluted using T buffer containing 10 mM maltose and 300 mM KCl.

[0028] Subsequently, the eluent was incubated with Ni-NTA resin at 4°C for 3 h under conditions containing 10 mM imidazole. After washing three times with T buffer containing 10 mM imidazole and 150 mM KCl, the protein was eluted with T buffer containing 200 mM imidazole and 150 mM KCl. Finally, the purified protein was concentrated and aliquoted, and stored at −80°C.

[0029] 6. Expression and purification of FIGNL1 The FIGNL1 coding sequence with a C-terminal Flag tag was cloned into the pESC-TRP-MBP vector to construct the pESC-MBP-FIGNL1-Flag plasmid, which was then transformed into a protease-deficient Saccharomyces cerevisiae strain. After overnight culture, the yeast culture was diluted 1:4 to 2.4 L of deletion medium supplemented with 2% galactose, 3% glycerol, and 3% lactate, and cultured at 30°C for 24 h. The yeast cells were then collected and stored at −80°C.

[0030] For protein purification, the cell pellet was resuspended in T buffer containing 500 mg KCl and a protease inhibitor, and cells were lysed using 0.5 mm glass beads and a BeadBeater (BioSpec). After filtration and centrifugation at 30,000 rpm for 1 h, the supernatant was collected and incubated with starch affinity beads at 4°C for 4 h. The beads were washed four times with T buffer containing 500 mg KCl and once with T buffer containing 300 mg KCl.

[0031] Protein eluted from starch affinity beads was then further purified using anti-FlagM2 affinity beads (Sigma-Aldrich) and incubated at 4°C for 4 h. The beads were washed three times with T buffer containing 300 mM KCl, followed by one wash with T buffer containing 150 mM KCl. Finally, the protein was eluted with T buffer containing 250 ng / μL Flag peptide and 150 mM KCl. The purified protein was concentrated, aliquoted, and stored at −80°C.

[0032] 7. Expression and purification of RAD51 Untagged RAD51 protein was expressed in E. coli and induced at 16°C with 0.1 mM IPTG for 16 h. After cell collection, the cells were sonicated in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 200 mM KCl, 2 mM MDTT, 10% sucrose) and supplemented with protease inhibitors.

[0033] After centrifugation at 13,000 rpm for 30 min, ammonium sulfate was added to the supernatant to a final concentration of 0.23 g / mL to precipitate RAD51 protein. The protein precipitate was resuspended in a dissolution buffer (30 mM Tris-HCl, pH 7.4, 10% glycerol, 0.5 mM EDTA, 0.5 mM DTT) and protease inhibitors were added. The soluble fraction was purified sequentially by QSepharose, Macro hydroxyapatite, MonoQ, and Superdex 200 gel filtration. The final RAD51 protein was concentrated, aliquoted, and stored at −80°C.

[0034] 8. Pull-down test To detect the direct interactions among MACIR, FIRRM, FIGNL1, and RAD51, as well as the interactions between the various truncated mutants of MACIR and FIRRM, purified proteins were mixed in T50 buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 50 mM KCl, 1 mM DTT, 0.01% Igepal) as needed and incubated at 4°C for 3 h. The protein mixture was then incubated with 15 μL of anti-FlagM2 affinity magnetic beads or Ni-NTA resin at 4°C with shaking for 1 h. After centrifugation, the supernatant was collected, the magnetic beads were washed five times with T50 buffer, and the mixture was boiled at 95°C with 1×SDS loading buffer for 5 min. The supernatant and eluted samples were analyzed by Western blot.

[0035] 9. Cell survival experiment Cell viability was assessed using the CCK-8 assay kit from Meilun Biotechnology. Briefly, after siRNA transfection, cells (3,000 cells / well) were seeded in 96-well plates and treated with different concentrations of the drug. After 72 h of culture, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 0.5–1.5 h. Absorbance at 450 nm was measured using an Infinite F50 Plus microplate reader (TECAN).

[0036] 10. Cloning experiment After siRNA transfection according to the experimental design, U2OS, SKOV3, and OVCAR8 cells were seeded in 60 mm culture dishes. After cell adhesion, the cells were treated with the specified concentrations of cisplatin, MMC, or formaldehyde for 24 h, followed by replacement with fresh culture medium. After culturing for 7–14 days, the cells were fixed and stained with a staining solution containing 0.1% Coomassie Brilliant Blue R-250 for 30 min, rinsed with distilled water to remove excess dye, and the number of clones was counted.

[0037] 11. Immunofluorescence assay After specific transfection into HeLa and U2OS cells, cells were seeded onto glass slides and treated with 3.3 mM cisplatin or 120 nM MMC for a specified time. Following treatment, cells were fixed with 4% paraformaldehyde at room temperature for 15 min, then permeabilized with 0.25% Triton X-100 for 5 min. Subsequently, cells were blocked in PBS containing 3% BSA for 10 min, incubated with primary antibody at room temperature for 1 h, and then incubated with secondary antibody for 1 h. Finally, DAPI staining was performed for 5 min, and images were acquired using ZEN microscopy software.

[0038] 12. Comet Experiment HeLa cells transfected with siRNA were subjected to the basic comet assay with or without 120 nM MMC treatment. The simplified steps are as follows: Cells (3 × 10⁻⁶ cells / year) were... 5 Mix 0.5% low-melting-point agarose ( / mL) at a 1:1 ratio with a glass slide pre-coated with 1% ordinary agarose. Incubate the slide in freshly prepared lysis buffer at 4°C for 2 h. Then incubate in alkaline electrophoresis buffer for 20 min and perform electrophoresis at 25 V for 20 min. After electrophoresis, place the slide in neutralization buffer for 20 min, rinse with double-distilled water, and stain with 2.5 μg / mL propidium iodide for 20 min. Observe the comet tail using a 40× fluorescence microscope, counting 100 cells from each replicate slide under each condition. The procedure for the neutral comet experiment is basically the same, but a neutral electrophoresis buffer is used, and the neutralization step is not performed after electrophoresis.

[0039] 13. Quantitative PCR Total RNA was extracted from MCF-7 cells using TRIzol reagent, and cDNA was synthesized using the YEASEN cDNA synthesis kit according to the manufacturer's instructions. Real-time quantitative PCR was performed using the SYBR Green qPCR kit on an ABI instrument. Each sample was tested in triplicate, with ACTB used as an internal control gene, and the relative expression level was calculated using the 2⁻ΔΔCt method. Primers were synthesized by Ribobio.

[0040] 14. Nearest Neighbor Analysis (PLA) Following siRNA transfection, HeLa cells stably expressing SFB-MACIR or MBP-FIRRM-Flag were treated with 120 nM MMC for 2 h to induce ICL, followed by a 12 h recovery period. Cells were fixed, permeabilized, and blocked according to the immunofluorescence protocol, and then incubated with primary antibody in a 37°C humidified chamber for 1 h. Subsequent PLA probe incubation, ligation, and amplification procedures were performed according to the Duolink in situ PLA kit (Sigma-Aldrich) instructions. Finally, DAPI staining was applied for 5 min, and images were acquired using a Leica microscopy system.

[0041] 15. Separation of soluble components from chromatin components HeLa cells were transfected with siRNA and treated with 120 nM MMC, then collected from 10 cm culture dishes. 5% of the cells were used to prepare whole-cell lysis buffer using CLB-400 lysis buffer. The remaining cells were lysed on ice with EBC Buffer A for 5 min, followed by centrifugation at 1000 × g, 4°C for 5 min, and the supernatant was collected as the soluble fraction. The precipitate was washed twice with EBC Buffer A, resuspended in EBC Buffer B, sonicated, and incubated in a 30°C water bath for 10 min. Subsequently, it was centrifuged at 21000 × g, 4°C for 20 min, and the supernatant was collected as the chromatin-binding protein fraction. Each fraction was analyzed by SDS-PAGE and Western blot.

[0042] 16. RAD51 filament depolymerization experiment 5′-Biotin-labeled 60 nt ssDNA (dT60) was immobilized on streptavidin agarose magnetic beads. After washing in buffer A containing 100 mM KCl, the beads were incubated with RAD51 in buffer A containing ATP at room temperature for 30 min to form RAD51 filaments. The beads were then washed, and the mixture was incubated at 37°C with MACIR, FIRRM-FIGNL1, or FIGNL1-FIRRM-MACIR (with or without ssDNA capture agent) for 15 min. The supernatant was collected after centrifugation as dissociated RAD51, and the bead-bound fraction was eluted with SDS loading buffer. The distribution of RAD51 was analyzed by Western blot.

[0043] 17. DNA Binding Experiment Different concentrations of protein were incubated with 10 nMy5-labeled ssDNA (90 nt) or dsDNA (90 bp) in binding buffer at 37°C for 15 min. The reaction products were electrophoresed on a 4% non-denaturing polyacrylamide gel, and the fluorescence signal was detected using a G:Box imaging system.

[0044] 18. HR and NHEJ reporter gene experiments U2OS cells carrying DR-GFP (HR) or EJ5-GFP (NHEJ) reporter systems were transfected with siRNAs targeting BRCA2, 53BP1, or MACIR, and then transfected with the I-SceI expression plasmid to induce DSB. After 48 h, cells were collected and the proportion of GFP-positive cells was detected by flow cytometry to assess the HR and NHEJ repair efficiency.

[0045] 19. Laser Micro-Irradiation Experiment U2OS cells transfected with the GFP-MACIR expression plasmid were induced to undergo DNA damage using a 355 nm UV laser (30% intensity, 5 Hz, 200 ms each time). GFP fluorescence imaging was performed using Nikon NIS-Elements software, with reference images acquired before irradiation, followed by time-series acquisitions every 10 s for 10 min. The fluorescence intensity at the damage sites was quantitatively analyzed using ImageJ software.

[0046] 20. Virtual screening based on molecular docking The FIGNL1 ATPase site was virtually screened using Schrödinger Maestro 12.8, and its 3D structure was visualized using PyMOL. The FIGNL1 structure (PDBID: 8R64) was downloaded, and hydrogen atom addition and side chain repair were performed using ProteinPreparationWizard, followed by energy minimization using the OPLS2005 force field. An acceptor grid was generated centered on the ATP molecule. The SpecsHTS compound library (200,382 compounds) was processed using LigPrep, and then high-throughput virtual screening, standard precision (SP), and high precision (XP) docking were performed using Glide to ultimately select the top 200 candidate compounds.

[0047] 21. Microthermophoresis (MST) Experiment The purified FIGNL1 was fluorescently labeled using the RED-NHS protein labeling kit. The labeled FIGNL1 was incubated with different concentrations of candidate compounds in MST buffer for 15 min, then loaded into capillaries and detected using a Monolith NT.115 instrument. The binding constant Kd was calculated.

[0048] 22. ATPase activity detection 50 nM protein was mixed with 1 mM ATP in reaction buffer and incubated at 37°C for 1 h. The inorganic phosphorus content released by ATP hydrolysis was determined using the malachite green assay.

[0049] 23. Quantitative and Statistical Analysis All experiments were performed at least three times independently. Statistical analyses were performed using GraphPadPrism8 software. Immunofluorescence data were analyzed using the Mann-Whitney rank-sum test, and other data were analyzed using the two-tailed unpaired Student's test. p < 0.05 was considered statistically significant.

[0050] II. Experimental Results 1. Identification of MACIR / C5orf30 as a novel component of the FIRRM / C1orf112-FIGNL1 anti-recombinase complex. Previous studies and work by other research teams have demonstrated that the anti-recombinase complex FIRRM / C1orf112-FIGNL1 promotes ICL repair during the HR repair phase of the FA pathway by dissociating the RAD51 filament structure. To further elucidate the molecular mechanism of FIRRM-FIGNL1 dissociating the RAD51 filament structure, we attempted to identify novel interacting proteins of this complex. First, we analyzed the potential interacting proteins of FIRRM and FIGNL1 using the GeneMANIA database, and ranked and plotted the candidate interacting proteins according to a weighted score ratio. Notably, among the predicted interacting proteins of FIRRM, a previously undefined protein, C5orf30 (also known as MACIR), ranked first, followed by FIGNL1; while in the FIGNL1 interaction network, MACIR did not appear, but FIRRM was included. Consistently, in the MACIR interaction protein network, FIRRM was predicted as the strongest interactor, while FIGNL1 was also not detected.

[0051] To verify the reliability of the above predictions, we further analyzed the interaction network of FIRRM, FIGNL1, and MACIR using the BioGRID database. Comparative analysis of the GeneMANIA and BioGRID datasets revealed consistent identification of an interaction between MACIR and FIRRM. These results suggest that MACIR may be integrated into the FIRRM-FIGNL1 complex through direct interaction with FIRRM. Therefore, we selected MACIR as a candidate molecule to further investigate its functional mechanism in the FIRRM-FIGNL1 anti-recombinase complex.

[0052] To investigate the interaction between MACIR and FIRRM / FIGNL1, we performed a series of co-immunoprecipitation (Co-IP) experiments. The results are as follows: Figure 1 The results show that exogenously expressed MACIR can effectively suppress endogenous FIRRM and FIGNL1. However, as Figure 2 As shown, exogenously expressed FIRRM or FIGNL1 failed to suppress endogenous MACIR. This seemingly contradictory result may stem from differences in the abundance of endogenous proteins: FIRRM and FIGNL1 may be relatively abundant in cells, allowing excess endogenous FIRRM / FIGNL1 to bind to exogenous MACIR; while the endogenous MACIR content is low, failing to adequately bind overexpressed FIRRM / FIGNL1. To verify this hypothesis, we co-expressed MACIR with FIRRM or FIGNL1 and performed Co-IP experiments. Figure 3 As shown, the results were as expected; both exogenously expressed FIRRM and FIGNL1 successfully pulled down the exogenous MACIR. Furthermore, as... Figure 4 As shown, Co-IP using antibodies against endogenous proteins also confirmed the interaction between endogenous FIRRM / FIGNL1 and MACIR.

[0053] Our previous research has shown that FIRRM and FIGNL1 maintain their protein stability through interaction. Therefore, we further investigated whether MACIR also affects the stability of the FIRRM-FIGNL1 complex. The results are as follows: Figure 5 The results show that knocking down MACIR did not change the protein levels of FIRRM or FIGNL1, suggesting that MACIR does not function as a stabilizing factor for this complex.

[0054] In summary, these results identify MACIR as a binding partner of the FIRRM-FIGNL1 complex, suggesting that MACIR may act as a potential regulator involved in the function of this anti-recombinase complex.

[0055] 2. MACIR forms a ternary complex with FIRRM and FIGNL1 through direct interaction with FIRRM. The Co-IP experiment described above showed that MACIR interacts with FIRRM and FIGNL1 in cells. To further determine whether these interactions are direct, we performed in vitro pull-down experiments using purified proteins. First, we expressed and purified MACIR, FIRRM, FIGNL1, and RAD51 proteins in bacteria or yeast, respectively, using affinity chromatography or ion exchange chromatography. Figure 6 As shown in the figure. Subsequently, pull-down experiments were performed using these purified proteins. The results are as follows. Figure 7 The results showed that MACIR could efficiently pull down FIRRM in vitro, indicating a direct interaction between the two; the results are as follows. Figure 8 As shown, MACIR cannot pull down FIGNL1, suggesting that the interaction between MACIR and FIGNL1 in cells is likely indirect.

[0056] Previous studies have confirmed that the FIRRM-FIGNL1 complex can directly bind to RAD51, primarily mediated by the FIGNL1 subunit. To investigate whether MACIR can also directly interact with RAD51, we conducted a pull-down experiment between MACIR and RAD51. The results showed no direct interaction between the two, as described below. Figure 9 As shown.

[0057] Based on the above results, we propose that MACIR, FIRRM, and FIGNL1 form a ternary complex that directly binds to RAD51 and participates in anti-recombinase activity. To verify this hypothesis, we co-incubated the three proteins in vitro and performed sequential pull-down experiments using different affinity tags fused to FIGNL1 and MACIR, respectively. The results are as follows: Figure 10 The results showed that FIRRM, MACIR, and FIGNL1 were detected simultaneously in the final elution product, indicating that the three exist in the same complex. Consistently, sequential Co-IP experiments performed in cells yielded the same conclusion, such as... Figure 11 As shown. In summary, these results support the direct interaction of MACIR with FIRRM and its formation of a ternary complex with FIRRM and FIGNL1 through mutual interaction.

[0058] 3. MACIR promotes ICL repair and maintains genome stability Our previous and other studies have shown that the FIRRM-FIGNL1 complex plays an important role in ICL repair. Given that MACIR can form a complex with FIRRM-FIGNL1, we further investigated whether MACIR also participates in ICL repair. First, we used a CCK-8 assay to assess the effect of MACIR deficiency on cell survival under ICL inducer treatment. Consistent with the FIRRM knockdown phenotype, MACIR knockdown significantly enhanced cell sensitivity to cisplatin and mitomycin C (MMC), such as... Figure 12 As shown.

[0059] Aldehydes (including formaldehyde, acetaldehyde, and malondialdehyde) are known to induce synovial fluid (ICL) damage. Notably, studies have reported significantly elevated levels of acetaldehyde and malondialdehyde in the synovial fluid of rheumatoid arthritis (RA) patients, and MACIR mutations are thought to promote the development and progression of RA. This prompted us to investigate whether MACIR plays a role in protecting against aldehyde-induced damage. The results are as follows... Figure 13 The results showed that MACIR-deficient cells exhibited higher sensitivity to all three aldehydes tested. Furthermore, clonogenic assays demonstrated that the clonogenic ability of MACIR-deficient cells was significantly reduced after treatment with platinum-based drugs or aldehydes. Figure 14 As shown in the figure. These results suggest that MACIR helps cells cope with platinum- or aldehyde-induced ICL damage.

[0060] To further investigate the role of MACIR in ICL injury and repair, we performed immunofluorescence staining for γH2AX (a DNA double-strand break marker). Twelve hours after cisplatin withdrawal, there was no significant difference in the number of γH2AX sites between the control group and MACIR or FIRRM knockdown cells. Figure 15 As shown; however, γH2AX sites persisted in MACIR or FIRRM-deficient cells 48 h after treatment, as shown. Figure 15 As shown, these knockdowns did not increase ICL lesion formation, but significantly impaired its repair, leading to DSB accumulation. Given that MACIR and FIRRM bind directly and coexist in the same complex, we further investigated whether they function in the same ICL repair pathway. To this end, we conducted a dual knockdown experiment of FIRRM and MACIR. The results are as follows... Figure 16 The results showed that further knockdown of MACIR in the absence of FIRRM did not further increase the level of γH2AX sites, suggesting that MACIR and FIRRM play a role in the same pathway.

[0061] When DNA damage is not effectively repaired, nuclear DNA may break and leak into the cytoplasm. Therefore, we used a comet assay to assess the nuclear DNA integrity of MACIR-deficient cells under ICL-damaged conditions. Both neutral and basic comet assays showed that after 48 hours of MMC treatment, the comet tail length of MACIR-deficient cells significantly increased, such as... Figure 17 As shown, this indicates the accumulation of dsDNA and ssDNA damage and fragmentation. We also examined spontaneous DNA damage under conditions without exogenous treatment. The results are as follows. Figure 18 The results showed that spontaneous DNA damage and breakage significantly increased at 48 h and 72 h after MACIR knockdown. Further immunofluorescence experiments showed that at 72 h after knockdown, cytosolic DNA accumulated significantly in MACIR-deficient cells, to a degree similar to that in FIRRM and the positive control MRE11-deficient cells. Figure 19 As shown.

[0062] Previous studies have shown that the accumulation of cytoplasmic DNA can activate the cGAS–STING pathway and promote the development of rheumatoid arthritis. MACIR is considered a negative regulator of inflammatory damage in rheumatoid arthritis. Based on this, we hypothesize that MACIR deficiency may activate the cGAS–STING pathway through DNA damage accumulation. Consistent with this hypothesis, the expression levels of multiple inflammatory factors (including IFN-β, IL-6, CXCL-10, and TNF-α) were significantly increased in MACIR-deficient cells, such as... Figure 20 As shown, MACIR loss supports the idea that DNA damage accumulation can indirectly promote the progression of RA. In summary, these results indicate that MACIR plays a crucial role in promoting ICL repair and maintaining genomic DNA integrity.

[0063] 4. MACIR promotes the dissociation of RAD51 filaments during the HR repair phase of the FA pathway. Subsequently, we systematically investigated the specific molecular mechanism by which MACIR participates in ICL repair. First, we used immunofluorescence assays to detect the subcellular localization of MACIR under ICL injury conditions. After MMC treatment, we observed a significant translocation of MACIR to the nucleus, such as... Figure 21 As shown, this suggests that MACIR may enter the nucleus from the cytoplasm and directly participate in ICL repair.

[0064] To detect whether nuclear-localized MACIRs are directly recruited to DNA damage sites, we conducted laser micro-irradiation experiments. Figure 22 As shown, MACIR gradually accumulates on laser-induced DNA damage bands over time. These results indicate that ICL-induced ATM activation initiates nuclear translocation of MACIR and promotes its recruitment to DNA damage sites.

[0065] Our previous research showed that the FIRRM-FIGNL1 complex can bind to and disassemble the RAD51 filamentary structure. Therefore, we hypothesized that MACIR might also be recruited to the vicinity of RAD51 and participate in its dissociation. To verify this, we first used proximity connectivity analysis (PLA) to detect the spatial proximity relationship between MACIR and RAD51. After MMC-induced ICL, the proximity signal between MACIR and RAD51 was significantly enhanced, such as... Figure 23 As shown. It is noteworthy that under FIRRM knock-down conditions, this proximity signal is significantly reduced, as... Figure 23 As shown, this indicates that FIRRM mediates the recruitment of MACIR to the vicinity of RAD51. Subsequently, we analyzed the effect of MACIR on the RAD51 focal point using immunofluorescence experiments. The results are as follows. Figure 24The results showed that MACIR knockdown did not affect RAD51 focal formation 12 h after ICL treatment, but significantly inhibited RAD51 focal regression at 48 h. These results indicate that MACIR, similar to FIRRM, is involved in the dissociation of intracellular RAD51 filaments.

[0066] To further evaluate the effect of MACIR on the function of the FIRRM-FIGNL1 anti-recombinase complex, we examined the effect of MACIR knockdown on PLA signaling between FIRRM and RAD51. The results are as follows: Figure 25 The results showed that MACIR knockdown further enhanced the PLA signal between FIRRM and RAD51, suggesting that MACIR may promote the dynamic turnover of the FIRRM-FIGNL1 complex on the RAD51 filamentous structure. Consistently, chromatin dissociation experiments showed that MACIR knockdown significantly increased the levels of chromatin-bound FIRRM and FIGNL1, such as... Figure 26 As shown. Based on the above results, MACIR may be recruited to the vicinity of RAD51 via FIRRM, thereby enhancing the dissociation activity of FIRRM-FIGNL1 on the RAD51 filamentous structure.

[0067] To further clarify the role of MACIR in the selection of DSB repair pathways, we used the DR-GFP and EJ5-GFP reporter systems for analysis. In the DR-GFP assay, MACIR knockdown significantly reduced the proportion of GFP-positive cells, to a degree comparable to the loss of the classic HR factor BRCA2, indicating that MACIR promotes HR repair. Conversely, in the EJ5-GFP assay, unlike the NHEJ factor 53BP1, MACIR loss had no significant effect on the NHEJ pathway. These results suggest that MACIR specifically participates in HR pathway-mediated DSB repair, rather than NHEJ, which is highly consistent with its function in regulating RAD51.

[0068] 5. MACIR directly enhances the depolymerization effect of the FIRRM-FIGNL1 complex on the RAD51 filament structure in vitro. To directly investigate the effect of MACIR on the depolymerization of RAD51 filaments, we reconstructed the assembly and depolymerization process of RAD51 filaments in vitro using purified MACIR, FIRRM, FIGNL1, RAD51 protein, and DNA substrate. Our previous studies have demonstrated that the FIRRM-FIGNL1 complex can promote the depolymerization of RAD51 filaments in vitro. Consistent with this, we successfully reproduced the depolymerization activity of FIRRM-FIGNL1 on RAD51 filaments in the reconstructed system, validating the reliability of the experimental system. Subsequently, we tested whether MACIR itself possesses the ability to depolymerize RAD51 filaments. Figure 27As shown, the addition of MACIR alone did not exhibit RAD51 filament disintegration activity. Based on this result, we hypothesize that MACIR may exert its effect by enhancing the anti-recombinase activity of the FIRRM-FIGNL1 complex. To verify this hypothesis, we added MACIR to a reaction system containing FIRRM and FIGNL1 and examined its effect on the depolymerization of the RAD51 filament structure. Figure 28 As shown, the addition of MACIR further improved the depolymerization efficiency of FIRRM-FIGNL1 on the RAD51 filament structure, indicating that MACIR can enhance the anti-recombinase activity of the complex.

[0069] Our previous research showed that FIGNL1 can bind single-stranded DNA (ssDNA) but not double-stranded DNA (dsDNA), while FIRRM did not detect significant DNA binding ability. Therefore, we further analyzed the DNA binding characteristics of MACIR through in vitro DNA binding experiments. Figure 29 As shown, MACIR can bind both ssDNA and dsDNA simultaneously, with a stronger affinity for ssDNA. Notably, MACIR's DNA-binding activity is higher than FIGNL1 or RAD51, such as... Figure 29 Given the strong affinity of MACIR for ssDNA, we hypothesized that MACIR would more effectively promote the depolymerization of RAD51 filaments in the absence of ssDNA traps (used to capture free proteins). To verify this hypothesis, we repeated the RAD51 depolymerization experiment without the addition of ssDNA traps. The results were as expected; in the absence of ssDNA traps, the enhancing effect of MACIR on the anti-recombinase activity of FIRRM-FIGNL1 was more significant. Figure 30 As shown, compared to the reaction system containing ssDNAtrap ( Figure 28 The ratio changes from 1.50 to 1.11.

[0070] In summary, our in vitro biochemical experiments show that MACIR has a strong DNA binding capacity and can directly enhance the depolymerization activity of FIRRM-FIGNL1 on RAD51 filamentous structures.

[0071] 6. The direct interaction between MACIR, FIRRM, and DNA is crucial for ICL repair. To further elucidate the molecular mechanism by which MACIR enhances FIRRM-FIGNL1-mediated depolymerization of the RAD51 filamentous structure, we first identified the functional domains in MACIR responsible for binding to FIRRM and DNA. Based on the MACIR structure predicted by AlphaFold, we constructed three truncated mutants: ΔN-terminal (deletion of amino acids 1–80), ΔC-terminal (deletion of amino acids 151–206), and ΔMiddle (deletion of amino acids 81–150), and purified these proteins in *E. coli*. In vitro DNA binding assays showed that deletion of the middle region had the most significant impact on MACIR binding to ssDNA, followed by N-terminal deletion, while C-terminal deletion had no significant effect on DNA binding ability. Figure 31 As shown in the figure. These results indicate that MACIR binds to ssDNA through multiple regions, with the middle region playing a major role and the N-terminal region playing a secondary role. Subsequently, we evaluated the interaction between various truncated mutants of MACIR and FIRRM using in vitro pull-down experiments. Figure 32 As shown, the binding of ΔMiddle to FIRRM was significantly weakened, while ΔN-terminal and ΔC-terminal retained their interaction ability. Intracellular Co-IP experiments also confirmed this result, such as... Figure 33 As shown.

[0072] To further distinguish between the DNA binding site and the FIRRM binding site within the MACIR intermediate region, we constructed two additional truncated mutants, ΔM1 (deletion of amino acids 80–113) and ΔM2 (deletion of amino acids 114–149), and purified the proteins, as follows: Figure 34 As shown in the figure. DNA binding experiments showed that, compared with ΔMiddle, both ΔM1 and ΔM2 partially recovered ssDNA binding capacity, such as... Figure 35 As shown, both regions M1 and M2 contain DNA binding sites. Furthermore, pull-down and Co-IP experiments revealed that both ΔM1 and ΔM2 retained their binding ability to FIRRM, as... Figure 36 , Figure 37 As shown. These results indicate that the middle region of MACIR interacts directly with FIRRM and DNA through multiple sites. To assess the functional significance of this region in ICL damage repair, we re-expressed the ΔMiddle mutant in MACIR-deficient cells for functional salvage experiments. Immunofluorescence results showed that, unlike wild-type MACIR, ΔMiddle failed to reduce γH2AX site levels after ICL induction, as shown. Figure 38 As shown. In contrast, ΔN-terminal can partially reduce the γH2AX site, such as Figure 38As shown, this is consistent with the result that its DNA binding ability is between that of wild type and ΔMiddle. Figure 31 In summary, these results indicate that the region mediating MACIR-FIRRM / DNA interaction is essential for its ICL repair function.

[0073] Our previous research has also shown that the highly conserved WCF tripeptide sequence within the DUF4487 domain of FIRRM is crucial for its interaction with FIGNL1. In this study, we further analyzed the region of FIRRM responsible for MACIR binding. We overexpressed wild-type and truncated mutant FIRRM and performed Co-IP analysis. Figure 39 As shown, N-terminal deletion significantly weakens the interaction between FIRRM and MACIR. To assess the functional significance of this interaction in ICL repair, we reintroduced the ΔN mutant in FIRRM-deficient cells and examined the ICL-induced γH2AX site. The results showed that the ΔN mutant could not rescue ICL repair defects, as... Figure 40 As shown, the interaction between the FIRRMN end and MACIR is crucial for its ICL repair function.

[0074] In summary, our research demonstrates that direct binding between MACIR and FIRRM / DNA through specific interaction regions is crucial for efficient ICL repair.

[0075] 7. Targeting MACIR can enhance the sensitivity of ovarian cancer cells to platinum-based chemotherapy drugs. If ICL damage induced by aldehydes or other factors is not effectively repaired, it can lead to genomic instability and ultimately promote tumorigenesis. On the other hand, platinum-based chemotherapy drugs primarily exert their anti-tumor effects by inducing ICL damage. Therefore, we further explored the potential association between MACIR and tumors. Through analysis of the Kaplan–Meierplotter database, we found that MACIR, FIRRM, and FIGNL1 are highly expressed in various tumor types, including breast and ovarian cancer, such as… Figure 41 As shown. Given that platinum-based chemotherapy is one of the first-line standard treatments for ovarian cancer, we further analyzed the relationship between MACIR expression levels and clinical outcomes in ovarian cancer patients. Kaplan–Meierplotter data analysis showed that high expression of MACIR, FIRRM, and FIGNL1 was significantly associated with shorter progression-free survival (PFS) and overall survival (OS) in ovarian cancer patients. Notably, high expression of these three genes was also associated with poor prognosis in patients receiving platinum-based chemotherapy, such as… Figure 42As shown, high expression of MACIR, FIRRM, and FIGNL1 may confer resistance to platinum-based drugs in ovarian cancer cells.

[0076] Based on the above observations, we hypothesize that targeting MACIR, FIRRM, and FIGNL1 may enhance the sensitivity of ovarian cancer cells to platinum-based chemotherapy. To verify this hypothesis, we knocked down MACIR, FIRRM, or FIGNL1 in the ovarian cancer cell lines SKOV3 and OVCAR8, and assessed their sensitivity to commonly used platinum-based drugs (cisplatin and carboplatin) using cell survival assays. Figure 43 and Figure 44 As shown, knocking down any one gene significantly increased the sensitivity of ovarian cancer cells to platinum-based drugs. Furthermore, clonogenic assays indicated that silencing MACIR or FIRRM significantly weakened the clonogenic ability of ovarian cancer cells under platinum-based drug treatment. Figure 45 and 46 As shown in the figure. In summary, these results indicate that high expression of FIGNL1-FIRRM-MACIR is closely associated with poor prognosis in ovarian cancer patients, and that targeted inhibition of this complex can enhance the killing effect of platinum-based chemotherapy drugs on ovarian cancer cells.

[0077] 8. Screening of inhibitors targeting the FIGNL1-FIRRM-MACIR complex and their role in enhancing the sensitivity of platinum-based chemosensitivity in ovarian cancer. FIGNL1 is the catalytic subunit of the FIGNL1-FIRRM-MACIR anti-recombinase complex, exerting its RAD51 destructive activity through ATP hydrolysis and binding to RAD51. Given that the structure of FIGNL1 has recently been resolved, and its ATPase domain possesses structural features suitable for small molecule targeting, we conducted structure-based virtual screening to identify potential complex inhibitors.

[0078] To evaluate the impact of the top 20 candidate compounds obtained from virtual screening on FIGNL1 ATPase activity, we performed ATPase activity assays. Figure 47 As shown, five compounds (compounds 5, 8, 10, 15, and 19) significantly inhibited the ATPase activity of FIGNL1. Subsequently, we used microscale thermophoresis (MST) to detect whether these compounds could directly bind to FIGNL1. The results showed that all five compounds had a strong direct binding ability to FIGNL1, with compound 10 exhibiting the highest affinity. Figure 48 As shown.

[0079] Next, to verify whether these compounds inhibit ICL repair in cells, we detected γH2AX site levels by immunofluorescence after cisplatin treatment. Figure 49As shown, 48 hours after cisplatin withdrawal, inhibitor treatment resulted in varying degrees of persistence of γH2AX sites, with compounds 8 and 10 exhibiting the most significant effects, suggesting their interference with ICL repair. To further confirm whether compounds 8 and 10 inhibit ICL repair by specifically targeting FIGNL1, we examined the γH2AX site levels in FIGNL1 knockdown cells after the addition of these compounds. Figure 50 and 51 As shown, in the absence of FIGNL1, compound treatment did not produce a cumulative effect, indicating that the inhibitory effects of compounds 8 and 10 are specifically dependent on FIGNL1. Furthermore, we analyzed the binding modes of compounds 8 and 10 to FIGNL1 through molecular docking, revealing that they interact with multiple binding sites of FIGNL1 through various interaction mechanisms, including hydrogen bonding, salt bridging, and metal coordination.

[0080] Finally, we evaluated the effects of compounds 8 and 10 on the cytotoxic effects of platinum-based chemotherapy drugs in ovarian cancer cells using cell viability and colony formation assays. CCK-8 results showed that compounds 8 and 10 significantly enhanced the sensitivity of ovarian cancer cells (SKOV3 and OVCAR8) to cisplatin and carboplatin, such as... Figure 52 As shown. Consistently, clonogenic assays further demonstrated that compounds 8 and 10 significantly inhibited the clonogenic ability of ovarian cancer cells under cisplatin or carboplatin treatment conditions, such as Figure 53 As shown, this indicates that it can significantly enhance the killing effect of platinum-based drugs on ovarian cancer cells.

[0081] In summary, compounds 8 and 10 can act as selective inhibitors of the catalytic subunit of the FIGNL1-FIRRM-MACIR complex, and when used in combination with platinum-based chemotherapy drugs, they can effectively inhibit the growth of ovarian cancer cells.

[0082] Compound 8 is named 4-{2-[(5-chloro-2-methoxyaniline)carbonyl]aniline}-4-oxobutyric acid, with the molecular formula C18H17ClN2O5 and the structural formula as follows: .

[0083] Compound 10 is named 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamide, with the molecular formula C17H21N3O5S and the structural formula as follows: .

[0084] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0085] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. Application of inhibitors targeting the FIGNL1-FIRRM-MACIR complex in the preparation of tumor drugs.

2. The application according to claim 1, characterized in that, The tumor is a malignant tumor that develops platinum-based drug resistance due to ICL damage mechanisms.

3. The application according to claim 1, characterized in that, The tumor is ovarian cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, or bladder cancer.

4. The application according to claim 1, characterized in that, The inhibitor is 4-{2-[(5-chloro-2-methoxyaniline)carbonyl]aniline}-4-oxobutyric acid or 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamide.

5. The application of inhibitors targeting the FIGNL1-FIRRM-MACIR complex in the preparation of platinum-based chemotherapy combination drugs for tumors, characterized in that, The combination therapy includes the inhibitor and a platinum-based chemotherapy drug.

6. The application according to claim 5, characterized in that, The platinum-based chemotherapy drugs are one or more of cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin.

7. The application according to claim 5, characterized in that, The inhibitor is 4-{2-[(5-chloro-2-methoxyaniline)carbonyl]aniline}-4-oxobutyric acid or 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamide.

8. A combination drug composition for enhancing the sensitization of platinum-based chemotherapy in tumors, characterized in that, It contains inhibitors of the FIGNL1-FIRRM-MACIR complex and platinum-based chemotherapy drugs, as well as pharmaceutically acceptable carriers, diluents, or excipients.

9. The combination drug composition according to claim 8, characterized in that, The platinum-based chemotherapy drugs are one or more of cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin.

10. The combination drug composition according to claim 8, characterized in that, The inhibitor is 4-{2-[(5-chloro-2-methoxyaniline)carbonyl]aniline}-4-oxobutyric acid or 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamide.

11. The application of 4-{2-[(5-chloro-2-methoxyaniline)carbonyl]aniline}-4-oxobutyric acid or 2-{[5-(2-ethoxyethyl)-4-hydroxy-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamide in the preparation of tumor drugs.

12. The application according to claim 11, characterized in that, The tumor is a malignant tumor that develops platinum-based drug resistance due to ICL damage mechanisms.

13. The application according to claim 12, characterized in that, The tumor is either ovarian cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, or bladder cancer.

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