Electrophiles and electrophile prodrugs as RAD51 inhibitors

Abstract

1. A method comprising co-administering to a subject having, suspected of having, or at risk of developing cancer a therapeutically effective amount of at least one compound (a) selected from (a)(i) a nitroalkene fatty acid, (a)(ii) an unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between the electron-withdrawing group and the leaving group, (a)(iii) a thiolated nitro fatty acid, or (a)(iv) a dicarboxylic acid compound containing an electron-withdrawing group, and a therapeutically effective amount of at least one anti-neoplastic agent (b), wherein the cancer is a cancer associated with a genetic etiology of defects in DNA repair genes, a cancer with a high frequency of natural genomic instability, a cancer that responds well to DNA damaging agents, or a cancer that responds well to a combination of DNA damaging agents and immunotherapy.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 767,424, filed November 14, 2018, which is incorporated herein by reference.

[0002] Government support acknowledged This invention was made with Government support under Grant Nos. HL058115, DK072506, HL103455, and HL064937 awarded by the National Institutes of Health. The Government has certain rights in the invention. [Background technology]

[0003] background Triple-negative breast cancer (TNBC) constitutes approximately 20% of all breast cancers and is the most aggressive subtype of breast cancer. Due to the lack of estrogen and progesterone receptors and the receptor tyrosine kinase ERB2 (HER2), which define the majority of breast cancers, there are no targeted therapies for patients with TNBC. This, combined with a high rate of metastasis and a lower 5-year survival rate than other breast cancer phenotypes, means that there is a significant unmet need for new therapeutic strategies.

[0004] Fatty acid nitroalkenes are endogenously detectable products of nitric oxide- and nitrite-dependent metabolic and inflammatory responses with unsaturated fatty acids. Due to their electrophilic nature, fatty acid nitroalkenes mediate post-translational modifications (PTMs) of reactive nucleophilic cysteine ​​thiols in proteins including p65, Keap1, PPARγ, STING, and 5-lipoxygenase in NF-κB, thus regulating protein structure and function to mediate multifaceted cytoprotective and anti-inflammatory signaling responses.

[0005] Numerous exogenous and endogenous stimuli DNA damage events require that DNA damage be carefully detected and efficiently repaired. Several DNA repair mechanisms have been identified that ameliorate deleterious genomic perturbations, such as direct restoration, mismatch repair, nucleotide excision repair, base excision repair, and double-strand break (DSB) repair. DNA DSBs are particularly pathogenic because loss and mutation of genomic material promotes genomic variation and imbalance.

[0006] DNA DSB repair consists of four pathways: classical non-homologous end joining (c-NHEJ), alternative end joining (ALT-EJ), single-strand annealing (SSA), and homologous recombination-directed repair (HDR). NHEJ does not require homology to bridge DSBs before ligation, while ALT-EJ is mediated by short stretches of homology to bridge DSBs before ligation. SSA uses neighboring homology to bridge DNA lesions, causing deletions between repeat sequences. HDR is the primary event in DSB repair that involves invasion by at least one strand from the DSB into a homologous template, which serves as a template for nascent DNA synthesis to form an extended strand that can form a bridge to the other DSB end. The homologous strand invasion step is catalyzed by the recombinase Rad51. End resection is the processing of chromosomal DSBs generating 3' single-stranded DNA. Each of these pathways involves a specific set of DNA repair proteins (Figure 14A).

[0007] There are two main pathways to repair DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is faster and more frequently used, while HR repair mechanisms maintain the highest fidelity of the genome. HR repair protects cells from deleterious genomic instability caused by DSBs by correcting genetic material loss through homologous template search that maintains the genomic landscape. RAD51 is a critical component of HR, facilitating homology search and strand exchange to repair DSBs. RAD51 and structurally similar proteins XRCC2, XRCC3, RAD51B, RAD51C, RAD51D, DMC1, and SWSAP1 all act in concert to promote HR. Thus, reduced RAD51 and paralog activity is associated with oncogenesis. Although RAD51 is essential for high-fidelity repair of DSBs that maintain genomic homeostasis, overexpression of RAD51 in cancer may have deleterious consequences. The degree of RAD51 overexpression correlates with breast cancer tumor grade and has been identified in triple-negative breast cancer (TNBC) cell lines and metastatic patient samples. RAD51 overexpression inhibits chemotherapy efficacy in cancer patients by making cancer cells more resistant to DNA-damaging agents. Response to neoadjuvant chemotherapy inversely correlates with the number of BRCA1, γH2AX, and RAD51 foci before treatment, and RAD51 foci after treatment. Summary of the Invention [Means for solving the problem]

[0008] Abstract Subjects who have, are suspected of having, or are at risk for developing cancer, or are in remission from cancer, (a) a therapeutically effective amount of at least one compound (a) selected from: (a)(i) a nitroalkene fatty acid; (a)(ii) an unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between the electron-withdrawing group and the leaving group; (a)(iii) a thiolated nitro fatty acid; or (a)(iv) a dicarboxylic acid compound containing an electron-withdrawing group; a therapeutically effective amount of at least one anti-neoplastic agent (b); and the cancer is a cancer with a genetic etiology of defects in DNA repair genes, a cancer with a high frequency of natural genomic instability, a cancer that is treated with a DNA damaging agent, or a cancer that is treated with a combination of a DNA damaging agent and immunotherapy; A method is disclosed herein.

[0009] Illustrative cancers include breast cancer, particularly triple-negative breast cancer, colon cancer, prostate cancer, ovarian cancer, brain cancer (glioblastoma), pancreatic cancer, and skin cancer (e.g., melanoma). Cancer cells that have acquired the ability to spread (metastasize) and are highly genomically unstable are particularly suitable for the combination treatments disclosed herein.

[0010] Also disclosed herein are methods that include administering to a subject having, suspected of having, or at risk of developing triple-negative breast cancer, a therapeutically effective amount of at least one compound (a) selected from (a)(i) a nitroalkene fatty acid, (a)(ii) an unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between the electron-withdrawing group and the leaving group, (a)(iii) a thiolated nitro fatty acid, or (a)(iv) a dicarboxylic acid compound containing an electron-withdrawing group.

[0011] (a) a therapeutically effective amount of at least one compound (a) selected from: (a)(i) a nitroalkene fatty acid; (a)(ii) an unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between the electron-withdrawing group and the leaving group; (a)(iii) a thiolated nitro fatty acid; or (a)(iv) a dicarboxylic acid compound containing an electron-withdrawing group; a therapeutically effective amount of at least one anti-neoplastic agent (b); Further disclosed herein is a pharmaceutical composition comprising:

[0012] The foregoing will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0013] [Figure 1-1]Figures 1A-1K show that NO2-OA (10-nitro-octadec-9-enoic acid) inhibits TNBC cell growth, RAD51 foci formation, and sensitivity to ionizing radiation. Figure 1A. MDA-MB-231 (MM231) cells (0.5 × 106) were orthotopically injected into 6-week-old mice, and when tumors reached a volume of 100 mm3, they were gavaged with 15 mg / kg oleic acid (OA) (black) or NO2-OA (red) for 4 weeks. Figure 1B. Tumor γH2AX expression was increased in NO2-OA-treated mice compared to OA control mice based on immunoblot (n = 6-7 per group). Figure 1C. MM231 (red), BT549 (blue), or HS578T (green) cells were treated with increasing concentrations of NO2-OA, and relative growth was measured by quantifying luminescent ATP levels (CellTiter-Glo). EC50 values ​​show the mean + SEM, n = 3. Figure 1D-1F. MM231 (red), BT549 (blue), or HS578T (green) cells were treated with increasing concentrations of doxorubicin, cisplatin, or olaparib ± NO2-OA and measured as above. Figure 1G. MM231 (red) cells were treated with increasing concentrations of daily olaparib + vehicle, OA, or NO2-OA and measured as above. Figure 1H-1I. NO2-OA reduced RAD51 foci formation (green) and increased γH2AX (red) in MM231 cells after 5 Gy irradiation. Overlaid samples include DAPI stained nuclei (blue). Cells on 16-well coverslips were administered 5 Gy and then treated with 5 μM NO2-OA or vehicle for 6 h prior to IF processing. The average percentage of cells with 5 or more foci from confocal z-stack images is shown from 0 or 5 Gy samples + SEM. Figure 1J. NO2-OA reduces Rad51 foci in TNBC cell line MDA-MB-231 and has no effect on irradiated benign breast epithelial cells (MCF-10A) due to the distinctly different metabolism and PK of NO2-OA in non-tumorigenic ductal epithelial cells compared to TNBC cells. Rad51 foci were counted and γH2AX intensity was evaluated by ImageJ (histogram) in 5 Gy irradiated cells analyzed by immunofluorescence (IF). Figure 1K, top.NO2-OA shows a lower IC50 and better combination index when combined with talazoparib compared to NO2-OA. Growth inhibition (relative to the number of cells) of MDA-MB-231 cells treated daily for 3 days with talazoparib (0.01–75 μM) and NO2-OA (0.2–12 μM) was determined by luminescent detection of ATP (CellTite-Glo). Figure 1K, bottom. Combination index (CI) was calculated by the Chou-Talalay method. CI<1 is synergistic. Data represent the mean of three independent experiments. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 1-9] Same as above.

[0014] [Figure 2-1]2A-2D: Inhibition of homologous recombination but not non-homologous end joining by NO2-OA. 2A-2B: U2OS cells containing the HR reporter construct DR-GFP were transfected with I-SceI plasmid and treated with vehicle (gray), 5 μM OA (green), or 5 μM NO2-OA (red). I-SceI was absent in negative control cells. Mean + SEM, n > 3. 2A: The number of GFP-positive cells was detected by flow cytometry at 48 hours. 2B: The appearance of GFP-positive cells over 68 hours was quantified using live cell fluorescence microscopy. The number of GFP-positive cells was normalized to cell confluency and compared. 2C-2D: U2OS cells containing the NHEJ reporter construct EJ5-GFP were transfected with I-SceI plasmid and treated with vehicle (gray), 5 μM OA (green), or 5 μM NO2-OA (red). I-SceI was absent in negative control cells. Mean + SEM, n = 3. Figure 2C. The number of GFP-positive cells was detected by flow cytometry at 48 hours. Figure 2D. The appearance of GFP-positive cells over 68 hours was quantified using live cell fluorescence microscopy. The number of GFP-positive cells was normalized to cell confluency and compared. [Figure 2-2] Same as above.

[0015] [Figure 3-1]3A-3I: NO2-OA covalently binds RAD51 at Cys319 and blocks ABL heterodimerization. FIG. 3A. U2OS cells containing the HR reporter construct DR-GFP were stably transfected with control or RAD51 overexpression plasmids, and cells were probed as above with 5 μM NO2-OA in control (red) or RAD51 overexpressing cells (red stripes). FIG. 3B. NO2-OA binds RAD51 in vitro. Purified RAD51 proteins were incubated with control, biotinylated OA, NO2-OA, or nitrated stearate NO2-SA for 1 h, precipitated with streptavidin-coated agarose, and then detected by immunoblotting. FIG. 3C. NO2-OA covalently reacts with RAD51 Cys319 in cells expressing RAD51. 293T cells expressing WT or cysteine-mutated RAD51 were incubated with biotinylated NO2-OA for 1 h, precipitated, and detected. Three independent experiments were quantified and analyzed by one-way ANOVA, *p<0.05. Figure 3D. Alexa Fluor488-conjugated DNA was incubated with purified RAD51, ATP, and 5 μM OA (black), 5 μM NO2-OA (gray), or 10 μM NO2-OA (white), and fluorescence polarization was quantified and normalized to a control lacking ATP. Figure 3E. Molecular modeling of RAD51 (blue) and NO2-OA (purple). The covalent bond of NO2-OA to the Cys319 residue of RAD51 (gold) is predicted to be further stabilized by hydrophobic interactions with Pro318 and possible hydrogen bonding with Glu322 of RAD51. Figure 3F. NO2-OA disrupts ABL binding to RAD51 in vitro. Purified RAD51 and ABL core proteins were incubated with 0, 100, or 500 nM OA-NO2 for 1 h, and ABL was precipitated. The amount of bound RAD51 was detected by immunoblotting. Figure 3G. NO2-OA disrupts the interaction of RAD51 with ABL and reduces RAD51 Y315 phosphorylation as assessed by immunoprecipitation assay (IP).293T cells were transfected with FLAG-RAD51 and ABL core protein and then treated with NO2-OA for 1 h. The interaction of RAD51 with ABL and phosphorylated RAD51 Y315 were probed by IP and immunoblot. Figure 3H. NO2-OA binds to RAD51 in MM231 or MM468 cells. Cells were incubated with biotinylated NO2-OA, and then lysates were precipitated with streptavidin-coated agarose and detected by immunoblot. Figure 3I. NO2-OA impairs HR by inhibiting RAD51 filament formation, causing genomic instability and death in TNBC cells. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above. [Figure 3-5] Same as above.

[0016] [Figure 4-1] Figures 4A-4F show that NO2-OA inhibits TNBC cell growth in vitro and in vivo. (Figure 4A) Chemical structures of nitro-oleic acid (NO2-OA; 10-nitro-octadec-9-enoic acid) and non-electrophilic 10-nitrostearic acid (NO2-SA; 10-nitro-octadecanoic acid), and oleic acid (OA; octadec-9-enoic acid). * indicates electrophilic carbon. (Figures 4B-4D). Effect of NO2-OA on the growth of MDA-MB-231, MDA-MB-468, and MCF7 compared to MCF-10A cells. Data are shown as percentage of untreated control cells (mean ± SD). *p<0.05 indicates significant difference between the two cell types in each treatment. Three independent experiments were performed (n=5 each). (Figure 4E) IC50 values ​​of NO2-OA in each breast cancer cell line. (FIG. 4F) Effect of NO2-OA (7.5 mg / kg daily) on MDA-MB-231 xenograft tumor growth (mean±SEM). *, p<0.05 vs. vehicle group within the treatment period. Significance was determined by two-way ANOVA followed by Tukey's post-hoc test. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0017] [Figure 5-1] 5A-5F show that NO2-OA promotes cell cycle arrest and apoptosis in TNBC cells. The percentage of cell population in each phase of the cell cycle (G0 / G1, S, and G2 / M) is shown for (FIG. 5A) MDA-MB-231, (FIG. 5B) MDA-MB-468, and (FIG. 5C) MCF-10A cells treated with NO2-OA (5 μM) for 24 h. Cells were harvested and analyzed by fluorescence-activated cell sorting. Significance was determined by one-way ANOVA followed by Tukey's post-hoc test. Data are mean ± SD. n=3. *, p<0.05 vs. control. (Fig. 5D) Immunoblot analysis of cyclin D1 and p21 in MCF-10A, MDA-MB-231, and MDA-MB-468 cells treated with OA (7.5 μM), NO2-SA (7.5 μM), or NO2-OA (5 μM) for 24 h. (Fig. 5E) Immunoblot analysis of PARP-1 cleavage in MCF-10A, MDA-MB-231, and MDA-MB-468 cells treated with OA (7.5 μM), NO2-SA (7.5 μM), or NO2-OA (5 μM) for 24 h. (Fig. 5F) Immunoblot analysis of caspase-8 and caspase-9 cleavage in MDA-MB-231 and MDA-MB-468 cells treated with or without NO2-OA (5 μM) for 24 h. β-actin was used as a loading control. Data in Figures 5D-5F are representative of three independent experiments. [Figure 5-2] Same as above. [Figure 5-3] Same as above.

[0018] [Figure 6-1]Figures 6A-6H show that multidrug resistance protein 1 (MRP1) affects NO2-OA transport and signaling in TNBC cells. (Figure 6A) Transport of NO2-OA-SG by MCF-10A, MDA-MB-231, and MDA-MB-468 cells was measured by LC-MS / MS analysis. The relative extent of NO2-OA-SG transport is reported as the ratio of NO2-OA-SG to the exogenously added 15NO2-d4-OA-SG standard. *p<0.05 vs. MCF-10A, n=(Mann-Whitney U test). (Figure 6B) Representative immunoblots of endogenous MRP1 protein expression in MCF-10A, MDA-MB-231, and MDA-MB-468 cells. (Figure 6C) Inhibition of MRP1 activity and (Figure 6D) MRP1 expression increased intracellular NO2-OA-SG adduct concentrations in MCF-10A cells. Relative abundance represents the relative abundance of NO2-OA-SG to the 15NO2-d4-OA-SG standard, normalized to the protein concentration from each NO2-OA-treated sample divided by the abundance of the control or scrambled sample. *p<0.05 vs. control (n=6) or scrambled (n=9) as determined by Mann-Whitney U test. siRNA knockdown efficiency of MRP1 was assessed by q-RT-PCR, n=4. (Figure 6E) Effect of probenecid on growth inhibition of MCF-10A cells by NO2-OA. Cells were pretreated with or without probenecid (0.25 mM) for 1 h and then combined with 0–25 μM NO2-OA for 48 h. FluoReporter dsDNA staining assay was performed to measure the number of cells. Data are shown as a percentage of untreated control cells (n=3), *, p<0.05, n=3 (unpaired Student's t-test). (FIG. 6G) Immunoblot analysis of cyclin D1 and p21 in MCF-10A cells treated with NO2-OA (5 μM) in the presence or absence of probenecid (1 mM was used for this 24 h incubation). (FIG. 6H) Immunoblot analysis of caspase-3 and PARP-1 cleavage in MCF-10A cells treated with NO2-OA (5 μM) in the presence or absence of probenecid (1 mM) for 24 h.Full length (FL) and cleaved (C) PARP-1 protein levels as well as procaspase-3 protein levels are shown. All data are means ± SD. All immunoblots are representative of three independent experiments. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0019] [Figure 7-1] Figures 7A-7B show that NO2-OA depletes GSH levels and enhances GSSG formation in TNBC cells. Cellular (Figure 7A) GSH and (Figure 7B) GSSG responses to NO2-OA in MCF-10A (black bars), MDA-MB-231 (gray bars), and MDA-MB-468 (white bars) cells are shown. Cells were treated with NO2-OA (5 μM) for the indicated times (hours). GSH and GSSG were extracted from cells (3 × 106 cells / ml) and quantified by LC-MS / MS. *p<0.05 vs. 0 hours by unpaired two-tailed Student's t-test. Data are presented as mean ± SD, n=5. [Figure 7-2] Same as above.

[0020] [Figure 8-1]Figures 8A-8D show that NO2-OA inhibits TNFα-induced TNBC cell migration and invasion. (Figure 8A) Schematic of the experiment and representative images of crystal violet-stained migrating MDA-MB-231 or MDA-MB-468 cells. Cells (1x105) were placed in the upper chamber containing serum-free medium with the indicated treatment conditions. Migrating cells were photographed at 100x using a light microscope. (Figures 8B and 8C) Quantification of migrating cells from Figure 4A was performed by crystal violet solubilization and spectrophotometric analysis at A573 nm. The percentage of migrating cells in each treatment group was compared to the number of migrating cells in the absence of TNFα stimulation (serum control). *, p<0.05 vs serum control; **, p<0.05 vs TNFα alone. (FIG. 8D) To test the effect of NO2-OA on TNBC cell invasion, MDA-MB-468 cells were incubated in serum-free medium containing ng / mL TNFα and cell migration through the Matrigel matrix toward 5% FBS chemoattractant was performed for 24 hours. The percentage of cells invading in each treatment was relative to serum control. *p<0.05 ns vs. TNFα alone, not significant. Significance was determined by one-way ANOVA followed by Tukey post-hoc test. All data are mean ± SD. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0021] [Figure 9-1]Figures 9A-9I show that NO2-OA inhibits TNFα-induced NF-κB transcriptional activity in TNBC cells. The effect of NO2-OA on TNFα-induced activation of NF-κB-dependent reporter gene transcription was measured in (Figure 9A) MDA-MB-231 or (Figure 9B) MDA-MB-468 cells transfected with an NF-κB-luciferase reporter. *, p<0.05 versus TNFα alone. (n=3) Significance was determined by Kruskal-Wallis test followed by Dunn's post-hoc test with Bonferroni correction for multiple comparisons. (Figure 9C) Determination of NF-κB target genes downregulated by NO2-OA in MDA-MB-468 cells using human NF-κB target PCR arrays. Histograms represent the percentage of mRNA expression in NO2-OA-treated cells relative to untreated cells. GAPDH was used as an internal control (black bars). Effect of NO2-OA on the expression levels of (Fig. 9D) ICAM-1, (Fig. 9E) uPA, or (Fig. 9F) RelA genes in TNFα-induced MDA-MB-231 cells. Similarly, effect of NO2-OA on the expression levels of (Fig. 9G) ICAM-1, (Fig. 9H) uPA, or (Fig. 9I) RelA genes in TNFα-induced MDA-MB-468 cells. Fold increase over untreated control is represented. *, p<0.05 vs. untreated control, **, p<0.05 vs. TNFα alone. ns, not significant. Significance was determined by one-way ANOVA followed by Tukey post-hoc test. All data are presented as mean ± SD, n=5. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above.

[0022] [Figure 10-1]Figures 10A-10F show that NO2-OA inhibits TNFα-induced IKKβ phosphorylation and IκBα degradation, covalently attaching to IKKβ. MDA-MB-231 and MDA-MB-468 cells were used in all studies. (Figure 10A) Representative immunoblots of IKKβ (Ser180) phosphorylation, total IKKβ levels, and relative phosphorylated IKKβ levels, where all phosphorylated IKKβ levels normalized to total IKKβ were quantified. (Figure 10B) Representative immunoblots of IκBα protein levels are shown, quantifying relative total IκBα levels (normalized to total β-actin) in response to NO2-SA, NO2-OA, and the NF-kB inhibitor BAY11-7082. (FIG. 10C) Representative immunoblots of IκBα (Ser32) phosphorylated and total IκBα in response to NO2-SA, NO2-OA, and the NFkB inhibitor BAY11-7082 are shown. (FIGS. 10D-10F) NO2-OA alkylates TNBC IKKβ protein. Biotinylated NO2-OA, NO2-SA, and OA, as well as attached proteins, were affinity purified from cell lysates by streptavidin agarose beads. Pulled-down IKKβ protein was then detected by immunoblotting. IKKβ and control β-actin immunoblots from the same input lysates used for affinity purification are shown below the panels. *p<0.05 versus TNFα alone. ns, not significant. Significance was determined by one-way ANOVA followed by Tukey post-hoc test. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above.

[0023] [Figure 11-1]11A-11C show that NO2-OA alkylates and destabilizes NF-κB RelA protein in TNBC cells. (FIG. 11A) MDA-MB-231 or MDA-MB-468 cells were treated with 5 μM Bt-NO2-OA, Bt-NO2-SA, or Bt-OA for 2 h. After cell lysis, biotinylated NO2-FA with adducts was affinity purified using streptavidin agarose beads. Pulled-down RelA protein was then detected by immunoblotting. RelA and control β-actin immunoblots from the same input lysates used for affinity purification are shown below the panel. (FIG. 11B) Endogenous RelA protein levels were detected by immunoblotting probing with anti-RelA antibody, using β-actin as a loading control. RelA levels relative to untreated controls (normalized by total β-actin) were quantified. *, p<0.05 versus untreated control. Significance was determined by one-way ANOVA followed by Tukey post-hoc test. (FIG. 11C) MDA-MB-231 or MDA-MB-468 cells were treated with vehicle (methanol), NO2-OA (5 μM), or NO2-SA (5 μM) for 6 h, and then cell lysates were harvested and immunoprecipitated with anti-RelA antibody, followed by immunoblotting. The pull-down levels of immunoprecipitated RelA protein are shown below the panels. [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0024] [Figure 12]12A-12B. Combination index determination. Growth inhibition (relative cell number) of MDA-MB-231 cells treated daily for a total of 3 days with talazoparib (0.01-75 μM) and OA-NO2 (FIG. 12A) and 7-NDA (FIG. 12B) (0.2-12 μM) was determined by luminescent detection of ATP (CellTiter-Glo). Combination index (CI) was calculated by the Chou-Talalay method (17). CI<1 is synergistic. Average of three independent experiments. 7-NDA has a better CI value compared to OA-NO2. Both enhance standard chemotherapy (PARPi, radiation, doxorubicin, platinum) either additively or synergistically to kill TNBC tumor cells.

[0025] [Figure 13-1] Figure 13A is a schematic diagram of different DNA DSB repair pathways. DNA DSB repair consists of four pathways: classical non-homologous end joining (c-NHEJ), alternative end joining (ALT-EJ), single-strand annealing (SSA), and homology-directed repair (HDR). NHEJ does not require homology (homologous sequences) to bridge DSBs before ligation, whereas ALT-EJ is mediated by short stretches of homology to bridge DSBs before ligation. SSA uses neighboring homology to bridge DNA lesions, causing deletions between repeat sequences. HDR is the primary event in DSB repair that involves invasion by at least one strand from the DSB into a homologous template, which serves as a template for nascent DNA synthesis to form an extended strand that can form a bridge to the other DSB end. The homologous strand invasion step is catalyzed by the recombinase Rad51. End resection is the processing of chromosomal DSBs generating 3' single-stranded DNA. Each of these pathways involves a specific set of DNA repair proteins (Figure 13A).

[0026] [Figure 13-2]Figure 13B is a GFP reporter assay to examine the effect of OA-NO2 on different DNA DSB repair pathways described in Figure 14A. U2OS cells containing GFP reporter construct DR-GFP were transfected with I-SceI plasmid and treated with vehicle (gray), 5 μM OA (green), or 5 μM OA-NO2 (red). The number of GFP positive cells was detected by flow cytometry at 48 hours. GFP reporter measuring NHEJ: n=3, SSA: n=3; HDR: n=3; Alt-EJ: n=1.

[0027] [Figure 14] Figure 14 shows the tumor volume of the combination of NO2-OA and talazoparib. MDA-MB-231 cells were orthotopically injected into the mammary fat pad of nude mice. Tumor volumes were measured over time by calipers in mice treated with OA (15 mg / kg) + vehicle (n=8), NO2-OA (15 mg / kg) + vehicle (n=7), OA + talazoparib (0.3 mg / kg) (n=10), or NO2-OA (15 mg / kg) + talazoparib (0.3 mg / kg).

[0028] [Figure 15] Figure 15 shows a schematic diagram of the synthetic lethality concept in which nitroalkenes (e.g. CP-1) and PARP inhibitors are combined. Tumor cells have overactive DNA repair mechanisms, and CP-1 inhibits double-stranded DNA break repair (homologous recombination). When combined with PARP inhibitors of single-stranded DNA break repair, CP-1+PARPi jointly induce synthetic lethality. This occurs by inhibiting both DSB and SSB repair.

[0029] [Figure 16]Figure 16A is a graph showing that nitroalkenes (e.g., methyl-ethyl-4-nitro-octa-4-enedioate (referred to as "CP-1")) provide a lower EC50 for inhibition of MDA-MB-231 TNBC cell growth than OA-NO2. Figure 16B is a graph showing inhibition of homologous recombination in the U2OS GFP reporter construct.

[0030] [Figure 17] Figures 17A and 17B are graphs showing that CP-1 sensitizes human high-grade serous ovarian cancer cells to PARP inhibitors and reduces the concentrations of the PARP inhibitors olaparib (Figure 17A) and talazoparib (Figure 17B) required for 50% tumor cell death. Cell growth / survival is determined from luminescent detection of cellular ATP concentration.

[0031] [Figure 18] Figure 18 shows that MDA-MB-231 TNBC cells are more sensitive to 7-NDA and 8-NDA when compared with OA-NO2. Growth inhibition is evaluated using ATP Celltiter Glo proliferation assay, and compounds are used at different concentrations to determine the EC50 of the compounds. CP-1 has the lowest EC50, followed by 7-NDA and 8-NDA. OA-NO2 has a higher EC50, and OA does not induce any growth inhibition (n=3).

[0032] Different metabolic pathways of 7-NDA and 8-NDA have been characterized in MDA-MB-231 TNBC cells. MDA-MB231 TNBC cells take up and metabolize 7-NDA and 8-NDA via beta-oxidation (Figure 19). Inactivation of the active 7-NDA and 8-NDA nitroalkene groups is achieved via reduction to nitroalkanes (Figure 20). Further metabolism includes reaction with glutathione and formation of cysteine ​​adducts (Figure 21), as well as omega-hydroxylation and carboxylation (Figure 22).

[0033] [Figure 19] FIG. 19 shows that treatment of MDA-MB231 TNBC cells with 7-NDA does not result in the formation of active nitroalkene-containing metabolites, as assessed after the formation of dinor and tetranor 7-NDA. Dinor 7-NDA corresponds to one beta-oxidation cycle, and tetranor corresponds to two beta-oxidation cycles. In contrast, treatment with 8-NDA results in active (structures with reactive nitroalkene moieties) beta-oxidation products. The formation of both dinor 8-NDA and tetranor 8-NDA is shown. The chromatograms correspond to the detection of the different compounds by HPLC-MSMS in negative ion mode using MRM on a triple quadrupole mass spectrometer.

[0034] [Figure 20] Figure 20 shows that MDA-MB231 TNBC cells inactivate both 7-NDA and 8-NDA to form reduced 7-NDA and reduced 8-NDA, respectively. Evidence was obtained for the formation of reduced tetranor 8-NDA, but the formation of reduced tetranor 7-NDA was not observed. The chromatograms correspond to the detection by HPLC-MSMS of the different compounds in negative ion mode using MRM on a triple quadrupole mass spectrometer.

[0035] [Figure 21] Figure 21 shows that MDA-MB231 TNBC cells metabolize both 7-NDA and 8-NDA to form cysteine ​​adducts detected in cell culture media and cell homogenates. Chromatograms correspond to HPLC-MSMS detection of different compounds using specific MRM transitions in a triple quadrupole mass spectrometer.

[0036] [Figure 22]Figure 22 shows that MDA-MB231 TNBC cells metabolize both 7-NDA and 8-NDA via omega hydroxylation and carboxylation. Both 7-NDA and 8-NDA dicarboxylic acids were detected in cell media and cell homogenates of cells treated with 10 uM 7-NDA or 8-NDA. The chromatograms correspond to the detection of different compounds by HPLC-MSMS using specific MRM transitions in a triple quadrupole mass spectrometer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] term The following explanations of terms and methods are provided to better describe the present compounds, compositions, and methods, and to guide those of ordinary skill in the art in practicing the present disclosure. It should also be understood that the terms used in this disclosure are only for the purpose of describing specific embodiments and examples, and are not intended to be limiting.

[0038] "Administration", as used herein, includes administration to a subject by another person or self-administration by the subject.

[0039] "Alkenyl" refers to a cyclic, branched, or straight-chain group containing only carbon and hydrogen and containing one or more double bonds, which may or may not be conjugated. Alkenyl groups can be unsubstituted or substituted. "Lower alkenyl" groups contain 1 to 6 carbon atoms.

[0040] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. An alkyl group may be a "substituted alkyl" in which one or more hydrogen atoms are replaced with a substituent, for example, halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl. For example, lower alkyl or (C1-C6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C3-C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C6)cycloalkyl(C1-C6)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (C1-C6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2-C6)alkenyl can be vinyl. aryl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2-C6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2-C6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2 ... -pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl; (C1-C6)alkanoyl can be acetyl, propanoyl, or butanoyl; halo(C1-C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(C1-C6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (C1-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, or alkoxycarbonyl. nyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C1-C6) alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; (C2-C6) alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

[0041] "Alkynyl" refers to a cyclic, branched, or straight-chain group containing only carbon and hydrogen and one or more triple bonds. Alkynyl groups can be unsubstituted or substituted.

[0042] The term "amine or amino" refers to the group -NRPRq, where Rp and Rq each independently represent hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, and (C1-C6)hydroxyalkyl groups.

[0043] "Animal" refers to a living multi-cellular vertebrate animal, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human subjects and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (e.g., dogs and cats), livestock (e.g., pigs, sheep, cows), and non-domestic animals, such as big cats. The term subject applies regardless of the stage in the life cycle of the organism. Thus, the term subject applies to the organism in utero or in ovo, depending on the organism (i.e., whether the organism is a mammal or an avian, such as poultry or wild fowl).

[0044] As used herein, "aryl" refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, such as phenyl or naphthyl. Unless otherwise indicated, an aryl group may be unsubstituted or substituted with one or more, particularly one to four, groups independently selected from, for example, halo, alkyl, alkenyl, OCF3, NO2, CN, OH, alkoxy, amino, CO2H, CO2 alkyl, aryl, and heteroaryl. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, and 2,4-methoxychlorophenyl.

[0045] The term "biological sample" refers to a tissue, a cell, a cell extract, or a homogenized tissue extract.

[0046] The term "co-administration" or "co-administering" refers to administration of a compound disclosed herein and at least one other therapeutic agent or therapy within the same general time period and does not require administration at the exact same moment (although co-administration includes administration at the exact same moment). Co-administration may therefore be on the same or different days, or in the same or different weeks. In some embodiments, co-administration of two or more agents or therapies is simultaneous. In other embodiments, a first agent / therapy is administered before a second agent / therapy. Those skilled in the art will appreciate that the formulations and / or routes of administration of the various agents or therapies used may vary. Appropriate dosages for co-administration can be readily determined by those skilled in the art. In some embodiments, when agents or therapies are co-administered, each agent or therapy is administered in a lower dosage than would be appropriate for administration alone. Co-administration is therefore particularly desirable in embodiments where co-administration of agents or therapies reduces the required dosage of a potentially harmful (e.g., toxic) agent and / or reduces the frequency with which a potentially harmful (e.g., toxic) agent is administered. "Co-administration" or "co-administering" includes administration of two or more active agents to a subject such that both active agents and / or their metabolites are present in the subject at the same time.Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active agents are present.Co-administration also includes delivery of a first agent via a first administration route and delivery of a second agent via a second administration route, where the first administration route and the second administration route are the same (e.g. both oral) or different (e.g. the first is oral and the second is topical).

[0047] The term "derivative" refers to a compound that is derived from a similar compound or that can be considered to result from another compound when one or more atoms are replaced with another atom or group of atoms.

[0048] The term "haloalkyl" refers to a C1-C8 alkyl group in which one or more hydrogen atoms in the C1-C8 alkyl group are replaced by halogen atoms, which may be the same or different. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, pentachloroethyl, and 1,1,1-trifluoro-2-bromo-2-chloroethyl.

[0049] The terms "halogen" and "halo" refer to -F, -Cl, -Br, or -I.

[0050] The term "heteroatom" is meant to include oxygen (O), nitrogen (N), and sulfur (S).

[0051] The term "heteroaryl" is used herein to refer to a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in the aromatic ring. Unless otherwise indicated, a heteroaryl group may be unsubstituted or substituted with one or more, preferably one to four, substituents selected from, for example, halo, alkyl, alkenyl, OCF3, NO2, CN, NC, OH, alkoxy, amino, CO2H, CO2 alkyl, aryl, and heteroaryl. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

[0052] The term "heterocycle" refers to a monocyclic, bicyclic, tricyclic, or polycyclic ring system, including bicyclic and tricyclic ring systems, that is either unsaturated or aromatic and contains 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, with the nitrogen and sulfur heteroatoms optionally oxidized, and the nitrogen heteroatom optionally quaternized. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined above. Representative examples of heterocycles include, but are not limited to, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzimidazolyl, isoindolyl, indazolyl, benzodiazolyl, benzotriazolyl, benzoxazolyl, benzoisoxazolyl, purinyl, indolyl, isoquinolinyl, quinolinyl, and quinazolinyl. Heterocycle groups may be unsubstituted or optionally substituted with one or more substituents.

[0053] "Heterocycloalkyl" refers to a monocyclic or bicyclic ring system containing one or two saturated or unsaturated rings and containing at least one nitrogen, oxygen, or sulfur atom in the ring. The term "cycloalkyl" refers to a monocyclic or bicyclic ring system containing one or two saturated or unsaturated rings.

[0054] The term "hydroxyalkyl" refers to an alkyl group having the indicated number of carbon atoms in which one or more of the alkyl group's hydrogen atoms have been replaced with an -OH group. Examples of hydroxyalkyl groups include, but are not limited to, -CHOH, -CHCHOH, -CHCHCHOH, -CHCHCHCHCHOH, -CHCHCHCHCHCHOH, -CHCHCHCHCHCHCHOH, and branched forms thereof.

[0055] The term "oxo" refers to an =O atom attached to a saturated or unsaturated (C3-C8) cyclic or (C1-C8) acyclic moiety. The =O atom can be attached to carbon, sulfur, and nitrogen atoms that are part of the cyclic or acyclic moiety.

[0056] The term "subject" includes both human subjects and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (e.g. dogs and cats), livestock (e.g. pigs, sheep, cows), and non-domestic animals, such as big cats. The term subject applies regardless of the stage in the life cycle of an organism. Thus, the term subject applies to organisms in utero or in ovo, depending on the organism (i.e., whether the organism is a mammal or an avian, such as poultry or wild fowl).

[0057] "Therapeutically effective amount" refers to the amount of a specified drug that is sufficient to achieve the desired effect in the subject being treated with the drug. Ideally, the therapeutically effective amount of a drug is an amount that is sufficient to inhibit or treat a disease or condition without causing substantial cytotoxic effects in the subject. The therapeutically effective amount of a drug may depend on the subject being treated, the severity of the disease, and the mode of administration of the therapeutic composition.

[0058] "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after the disease or pathological condition has begun to develop, or to the administration of a compound or composition to a subject who does not exhibit signs of a disease or who exhibits only early signs, with the aim of reducing the risk of developing a pathological condition or condition or reducing the severity of the pathological condition or condition. As used herein, the term "improving" in relation to a disease or pathological condition refers to any observable beneficial effect of treatment. A beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or other parameters known in the art that are specific to a particular disease. The phrase "treating a disease" refers, for example, to inhibiting the complete development of a disease in a subject at risk of the disease. "Preventing" a disease or condition refers to the prophylactic administration of a composition to a subject who does not exhibit symptoms of the disease or who exhibits only early symptoms, with the aim of reducing the risk of developing a pathology or condition or reducing the severity of the pathology or condition. In certain embodiments, treating a disease refers to inhibiting the metastasis of the disease.

[0059] A "pharmaceutical composition" is a composition containing an amount (e.g., a unit dosage amount) of one or more of the disclosed compounds together with one or more non-toxic pharma- ceutically acceptable excipients, including carriers, diluents, and / or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques, such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition).

[0060] The compounds of the present invention can exist in various isomeric forms, including configurational, geometric and conformational isomers, and can also exist in various tautomeric forms, particularly tautomeric forms that differ in the point of attachment of the hydrogen atoms. The term "isomer" is intended to encompass all isomeric forms of the compounds of the present invention, including tautomeric forms of the compounds.

[0061] Certain compounds described herein may have asymmetric centers and therefore may exist in different enantiomeric and diastereomeric forms. The compounds of the present invention may exist in optical isomeric or diastereomeric forms. Thus, the present invention encompasses compounds in the form of optical isomers, diastereoisomers, and mixtures thereof, including racemic mixtures. Optical isomers of the compounds of the present invention can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology, or through chemical separation of stereoisomers by utilizing optically active resolving agents. Unless otherwise indicated, "stereoisomer" refers to one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises more than about 80% by weight of the compound of one stereoisomer and less than about 20% by weight of the compound of the other stereoisomer, for example, more than about 90% by weight of the compound of one stereoisomer and less than about 10% by weight of the compound of the other stereoisomer, or more than about 95% by weight of the compound of one stereoisomer and less than about 5% by weight of the compound of the other stereoisomer, or more than about 97% by weight of the compound of one stereoisomer and less than about 3% by weight of the compound of the other stereoisomer.

[0062] The term "prodrug" refers to a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions, i.e., in vitro or in vivo, to provide an active compound, particularly the compound of the present invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of the compounds of the present invention that contain a biohydrolyzable group, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs (e.g., monophosphates, diphosphates, or triphosphates). For example, prodrugs of compounds with a carboxyl functional group are lower alkyl esters of the carboxylic acid. Carboxylic acid esters are conveniently formed by esterifying any of the carboxylic acid moieties present in the molecule. Prodrugs can typically be prepared using well-known methods, such as those described by BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY 6th ed. (Wiley, 2001), and DESIGN AND APPLICATION OF PRODRUGS (Harwood Academic Publishers Gmbh, 1985).

[0063] One embodiment disclosed herein comprises co-administering a therapeutically effective amount of compound (a) selected from (a)(i) nitroalkene fatty acid, (a)(ii) unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between the electron-withdrawing group and the leaving group, (a)(iii) thiolated nitro fatty acid, (a)(iv) dicarboxylic acid compound containing an electron-withdrawing group, or mixtures thereof, and a therapeutically effective amount of at least one anti-neoplastic agent (b) for treating cancer. Co-administration with compound (a) enhances the anti-proliferative and apoptosis-inducing effects of the anti-neoplastic agent. Co-administration is particularly useful for treating cancer.

[0064] In certain embodiments, the disclosed combination therapy may be useful for treating any type of neoplasm (e.g., cancer). Tumors or neoplasms include new growths of tissues in which the proliferation of cells is uncontrolled and progressive. Some such growths are benign, while others are termed "malignant" and result in the death of the organism. Malignant neoplasms or "cancers" are distinguished from benign growths in that, in addition to exhibiting vigorous cell proliferation, they also invade and metastasize into surrounding tissues. Furthermore, malignant neoplasms are characterized in that they exhibit a greater loss of differentiation (greater "dedifferentiation"), as well as a greater loss of their organization relative to each other and to the surrounding tissues. This characteristic is also called "anaplasia."

[0065] Illustrative neoplasms include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, and medullary carcinoma. , bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, and lymphoma.

[0066] In certain embodiments, the methods of the present disclosure are directed to methods for inhibiting cancer growth, including the processes of cell proliferation, invasiveness, and metastasis in a biological system. Preferably, the methods are used to inhibit or reduce cancer cell proliferation, invasiveness, metastasis, or tumorigenesis in a living animal, such as a mammal.

[0067] Also provided herein are methods of inducing cytotoxicity (cell death) or reducing the viability of cancer cells.

[0068] Particularly illustrative cancers include cancers related to genetic etiology of defective DNA repair genes, cancers with high frequency of natural genomic instability, cancers that respond well to DNA damaging agents, or cancers that respond well to the combination of DNA damaging agents and immunotherapy.Illustrative cancers include breast cancer, especially triple-negative breast cancer, colon cancer, prostate cancer, ovarian cancer, brain cancer (glioblastoma), pancreatic cancer, skin cancer (e.g. melanoma).Cancer cells that acquire the ability to spread (metastasize), are highly genomically unstable, and are particularly suitable for the combination treatment disclosed herein.

[0069] In certain embodiments, compound (a) is a RAD51 inhibitor, and therefore inhibits HDR, as well as being a potent inhibitor of SSA, as described in more detail below.

[0070] In certain embodiments, compound (a) is co-administered with a poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi), as described in more detail below. Compound (a)(i) - Nitroalkene fatty acid

[0071] In certain embodiments, (a)(i) the nitroalkene fatty acid is a compound that contains at least one carbon-carbon double bond and at least one nitro group. Certain nitroalkene fatty acids are described, for example, in U.S. Patent No. 7,776,916. An illustrative embodiment of a nitroalkene fatty acid has Formula I: [ka] [In the formula, R 1 is hydrogen, C1-C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 is alkynyl, R 2 , R 3 , R 7 , and R 8 are each independently hydrogen, oxygen, C1-C 24alkyl, NO2, OH, or OOH; R 4 is the terminal COOR 6 group (in the formula, R 6 is hydrogen, C1-C 24 alkyl, or a pharma- ceutically acceptable counterion; R 5 is hydrogen, C1-C 24 alkyl or R 4 and R 5 Together =C(R 9 )(R 10 )(wherein, R 9 is C1~C 24 Alkyl, C1-C 24 Alkenyl or C1-C 24 Contains alkynyl or R 9 is terminal COOR 6 R 10 is hydrogen, NO2, OH, or OOH), n is 1 to 24. is a nitroalkene fatty acid of Nitroalkene fatty acids contain at least one NO2 group.

[0072] In certain embodiments, R 3 and R 8 are each independently hydrogen, oxygen, C1-C 24 alkyl, NO2, CN, CHO, OH, or OOH; R 2 and R 7 are each independently hydrogen, oxygen, or C1-C 24 It is an alkyl.

[0073] In certain embodiments, R 1 is C1~C 24 Alkyl, more specifically C3-C 20 It is an alkyl.

[0074] In certain embodiments, R 2 is hydrogen.

[0075] In certain embodiments, R 3 or R 8 One of them is NO2, and R 3 or R 8 The other of these is hydrogen.

[0076] In certain embodiments, n is 3-20.

[0077] In certain embodiments, R 4 is -COOH.

[0078] In certain embodiments, R 5 is hydrogen.

[0079] In certain embodiments, R 7 is hydrogen.

[0080] In certain embodiments, R 4 is -COOH, R 5 is methyl, R 7 is methyl.

[0081] In certain embodiments, the nitroalkene fatty acid has formula II: [ka] (In the formula, R 1 , R 2 , R 3 , R 7 , R 8 , R 9 , and R 10 is the same as in formula I. In certain embodiments, R 1 , R 2 , and R 9 is the same as in formula I, R 3 , R 7 , R 8 , and R 10 are each independently hydrogen, oxygen, C1-C 24 It is alkyl, NO2, CN, CHO, OH, or OOH.

[0082] In certain embodiments, the nitroalkene fatty acid has formula III: [ka] [In the formula, R 1 is C1~C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 is alkynyl, R 2 and R 12 are each independently hydrogen, NO2, ONOO2, ONO, OH, or OOH; R 13 is the terminal COOR 6 group (in the formula, R 6 is hydrogen, C1-C 24 alkyl, or a pharma- ceutically acceptable counterion). is a nitroalkene fatty acid of Nitroalkene fatty acids contain at least one NO2 group.

[0083] In certain embodiments, R 2 and R 12 are each independently hydrogen, NO2, OH, or OOH.

[0084] In certain embodiments, the nitroalkene fatty acid is 10-nitro-octadec-9-enoic acid (NO2-OA).

[0085] In certain embodiments, the nitroalkene fatty acid is 7-NO2-nonadec-7-enoic acid (7-NDA) or 8-NO2-nonadec-7-enoic acid (8-NDA).

[0086] In certain embodiments, the nitroalkene fatty acid (referred to herein as "7-NDA") is [ka] It is.

[0087] In certain embodiments, the nitroalkene fatty acid (referred to herein as "8-NDA") is [ka] It is.

[0088] In certain embodiments, the nitroalkene fatty acid is 5-NO2-eicosa-5-enoic acid or 6-NO2-eicosa-5-enoic acid.

[0089] In certain embodiments, the nitroalkene fatty acid is substantially pure. In this aspect, the stereochemistry about the carbon-carbon double bond is substantially cis (or Z) or substantially trans (or E). Compound (a)(ii) - an unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between the electron-withdrawing group and the leaving group

[0090] In certain embodiments, (a)(ii) an unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between the electron-withdrawing group and the leaving group is described, for example, in PCT Publication No. WO2018 / 067709. In some embodiments, the unsaturated fatty acid can be a nitroxide of an activated fatty acid where the electron-withdrawing group is nitro (-NO2), and in certain embodiments, the unsaturated fatty acid can be a nitroxide of a nitroalkene where the electron-withdrawing group is a nitro group (-NO2) and the leaving group can be a nitroxide such as nitrate (-ONO2) or nitrito (-ONO). In certain embodiments, compound (a)(ii) is a prodrug where the nitrito or nitrate substituent is cleaved and the olefin is altered to produce an activated nitroalkene product. As used herein, "activated fatty acid" refers to a fatty acid in which at least one electron-withdrawing group is covalently attached to the saturated or unsaturated aliphatic chain of the fatty acid. Such activated fatty acids can include aliphatic chains substituted with any number of electron-withdrawing groups at any number of positions, and such electron-withdrawing groups may or may not be associated with a carbon-carbon double bond. Similarly, nitrogen oxide derivatives of nitroalkenes can include aliphatic chains with any number of double bonds, which may or may not be associated with an electron-withdrawing group. In certain embodiments, when the electron-withdrawing group is attached to the alpha (α) carbon, the leaving group can be located at the beta (β), gamma (γ), or delta (δ) carbon of the unsaturated aliphatic chain.

[0091] For example, compounds of some embodiments have the general formula IV: [ka] (In the formula, R 1 are -COH, -COR, -COOH, -COOR, -Cl, -F, -Br, -I, -CF3, -CN, -SO 3- , -SO2R, -SO3H, -NH3 + , -NH2R + , -NHR2 + , -NR3 + R is any electron withdrawing group, including, but not limited to, -NO2,2 is -OC(O)(C 1~4 ), -ONO2, -OPO(OH)2, -OSO3, and other inorganic esters, and X 1 and X 2 is -H, -COH, -COR, -COOH, -COOR, -COCF3, and -CF2R, and m and n are independently integers from 1 to 10), as well as compositions containing same.

[0092] In yet another embodiment, the compound has the general formula V: [ka] (In the formula, R 1 is -H or -COH, -COR, -COOH, -COOR, -Cl, -F, -Br, -I, -CF3, -CN, -SO3-, -SO2R, -SO3H, -NH3 + , -NH2R + , -NHR2 + , -NR3 + R is any electron withdrawing group, including, but not limited to, -NO2, 2 is -OC(O)(C 1~4 ), -ONO2, -OPO(OH)2, -OSO3, and other inorganic esters, and X 1 and X 2 is -H, -COH, -COR, -COOH, -COOR, -COCF3, and -CF2R, and m and n are independently integers from 1 to 10), as well as compositions containing same.

[0093] In certain embodiments, the nitrogen oxide of the nitroalkene has the general formula VI: [ka] (wherein m and n are independently an integer of 1 to 10), and compositions containing the same.

[0094] In some embodiments, the nitrogen oxide of the nitroalkene has formula VII: [ka] The nitrogen oxides may be

[0095] In certain embodiments, the nitroxide of the nitroalkene of formula VI is (E)-9-nitro-12-(nitrooxy)octadec-10-enoic acid, the nitroxide of conjugated linoleic acid, or NO2-NO3-CLA.

[0096] In such embodiments, the electron withdrawing group may be in either the E or Z configuration at the original double bond, or in a sp 3 It may be located with either R or S absolute stereochemistry relative to the chiral / stereocenter. For example, in one embodiment, the nitroxide derivative of the nitroalkene may have one electron-withdrawing group, and in another embodiment, the nitroxide derivative of the nitroalkene may be substituted with multiple electron-withdrawing groups at multiple positions along the hydrocarbon chain. The reversible nitroxide derivative of the nitroalkene may have an electron-withdrawing group located at any carbon along the aliphatic hydrocarbon chain between the carboxy terminal carbon and the terminal methyl (ω position), although in some embodiments, the electron-withdrawing group may be located within about 3 carbons from either the carboxy terminal carbon and / or the methyl terminal carbon, and in other embodiments, the electron-withdrawing group may be located within 5 carbons from either the carboxy terminal carbon and / or the methyl terminal carbon. In still other embodiments, the electron-withdrawing group may be located within 7 carbons from either the carboxy terminal carbon and / or the methyl terminal carbon, and in further embodiments, the electron-withdrawing group may be located within 9 carbons from either the carboxy terminal carbon and / or the methyl terminal carbon.

[0097] In certain embodiments, the electron-withdrawing group may be located at the carbon originating from the double bond of an activated fatty acid forming an "electron-withdrawing vinyl" group. The electron-withdrawing group of such a vinyl group may be on either side of the double bond. A fatty acid may have one or more than one electron-withdrawing vinyl group on any carbon of the aliphatic hydrocarbon chain, and there are several means by which an unsaturated fatty acid may have one electron-withdrawing group. In one embodiment, the reversible nitroxide of oleic acid (octadec-9-enoic acid, OA), originating from an 18-carbon omega-9 fatty acid (designated "18:1") with one double bond between the ninth carbon (C-9) and the tenth carbon (C-10), may have an electron-withdrawing group on either C-9 or C-10 and a leaving group at the other position. In another exemplary embodiment, the nitroxide of linoleic acid (octadeca-9,12-dienoic acid), originating from an 18-carbon omega-6 fatty acid (designated "18:2") having two double bonds between the omega-6 carbon (C-13) and the -7 carbon (C-12) and between the omega-9 carbon (C-10) and the 10 carbon (C-9), may have an electron-withdrawing group at C-9 or C-10, or C-12 or C-13, a leaving group at the corresponding other adjacent position, and at least one carbon-carbon double bond located between the electron-withdrawing group and the leaving group. In another embodiment, the reversible nitroxide of linoleic acid may have an electron-withdrawing group at C-9 or C-10, or C-12 or C-13, a leaving group at the corresponding other adjacent position, and at least one carbon-carbon double bond adjacent to the leaving group. Similarly, other polyunsaturated fatty acids naturally having three, four, five, six or more double bonds can have an electron-withdrawing group at either position on any of the native double bond carbons, a leaving group at the corresponding other position, and a carbon-carbon single bond between the electron-withdrawing group and the leaving group, including all possible permutations of positions and electron-withdrawing groups.

[0098] The term "electron-withdrawing group" is recognized in the art and refers to the tendency of a substituent to attract valence electrons from adjacent atoms, i.e., the substituent is electronegative with respect to adjacent atoms. The term "nucleophile" or "electron-donating group" is recognized in the art and refers to the tendency of a substituent to donate excess valence electrons from adjacent atoms, i.e., the substituent is electropositive with respect to adjacent atoms. A quantification of the level of electron-withdrawing power is given by the Hammett sigma (σ) constant (see, for example, J. March, Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1977 edition) pp. 251-259). Hammett constant values ​​are generally negative for electron-donating groups and positive for electron-withdrawing groups. For example, the Hammett constant (σ[P]) for para-substituted NH2 is about -0.7, and σ[P] for a nitro group is about +0.8.

[0099] Embodiments include any known electron-withdrawing group, including, but not limited to, aldehyde (-COH), acyl (-COR), carboxylic acid (-COOH), ester (-COOR), halide (-Cl, F, -Br, etc.), fluoromethyl (-CF3), fluoroalkyl (-CF n H 2-n R), cyano (-CN), sulfoxide (-SOR), sulfonyl (-SO2R), sulfonate (SO3R), 1°, 2°, and 3° ammonium (-NR3 + ), and nitro (-NO2), where each R can be independently hydrogen, methyl, or C2-C6 alkyl, alkenyl, or alkynyl. In some embodiments, the electron-withdrawing group can be a strong electron-withdrawing group having a σ of at least about 0.2, and in certain embodiments, the electron-withdrawing group can form a dipole. For example, in certain embodiments, the electron-withdrawing group can be nitro, ammonium, or sulfonyl.

[0100] The term "leaving group" is art-recognized and refers to the tendency of a substituent to leave a parent molecule along with a pair of electrons during heterolytic bond cleavage. Included leaving groups include, for example, -OC(O)(C 1~4 ), -ONO2, -OPO(OH)2, -OSO3, and other inorganic esters.

[0101] The fatty acid of the embodiments may be any unsaturated and polyunsaturated fatty acid known in the art. The term "fatty acid" refers to an aliphatic monocarboxylic acid. Various embodiments include activated fatty acids having an aliphatic hydrocarbon chain identical or similar to the identified naturally occurring fatty acids. For example, the aliphatic hydrocarbon chain of known naturally occurring fatty acids is generally unbranched and contains an even number of about 4 to about 24 carbons, while other embodiments include fatty acids having 12 to 18 carbons in the aliphatic hydrocarbon chain. In still other embodiments, the fatty acid may have more than 24 carbons in the aliphatic hydrocarbon chain. Embodiments encompass such naturally occurring fatty acids as well as non-naturally occurring fatty acids that may contain an odd number of carbons, and / or a non-naturally occurring linker that includes a heteroatom. Thus, some embodiments include fatty acids having an odd number of carbons, e.g., 5 to 23 carbons, and in other embodiments, 11 to 17 carbons. In still other embodiments, the fatty acid of the embodiments may have more than 23 carbons. Naturally occurring and non-naturally occurring fatty acids may also be branched at one or more locations along the hydrocarbon chain, and in various embodiments, each branch may comprise an aliphatic hydrocarbon chain of 1-24 carbons, 2-20 carbons, or 4-18 carbons, and each branch may have an even or odd number of carbons.

[0102] The aliphatic hydrocarbon chain of the fatty acid of various embodiments may be unsaturated or polyunsaturated. The term "unsaturated" refers to a fatty acid having an aliphatic hydrocarbon chain that includes at least one double bond and / or substituent. In contrast, a "saturated" hydrocarbon chain does not include any double bonds or substituents. Thus, each carbon of the hydrocarbon chain is "saturated" and has a maximum number of hydrogens. "Polyunsaturated" generally refers to a fatty acid having a hydrocarbon chain with more than one double bond. The double bonds of the unsaturated or polyunsaturated fatty acids of various embodiments may be located anywhere along the aliphatic hydrocarbon chain and may be in either a cis or trans configuration. The term "cis" refers to a double bond in which the carbons adjacent to the double bond are on the same side, and the term "trans" refers to a double bond in which the carbons adjacent to the double bond are on opposite sides. Typically, "cis" is the same as Z and "trans" is the same as E, although the IUPAC rules for naming compounds may give the reverse of this for non-carbon substituents, which is the typical case for nitroalkenes. For example, a nitroalkene can have two carbon groups in the "cis" configuration, but the two groups that get priority for naming the compound (the nitro group on one carbon of the alkene, and the carbon group on the other carbon of the alkene) are on opposite sides, and therefore E. Thus, a nitroalkene analog with a "cis" double bond is referred to as an E nitroalkene. Similarly, a nitroalkene analog with a "trans" double bond is referred to as a Z nitroalkene. Without wishing to be bound by theory, a double bond in the cis configuration along the carbon chain (a cis carbon chain, but an E nitroalkene) may induce a bend in the hydrocarbon chain. A double bond in the "trans" configuration along the carbon chain (a trans carbon chain, but a Z nitroalkene) may not bend the hydrocarbon chain. Embodiments may include reversible nitroxide derivatives of nitroalkenes with double bonds in either the E or Z configuration, and may include compositions that may include combinations of cis- and trans-containing nitroxide derivatives of nitroalkenes and positional isomers of nitroxide derivatives of nitroalkenes.

[0103] Many unsaturated and polyunsaturated fatty acids have been identified and are known to occur in nature. Such naturally occurring unsaturated or polyunsaturated fatty acids generally contain an even number of carbons in their aliphatic hydrocarbon chain. For example, naturally occurring unsaturated or polyunsaturated fatty acids may have 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, etc. carbons and may contain omega (ω)-3, ω-5, ω-6, ω-7, ω-9 carbon-carbon double bonds. Any such fatty acid may be useful in a compound. The symbol "ω" is used to refer to the terminal methyl carbon of the aliphatic hydrocarbon chain. The location of the double bond in an ω-X fatty acid is the carbon-carbon bond of the Xth carbon from the ω carbon. For example, an ω-6 fatty acid has a double bond between the 6th and 7th carbons counting backwards from the ω carbon, and an ω-3 fatty acid has a double bond between the 3rd and 4th carbons counting backwards from the ω carbon. Various embodiments include nitrated omega-3 fatty acids, including but not limited to linoleic acid, alpha linoleic acid, eicosapentanoic acid, docosapentaenoic acid, docosahexanoic acid, and stearidonic acid; nitrated omega-5 fatty acids, including but not limited to myristoleic acid; nitrated omega-6 fatty acids, including but not limited to linoleic acid, gamma linoleic acid, dihomo-gamma-linoleic acid, and arachidonic acid; nitrated omega-7 fatty acids, including but not limited to conjugated linoleic acid and palmitoleic acid; and nitrated omega-9 fatty acids, including but not limited to oleic acid and erucic acid. Of course, fatty acids may also be referred to using the IUPAC nomenclature where the location of the double bond is determined by counting from the carbon of the carboxylic acid, and "CX" represents a carbon in an aliphatic hydrocarbon using the IUPAC nomenclature where X is the number of the carbon counting from the carboxylic acid (including the carbonyl carbon itself). Embodiments also include synthetic equivalents of naturally occurring fatty acids and derivatives thereof.

[0104] Other embodiments include non-naturally occurring unsaturated or polyunsaturated fatty acids that may have an odd number of carbons, for example, 5, 7, 9, 11, 13, 15, 17, 19, 21, etc. As with naturally occurring fatty acids, the one or more double bonds associated with non-naturally occurring fatty acids may be located at any position along the aliphatic hydrocarbon chain, and the double bonds may be in either cis or trans configuration. In yet other embodiments, non-naturally occurring fatty acids may include one or more linker groups that interrupt the aliphatic hydrocarbon chain. For example, in some embodiments, activated fatty acids may have one or more non-carbon-carbon linkages, such as ester linkages, ether linkages, vinyl ether linkages, thioether linkages, amino linkages, imine linkages, etc., at any position within the aliphatic hydrocarbon chain.

[0105] In yet other embodiments, the carboxy terminus of the nitroxide of the activated fatty acid may be modified. For example, in some embodiments, the nitroxide of the activated fatty acid may include glycerol that is combined with the carboxy terminus of the fatty acid to produce a glycerolipid, and such glycerolipid may be a mono-, di-, or triglyceride, where at least one of the fatty acids of the di- or triglyceride may be an activated nitrate fatty acid, and any remaining fatty acid may be a saturated or unsaturated fatty acid. Similarly, in other embodiments, a carbohydrate may be combined with the carboxy terminus of the nitroxide-activated fatty acid to form a glycolipid. In such embodiments, any carbohydrate known in the art may be the carbohydrate moiety of the glycolipid, including, but not limited to, galactose and glucose. In yet other embodiments, carbohydrates may be attached to glycerides attached to the carboxy terminus of an activated nitrate fatty acid to form a glyceroglycolipid, which may have one or two activated fatty acids attached to the glycero portion of the glyceroglycolipid, and in embodiments where only one activated fatty acid is attached to the glyceroglycolipid, the remaining positions of the glycerol may contain saturated or unsaturated fatty acids, or hydrogen, alkyl, or functional groups such as alcohols, amines, phosphates, phosphonic acids, thiols, sulfonic acids, etc. In certain embodiments, the carboxy terminus of an activated fatty acid may be attached to a phosphate to form a phospholipid. In such embodiments, the phosphate may be linked directly to a fatty acid through the carboxy terminus, or to a diglyceride in which one or two activated fatty acids are attached to the glycerol moiety; in embodiments in which only one activated nitrate fatty acid is attached to the glycerol, the remaining positions of the glycerol may contain saturated or unsaturated fatty acids, or hydrogen, alkyl, or functional groups such as alcohols, amines, phosphates, phosphonic acids, thiols, sulfonic acids, and the like. In further embodiments, the carboxy terminus of the activated fatty acid may be linked to a cholesterol or other sterol moiety. In still other embodiments, the carboxy terminus may be modified by covalent attachment of a secondary active agent.In certain embodiments, the carboxy-terminal modification containing glycerol may not contain a nitro group. Without wishing to be bound by theory, modification of the carboxy-terminal end of activated nitrate fatty acid can enhance the distribution of activated fatty acid after administration, and can also improve the resilience of activated fatty acid after administration by inhibiting mitochondrial beta-oxidation. Compound (a)(iii)—Thiol-Added Nitro Fatty Acids (“Thiolated Fatty Acids”)

[0106] Potential barriers to the use of nitro-oleic acid as a drug candidate are the rapid metabolism of nitro-oleic acid as a result of beta-oxidation reactions and reduction of nitroalkene by prostaglandin reductase 1 in the first pass of the liver, and reversible addition to glutathione, followed by excretion. To be effective, the drug must survive first-pass metabolism. The active drug must be protected to adequately deliver an effective amount of the active drug to the circulation, as it may be metabolized in the gut microbiome and liver.

[0107] Modification of nitroalkene fatty acids by reversible thiolation of the nitroalkene prevents their metabolic inactivation, thus preserving the latent electrophilic character of the nitroalkene fatty acid. Upon dissociation of the thiol or polythiol substituent, the "activated" nitroalkene product becomes capable of targeting functionally critical nucleophilic residues in RAD51 or other DNA repair proteins.

[0108] Examples of thiolated fatty acids are described, for example, in PCT Publication No. WO2018 / 067705.

[0109] Some embodiments are generally directed to thiolated electrophilic unsaturated activated fatty acids, particularly thiolated unsaturated nitrated fatty acids. As used herein, "activated fatty acid" refers to a fatty acid in which at least one electron-withdrawing group is covalently attached to an unsaturated carbon of a saturated or unsaturated aliphatic chain of the fatty acid. Such activated fatty acids can include an aliphatic chain substituted with any number of electron-withdrawing groups at any number of positions of the hydrocarbon chain, and such electron-withdrawing groups may or may not be associated with a carbon-carbon double bond. Similarly, the thiolated activated fatty acids described herein can include an aliphatic chain having any number of double bonds, which may or may not be associated with an electron-withdrawing group, and a sulfur-containing group, i.e., a thiol group. In certain embodiments, when the electron-withdrawing group is attached to the alpha (α) carbon, the sulfur-containing group can be located at the beta (β), gamma (χ), or delta (δ) carbon of the unsaturated aliphatic chain.

[0110] The electrophilic double bond of the nitroalkene is converted to H(S) to form a thiolated activated fatty acid. x The thiolated activated fatty acid is then reversibly protected by R. Here, this thiolated activated fatty acid is a prodrug, avoiding metabolic processes during first pass. The electrophilic double bond is regenerated after loss of the protecting group, as shown below: [ka]

[0111] For example, the thiolated activated fatty acid of some embodiments has the general formula VIII: [ka] where R is hydrogen (-H), methyl, or a C2-C6 alkyl, alkenyl, or alkynyl, or (S)xR can be a sulfur-containing functional group such as sulfino (-SOOH), sulfo (-SOOOH), or thiocyanato (-SCN), where x is an integer from 1 to 5, and q and m are each independently an integer from 1 to 10. The compound of formula VIII contains a sulfur-containing group at the β carbon.

[0112] Other thiolated activated fatty acids include those of the general formula IX: [ka] where R is hydrogen (-H), methyl, or a C2-C6 alkyl, alkenyl, or alkynyl, or (S)xR can be a sulfur-containing functional group such as sulfino (-SOOH), sulfo (-SOOOH), or thiocyanato (-SCN), where x is an integer from 1 to 5, and q and m are each independently an integer from 1 to 10. Compounds of formula IX contain a sulfur-containing group at the δ carbon.

[0113] In some embodiments, R in formula VIII or IX can be a bifunctional alkyl, alkenyl, or alkynyl attached to the carboxyl of an activated fatty acid forming a bridged or cyclic structure. In such embodiments, the sulfur-containing moiety can be located at the β, χ, or δ carbon. For example, the general formulas Xa and Xb contain a cyclized bifunctional sulfur-containing moiety at the β carbon: [ka] wherein each Y is independently oxygen (O) or nitrogen (N), each x is independently an integer from 1 to 5, and q, m, p, and t are each independently an integer from 1 to 10.

[0114] In some embodiments, the sulfur-containing group may join two activated fatty acids. For example, various embodiments include those having the general formula XI: [ka] (In the formula, R1 and R2 are -H, -COH, -COR, -COOH, -COOR, -Cl, -F, -Br, -I, -CF3, -CN, -SO 3- , -SO2R, -SO3H, -NH3 + , -NH2R + , -NHR2 + , -NR3 +and -NO2, at least one of R1 and R2 is an electron withdrawing group, Y1 and Y2 are independently selected from -H, -COH, -COR, -COOH, and -COOR, at least one of R1 and R2 is an electron withdrawing group, x is an integer from 1 to 5, and q, m, p, and t are independently integers from 1 to 10, as well as compositions containing the same.

[0115] Various embodiments are represented by the general formulas XII, XIIb, and XIIc: [ka] (wherein x is an integer from 1 to 5, and q, m, p, and t are independently integers from 1 to 10), as well as compositions containing the same.

[0116] Other embodiments include those of formula XIII: [ka] (wherein x is an integer of 1 to 5), and compositions containing the same.

[0117] In some embodiments, the compound has formula XIV: [ka] Thiolated nitro-oleic acid (NO2-OA-S) x ) species.

[0118] The electron withdrawing groups may be in either the cis or trans configuration at the original double bond, or in the sp 3It may be located with either R or S absolute stereochemistry relative to the chiral / stereocenter. For example, in one embodiment, the thiolated activated fatty acid may have one electron-withdrawing group, and in another embodiment, the thiolated activated fatty acid may be substituted with multiple electron-withdrawing groups at multiple positions along the hydrocarbon chain. The thiolated activated fatty acid may have an electron-withdrawing group located at any carbon along the aliphatic hydrocarbon chain between the carboxy terminal carbon and the terminal methyl (ω position), but in some embodiments, the electron-withdrawing group may be located within about 3 carbons from either the carboxy terminal carbon and / or the methyl terminal carbon, and in other embodiments, the electron-withdrawing group may be located within 5 carbons from either the carboxy terminal carbon and / or the methyl terminal carbon. In still other embodiments, the electron-withdrawing group may be located within 7 carbons from either the carboxy terminal carbon and / or the methyl terminal carbon, and in further embodiments, the electron-withdrawing group may be located within 9 carbons from either the carboxy terminal carbon and / or the methyl terminal carbon.

[0119] In certain embodiments, the electron-withdrawing group may be located at the carbon originating from the double bond of an activated fatty acid forming an "electron-withdrawing vinyl" group. The electron-withdrawing group of such a vinyl group may be on either side of the double bond. A fatty acid may have one or more than one electron-withdrawing vinyl group on any carbon of the aliphatic hydrocarbon chain, and there are several means by which an unsaturated fatty acid may have one electron-withdrawing group. In one embodiment, a thiolated activated oleic acid (octadec-9-enoic acid) originating from an 18-carbon omega-9 fatty acid (designated "18:1") with one double bond between the ninth carbon (C-9) and the tenth carbon (C-10) may have an electron-withdrawing group on either C-9 or C-10 and a thiol (-SR) at the other position. In another exemplary embodiment, thiolated activated linoleic acid (octadeca-9,12-dienoic acid), originating from an 18-carbon ω-6 fatty acid (designated "18:2") with two double bonds between the ω-6 carbon (C-13) and the -7 carbon (C-12) and between the ω-9 carbon (C-10) and the 10 carbon (C-9), can have an electron-withdrawing group at C-9 or C-10, or C-12 or C-13, and a thiol (-SR) at the corresponding adjacent position on the other. Similarly, other polyunsaturated fatty acids that naturally have three, four, five, six, or more double bonds can have one electron-withdrawing at either position on any of the native double bond carbons, and a thiol (-SR) at the corresponding adjacent position on the other, including all possible permutations of positions and electron-withdrawing groups.

[0120] In other embodiments, mono- or polyunsaturated fatty acids may have two electron-withdrawing groups, and there are several ways in which unsaturated fatty acids can have two electron-withdrawing groups. For example, in one embodiment, thiolated activated linoleic acid (octadeca-9,12-dienoic acid), originating from an 18-carbon ω-6 fatty acid (represented as "18:2") with two double bonds between the ω-6 carbon (C-13) and the -7 carbon (C-12) and between the ω-9 carbon (C-10) and the 10 carbon (C-9), may have an electron-withdrawing group at any two of positions C-9, C-10, C-12, or C-13, with one or more thiols (-SR) at the corresponding adjacent positions on the other side, according to the following possible permutations: C-9 and C-12, C-9 and C-13, C-10 and C-12, or C-10 and C-13.

[0121] Similar to the previous description of compounds with one electron-withdrawing group or two electron-withdrawing groups, it is also possible to have three, four, five or more electron-withdrawing groups. Following the same logic as above, i.e., the logic in the previous description of compounds with one electron-withdrawing group or two electron-withdrawing groups, polyunsaturated fatty acids with three, four, five, six or more double bonds, conjugated or non-conjugated, can have multiple electron-withdrawing groups (three, four, five or more, if the positions available for substitution allow) at any position on any of the double bond carbons, including all possible permutations of positions, nucleophilic substituents and electron-withdrawing groups. In addition, in any embodiment, such as the embodiment described above, any number of non-electron-withdrawing groups may be covalently bonded to the carbon of the aliphatic chain of the activated fatty acid. For example, in some embodiments, the thiolated activated fatty acid may include one or more methyl, C2-C6 alkyl, alkenyl, or alkynyl, or amino covalently attached to one or more carbons of the aliphatic chain of the thiolated activated fatty acid.

[0122] The term "electron-withdrawing group" is recognized in the art and refers to the tendency of a substituent to attract valence electrons from adjacent atoms, i.e., the substituent is electronegative with respect to adjacent atoms. The term "nucleophile" or "electron-donating group" is recognized in the art and refers to the tendency of a substituent to donate excess valence electrons from adjacent atoms, i.e., the substituent is electropositive with respect to adjacent atoms. A quantification of the level of electron-withdrawing power is given by the Hammett sigma (σ) constant (see, for example, J. March, Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1977 edition) pp. 251-259). Hammett constant values ​​are generally negative for electron-donating groups and positive for electron-withdrawing groups. For example, the Hammett constant (σ[P]) for para-substituted NH2 is about -0.7, and σ[P] for a nitro group is about +0.8.

[0123] Embodiments include any known electron-withdrawing group, including, but not limited to, aldehyde (-COH), acyl (-COR), carboxylic acid (-COOH), ester (-COOR), halide (-Cl, F, -Br, etc.), fluoromethyl (-CF3), fluoroalkyl (-CF n H 2-n R), cyano (-CN), sulfoxide (-SOR), sulfonyl (-SO2R), sulfonate (SO3R), 1°, 2°, and 3° ammonium (-NR3 +), and nitro (-NO2), where each R can be independently hydrogen, methyl, or C2-C6 alkyl, alkenyl, or alkynyl. In some embodiments, the electron-withdrawing group can be a strong electron-withdrawing group having a σ of at least about 0.2, and in certain embodiments, the electron-withdrawing group can form a dipole. For example, in certain embodiments, the electron-withdrawing group can be nitro, ammonium, or sulfonyl. In other embodiments, the thiolated activated fatty acid can be further substituted with non-electron-withdrawing or electron-donating groups, including, for example, thiol (-SR), alcohol (-OH), reverse ester (-OOCR), alkyl, alkenyl, alkynyl, 1° and 2° amine (-NR2), N-containing heterocycles (-N=, -NR-), nitrate (-ONO2), nitrito (-ONO), and the like.

[0124] The fatty acid of the embodiments may be any unsaturated and polyunsaturated fatty acid known in the art. The term "fatty acid" refers to an aliphatic monocarboxylic acid. Various embodiments include activated fatty acids having an aliphatic hydrocarbon chain identical or similar to the identified naturally occurring fatty acids. For example, the aliphatic hydrocarbon chain of known naturally occurring fatty acids is generally unbranched and contains an even number of about 4 to about 24 carbons, while other embodiments include fatty acids having 12 to 18 carbons in the aliphatic hydrocarbon chain. In still other embodiments, the fatty acid may have more than 24 carbons in the aliphatic hydrocarbon chain. Embodiments encompass such naturally occurring fatty acids as well as non-naturally occurring fatty acids that may contain an odd number of carbons, and / or a non-naturally occurring linker that includes a heteroatom. Thus, some embodiments include fatty acids having an odd number of carbons, e.g., 5 to 23 carbons, and in other embodiments, 11 to 17 carbons. In still other embodiments, the fatty acid of the embodiments may have more than 23 carbons. Naturally occurring and non-naturally occurring fatty acids may also be branched at one or more locations along the hydrocarbon chain, and in various embodiments, each branch may comprise an aliphatic hydrocarbon chain of 1-24 carbons, 2-20 carbons, or 4-18 carbons, and each branch may have an even or odd number of carbons.

[0125] The aliphatic hydrocarbon chain of the fatty acid of various embodiments may be unsaturated or polyunsaturated. The term "unsaturated" refers to a fatty acid having an aliphatic hydrocarbon chain that includes at least one double bond and / or substituent. In contrast, a "saturated" hydrocarbon chain does not include any double bonds or substituents. Thus, each carbon of the hydrocarbon chain is "saturated" and has a maximum number of hydrogens. "Polyunsaturated" generally refers to a fatty acid having a hydrocarbon chain with more than one double bond. The double bonds of the unsaturated or polyunsaturated fatty acids of various embodiments may be located anywhere along the aliphatic hydrocarbon chain and may be in either a cis or trans configuration. The term "cis" refers to a double bond in which the carbons adjacent to the double bond are on the same side, and the term "trans" refers to a double bond in which the carbons adjacent to the double bond are on opposite sides. Typically, "cis" is the same as Z and "trans" is the same as E, although the IUPAC rules for naming compounds may give the reverse of this for non-carbon substituents, which is the typical case for nitroalkenes. For example, a nitroalkene can have two carbon groups in "cis", but the two groups that get priority for naming the compound (the nitro group on one carbon of the alkene, and the carbon group on the other carbon of the alkene) are on opposite sides, and therefore E. Thus, a nitroalkene analog with a "cis" double bond is referred to as an E nitroalkene. Similarly, a nitroalkene analog with a "trans" double bond is referred to as a Z nitroalkene. Without wishing to be bound by theory, a double bond in the cis configuration along the carbon chain (a cis carbon chain, but an E nitroalkene) may induce a bend in the hydrocarbon chain. A double bond in the "trans" configuration along the carbon chain (a trans carbon chain, but a Z nitroalkene) may not bend the hydrocarbon chain. Embodiments may include compositions that may include thiolate-activated fatty acids with double bonds in either the cis or trans configuration, and may include combinations of cis- and trans-containing thiolated activated fatty acids and positional isomers of thiolated activated fatty acids.

[0126] Many unsaturated and polyunsaturated fatty acids have been identified and are known to occur in nature. Such naturally occurring unsaturated or polyunsaturated fatty acids generally contain an even number of carbons in their aliphatic hydrocarbon chain. For example, naturally occurring unsaturated or polyunsaturated fatty acids may have 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, etc. carbons and may contain omega (ω)-3, ω-5, ω-6, ω-7, ω-9 carbon-carbon double bonds. Any such fatty acid may be useful. The symbol "ω" is used to refer to the terminal methyl carbon of the aliphatic hydrocarbon chain. The location of the double bond in ω-X fatty acids is the carbon-carbon bond of the Xth carbon from the ω carbon. For example, ω-6 fatty acids have a double bond between the 6th and 7th carbons counting backwards from the ω carbon, and ω-3 fatty acids have a double bond between the 3rd and 4th carbons counting backwards from the ω carbon. Various embodiments include nitrated omega-3 fatty acids, including but not limited to linoleic acid, alpha linoleic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, and stearidonic acid; nitrated omega-5 fatty acids, including but not limited to myristoleic acid; nitrated omega-6 fatty acids, including but not limited to linoleic acid, gamma linoleic acid, dihomo-gamma-linoleic acid, and arachidonic acid; nitrated omega-7 fatty acids, including but not limited to conjugated linoleic acid and palmitoleic acid; and nitrated omega-9 fatty acids, including but not limited to oleic acid and erucic acid. Of course, fatty acids may also be referred to using the IUPAC nomenclature, where the location of the double bond is determined by counting from the carbon of the carboxylic acid, and "CX" represents the carbon in the aliphatic hydrocarbon using the IUPAC nomenclature, where X is the number of the carbon counting from the carboxylic acid (including the carbonyl carbon itself). Some embodiments also include synthetic equivalents of naturally occurring fatty acids and their derivatives.

[0127] Other embodiments include non-naturally occurring unsaturated or polyunsaturated fatty acids that may have an odd number of carbons, for example, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, etc. As with naturally occurring fatty acids, the one or more double bonds associated with non-naturally occurring fatty acids may be located at any position along the aliphatic hydrocarbon chain, and the double bonds may be in either cis or trans configuration. In yet other embodiments, non-naturally occurring fatty acids may include one or more linker groups that interrupt the aliphatic hydrocarbon chain. For example, in some embodiments, activated fatty acids may have one or more non-carbon-carbon linkages, such as ester linkages, ether linkages, vinyl ether linkages, thioether linkages, amino linkages, imine linkages, etc., at any position within the aliphatic hydrocarbon chain.

[0128] Various embodiments include unsaturated or polyunsaturated fatty acids that may have a carbon-carbon double bond between any two carbons of the fatty acid's aliphatic chain, and any number of carbon-carbon double bonds may be present in such polyunsaturated fatty acids. For example, in some embodiments, polyunsaturated fatty acids may have two, three, four, five, six, or more carbon-carbon double bonds. In such embodiments, each of the more than one carbon-carbon double bonds may be individually in either cis or trans configuration. In some embodiments, thiolated activated fatty acids are derived from the reaction of at least one carbon-carbon double bond of a polyunsaturated fatty acid with an electron-withdrawing group attached thereto, and in other embodiments, more than one carbon-carbon double bond of such polyunsaturated fatty acids may have an electron-withdrawing group attached thereto. In addition, in such embodiments, the electron-withdrawing group may be attached to any carbon of the original carbon-carbon double bond or the carbon immediately adjacent to any carbon of the carbon-carbon double bond, and the thiol may be attached to the other carbon of the original carbon-carbon double bond or the carbon immediately adjacent to any carbon of the carbon-carbon double bond. For example, in some embodiments, an electron withdrawing group may be attached to the alpha (α) carbon of a former carbon-carbon double bond, and in other embodiments, an electron withdrawing group may be associated with the beta (β) carbon of a former carbon-carbon double bond. In each of those embodiments, a thiol may be attached to the beta (β) carbon of a former carbon-carbon double bond, and in other embodiments, an electron withdrawing group may be associated with the alpha (α) carbon of a former carbon-carbon double bond.

[0129] In certain embodiments, the unsaturated fatty acid having at least one electron-withdrawing group may be a conjugated fatty acid. In such embodiments, the two carbon-carbon double bonds in the aliphatic chain are adjacent to each other such that there is no methylene group between them. Such conjugated compounds are generally referred to as 1,3-dienes, or conjugated fatty acids. Such 1,3-dienes may contain one or more electron-withdrawing groups at any of six positions, i.e., at positions 1, 2, 3, and / or 4 of the 1,3-diene, and at the two carbons adjacent to the diene (positions 0 and 5 relative to positions 1, 2, 3, and 4 in the method of identifying the carbons in the 1,3-diene). For example, one associated electron-withdrawing group may be attached at any of the six positions identified above, i.e., at positions 1, 2, 3, or 4 of the diene, or at the carbon adjacent to the 1,3-diene (positions 0 or 5 as described above). In additional embodiments, two associated electron-withdrawing groups can be attached to any two of six possible positions, three associated electron-withdrawing groups can be attached to any two of six possible positions, four associated electron-withdrawing groups can be attached to any two of six possible positions, five associated electron-withdrawing groups can be attached to any two of six possible positions, and six associated electron-withdrawing groups can be attached to any two of six possible positions. In summary, any arrangement of electron-withdrawing groups attached to any of the six positions described above in a 1,3-diene is encompassed by multiple embodiments of the compound.

[0130] In certain embodiments, the thiolated activated fatty acid may undergo isomerization after preparation, such that the cis / trans configuration of the double bond, the location of the double bond in the carbon chain, or both may change.For example, in some embodiments, the thiolated activated fatty acid may be prepared from a carbon-carbon double bond with an electron-withdrawing group attached to the gamma carbon of the carbon-carbon double bond. After preparation, the carbon-carbon double bond may undergo isomerization, such that the electron-withdrawing group is now conjugated with the carbon-carbon double bond after isomerization.Such isomerization may occur naturally at any time after preparation, and may be initially prepared to contain a single type of thiolated activated fatty acid, and may then result in a composition containing a combination of isomers of the initially prepared activated fatty acid that is naturally produced.

[0131] In yet other embodiments, the carboxy terminus of the thiolated activated fatty acid may be modified. For example, in some embodiments, the thiolated activated fatty acid may include glycerol that is linked to the carboxy terminus of a fatty acid to produce a glycerolipid, and such a glycerolipid may be a mono-, di-, or triglyceride, where at least one of the fatty acids of the di- or triglyceride may be a thiolated activated fatty acid, and any remaining fatty acid may be a saturated or unsaturated fatty acid. Similarly, in other embodiments, a carbohydrate may be linked to the carboxy terminus of the thiolated activated fatty acid to form a glycolipid. In such embodiments, any carbohydrate known in the art may be the carbohydrate portion of the glycolipid, including, but not limited to, galactose and glucose. In yet other embodiments, carbohydrates may be attached to glycerides attached to the carboxy terminus of a thiolated activated fatty acid to form a glyceroglycolipid, which may have one or two activated fatty acids attached to the glycero portion of the glyceroglycolipid, and in embodiments where only one activated fatty acid is attached to the glyceroglycolipid, the remaining positions of the glycerol may contain saturated or unsaturated fatty acids, or hydrogen, alkyl, or functional groups such as alcohols, amines, phosphates, phosphonic acids, thiols, sulfonic acids, etc. In certain embodiments, the carboxy terminus of an activated fatty acid may be attached to a phosphate to form a phospholipid. In such embodiments, the phosphate may be directly linked to a fatty acid through the carboxy terminus, or to a diglyceride with one or two activated fatty acids attached to the glycerol moiety; in embodiments where only one thiolated activated fatty acid is attached to the glycerol, the remaining positions of the glycerol may contain saturated or unsaturated fatty acids, or hydrogen, alkyl, or functional groups, such as alcohols, amines, phosphates, phosphonic acids, thiols, sulfonic acids, and the like. In further embodiments, the carboxy terminus of the activated fatty acid may be linked to a cholesterol or other sterol moiety. In still other embodiments, the carboxy terminus may be modified by covalent attachment of a secondary activator. In certain embodiments, the carboxy terminus modification including glycerol may not include a nitro group.Without wishing to be bound by theory, modification of the carboxy terminus of a thiolated activated fatty acid can enhance the distribution of the activated fatty acid following administration and can also improve the resilience of the activated fatty acid following administration by inhibiting mitochondrial beta-oxidation.

[0132] The compounds increase the bioavailability of activated fatty acids present as dimers in thiolated molecules. Thiolation of electrophilic alkenes protects the molecules from first-pass metabolism in the intestine and liver. This protection is achieved by preventing reduction of the alkene by prostaglandin reductase 1 and by delaying addition to glutathione. Furthermore, the longer the polysulfide chain, the greater the stability of the molecule, allowing for prolonged release of the activated fatty acid in the circulation. An additional measure of protection is provided when thiolated nitro fatty acids release the nitro fatty acid and hydrogen sulfide. Compound (a)(iv) - Dicarboxylic acid compound containing an electron-withdrawing group

[0133] Compound (a)(iv) is a dicarboxylic acid compound containing an electron-withdrawing group, and in some embodiments, such compounds may further contain an alkene associated with the electron-withdrawing group. Various embodiments are directed to alkyl esters of dicarboxylic acid compounds containing an electron-withdrawing group, and in some embodiments, such compounds may further contain an alkene associated with the electron-withdrawing group. Various embodiments of the present invention are directed to compounds of formulas XV-XXIV. Such compounds are described, for example, in PCT Publication No. WO2017 / 151938. These electrophilic dicarboxylates can alkylate functionally critical thiols to inactivate RAD51 or other DNA repair proteins.

[0134] The compounds have the general formula XV or XVI: [ka] (wherein X is an electron-withdrawing group, [ka] may be individually a single bond or a double bond, and each m and n is independently an integer from 1 to 10. In certain embodiments, at least one of the compounds shown in Formulas XV and XVI may be [ka] is a double bond. In some embodiments, both of the groups shown in formulas XV and XVI are [ka] may be a single bond, and in other embodiments, both of the groups shown in formulas XV and XVI may be [ka] may be a double bond. In other embodiments, the compound has the general formulas XVII and XVIII: [ka] wherein X is an electron withdrawing group, and each m and n is independently an integer from 1 to 10.

[0135] Further embodiments include, for example, compounds of general formulas XIX and XX: [ka] wherein X is an electron withdrawing group, and each Y and Z is independently hydrogen or C1-C 10 is alkyl, [ka] is individually a single bond or a double bond, and each m and n is independently absent or an integer from 1 to 10. In certain embodiments, the present invention is directed to alkyl esters of dicarboxylic acid compounds containing electron-withdrawing groups, such as compounds of formulas XIX and XX: [ka] is a double bond. In some embodiments, both of the groups shown in formulas XIX and XX are [ka] may be a single bond, and in other embodiments, both of the groups shown in formulas XIX and XX may be [ka] may be a double bond. In some embodiments, the alkyl ester of a dicarboxylic acid compound containing an electron withdrawing group has the formulas XXI and XXII: [ka] wherein X is an electron withdrawing group, and each Y and Z is independently hydrogen or C1-C 10 and each m and n is independently an integer from 1 to 10. In certain embodiments, each Y and Z in the compounds of formulas XIX, XX, XXI, and XXII illustrated above can be methyl (C1 alkyl) or ethyl (C2 alkyl).

[0136] The alkylene formed by m and n in each of the compounds of formulas XV-XXII illustrated above is shown in formulas XVII, XVIII, XXI, and XXII, or in formulas XV, XVI, XIX, and XX. [ka] For example, compounds of some embodiments may include carbon-carbon double bonds in addition to the double bonds optionally present as indicated by formulas XXIII and XXIV: [ka] wherein X is an electron withdrawing group, and each Y and Z is independently hydrogen or C1-C 10each p and t is independently an integer from 1 to 10, each s is absent or is an integer from 1 to 10, and each r is an integer from 1 to 5. In certain embodiments, each Y and Z in the compounds of formulas XXIII and XXIV illustrated above can be methyl (C1 alkyl) or ethyl (C2 alkyl).

[0137] Additional embodiments include dicarboxylic acid compounds containing an electron withdrawing group, further comprising at least one alkene associated with an electron withdrawing group of formulas XV, XVII, XIX, XXI, and XXIII, wherein the at least one alkene associated with the electron withdrawing group is reduced by introduction of a nucleophile "A" via a Michael addition reaction to produce dicarboxylic acid compounds of formulas XVA, XVIIA, XIXA, XIXB, XXIA, and XXIIIA. [ka] wherein X is an electron withdrawing group, A is a nucleophile, and each Y and Z is independently hydrogen or C1-C 10 and each m, n, p, and t is independently an integer from 1 to 10, each s is absent or is an integer from 1 to 10, and each r is an integer from 1 to 5. In certain embodiments, each Y and Z can be methyl (C1 alkyl) or ethyl (C2 alkyl).

[0138] It is believed that the compounds of formulas XVA, XVIIA, XIXA, XIXB, XXIA, and XXIIIA may be useful either as prodrugs of the compounds of formulas XV, XVII, XIX, XXI, and XXIII, or as active therapeutic agents themselves. When used as prodrugs, it is believed that the compounds of formulas XVA, XVIIA, XIXA, XIXB, XXIA, and XXIIIA are metabolized in vivo after administration to a patient in need thereof to provide a therapeutically effective amount of the active agent according to formulas XV, XVII, XIX, XXI, and XXIII.

[0139] The term "nucleophile" is recognized in the art and refers to a chemical species that donates an electron pair to an electrophile to form a chemical bond involved in a reaction. Any molecule or ion that has a free electron pair or at least one π bond can act as an electrophile. Nucleophiles, or A, can include, but are not limited to, enols, hydroxide anions, alcohols, alkoxide anions, hydrogen peroxide, carboxylate anions, hydrogen sulfide, thiols, thiolate anions, anions of thiocarboxylic acids, anions of dithiocarbonates, ammonia, azides, amines, and nitriles.

[0140] The term "electron-withdrawing group" is recognized in the art and refers to the tendency of a substituent to attract valence electrons from neighboring atoms, i.e., the substituent is electronegative with respect to neighboring atoms. A quantification of the level of electron-withdrawing power is given by the Hammett sigma (σ) constant (see, for example, J. March, Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1977 edition) pp. 251-259). Hammett constant values ​​are generally negative for electron-donating groups and positive for electron-withdrawing groups. For example, the Hammett constant (σ[P]) for a para-substituted NH2 is about -0.7, and the σ[P] for a nitro group is about 0.8. Electron-withdrawing groups include, but are not limited to, aldehydes (-COH), acyl (-COR), carbonyls (-CO), carboxylic acids (-COOH), esters (-COOR), halides (-Cl, -F, -Br, etc.), fluoromethyl (-CF n ), cyano (-CN), sulfonyl (-SO n ), sulfone (-SO2R), sulfonic acid (-SO3H), 1°, 2°, and 3° ammonium (-NR 3+), as well as nitro (-NO2). In some embodiments, the electron-withdrawing group may be a strong electron-withdrawing group having a σ of at least about 0.2, and in certain embodiments, the electron-withdrawing group may form a dipole. For example, in certain embodiments, the electron-withdrawing group may be nitro, ammonium, or sulfonyl.

[0141] In certain embodiments, the dicarboxylic acid compound has the structure: [ka] [In the formula, m is 1 to 10, n is 1 to 10; The double bond is cis to trans; X is -NO2, -CN, halide, C x F 2x+1 (wherein x is 1 to 5), SOR (wherein R is H or C1 to C6 alkyl), SO2R (wherein R is H or C1 to C6 alkyl), or SO3R (wherein R is H or C1 to C6 alkyl). has.

[0142] In certain embodiments, X is -NO2.

[0143] In certain embodiments, the compound is: [ka] [In the formula, m is 1 to 10, n is 1 to 10; The double bond is cis to trans; Y and Z are each independently C1 to C 10 alkyl, alkenyl, or alkynyl; X is -NO2, -CN, halide, C x F 2x+1(wherein x is 1 to 5), SOR (wherein R is H or C1 to C6 alkyl), SO2R (wherein R is H or C1 to C6 alkyl), or SO3R (wherein R is H or C1 to C6 alkyl). It is an alkyl ester of a dicarboxylic acid having the structure:

[0144] In certain embodiments, m is 2 and n is 2.

[0145] In certain embodiments, Y and Z are each independently C1-C6 alkyl, more particularly methyl or ethyl.

[0146] In certain embodiments, X is -NO2.

[0147] In certain embodiments, the compound is: [ka] [In the formula, m is 1 to 10, n is 1 to 10; The double bond is cis to trans; Y and Z are each independently C1 to C 10 alkyl, alkenyl, or alkynyl; X is -NO2, -CN, halide, C x F 2x+1 (wherein x is 1 to 5), SOR (wherein R is H or C1 to C6 alkyl), SO2R (wherein R is H or C1 to C6 alkyl), or SO3R (wherein R is H or C1 to C6 alkyl). It is an alkyl ester of a dicarboxylic acid having the structure:

[0148] In certain embodiments, m is 2 and n is 2.

[0149] In certain embodiments, Y and Z are each independently C1-C6 alkyl, more particularly methyl or ethyl.

[0150] In certain embodiments, X is -NO2.

[0151] In certain embodiments, the compound is: [ka] It is. Compound (b) - Antineoplastic Agent

[0152] In certain embodiments, the anti-neoplastic agent is a DNA damaging agent or treatment. Illustrative anti-neoplastic agents include doxorubicin, cisplatin, olaparib, rucaparib, niraparib, talazoparib, veliparib, camptothecin, and radiation treatment (IR).

[0153] In certain embodiments, co-administration of compound (a) with antineoplastic agents doxorubicin, cisplatin, IR, or olaparib enhances the cell killing, anti-proliferative, anti-tissue invasion, and / or anti-metastatic effects of these DNA-damaging therapeutic strategies in TNBC cells. NO2-OA was identified to suppress IR-induced RAD51 foci formation, inhibit RAD51 binding to ssDNA, reduce HR, induce phosphorylation of Ser139 H2AX (γH2AX), disrupt RAD51-ABL heterodimerization, and reduce RAD51 Tyr315 phosphorylation. These observations reinforce the idea that reactive species induce genomic perturbations in part through disruption of HR and reveal a novel therapeutic strategy - redox-derived soft electrophiles sensitize cancer cells to DNA-directed therapeutic strategies such as IR, cisplatin, and doxorubicin.

[0154] Fatty acid nitroalkenes are endogenously produced products of nitric oxide- and nitrite-dependent nitration of unsaturated fatty acids. Due to kinetically rapid reversible Michael addition, fatty acid nitroalkenes can mediate PTM of susceptible Cys residues in proteins, potentially modifying protein function to induce signaling responses through pleiotropic mechanisms. Results disclosed herein show that NO2-OA reduces proliferation of TNBC cells, especially when co-administered with clinically relevant DNA-directed therapeutic strategies (Figures 1A-1K). NO2-OA also amplified the induction of DSBs by IR or I-SceI DNA cleavage, specifically via inhibiting HR but not NHEJ, by limiting IR-induced nuclear RAD51 foci formation and DNA recombination (Figures 2A-2D). The functionally critical Cys319 of RAD51 was targeted by NO2-OA but not by non-electrophilic native nitroalkane-substituted control fatty acids (Figures 3A-3I). Cys319 alkylation by NO2-OA disrupted the dimerization of RAD51 with ABL and reduced ABL-induced phosphorylation of RAD51 Tyr315 (Figure 3A-3I). NO2-OA target proteins other than RAD51 may further play a role in inhibiting HR. The reduction of RAD51 foci and induction of gamma-H2AX staining (a marker of DNA DSB breaks) by NO2-OA are cancer cell selective, as they do not occur in irradiated benign (non-tumorigenic) breast epithelial cells MCF-10A (Figure 1J).

[0155] The potent antiproliferative effect of olaparib indicates pharmacological induction of BRCAness phenotype by NO2-OA when administered in combination with NO2-OA in TNBC cell lines (Figures 1A-1K). Inhibition of HR-mediated DNA DSB repair by NO2-OA is cancer cell-specific and reflects loss-of-function mutations in BRCA genes that cause defective DNA repair capacity via impaired HR. Because increased expression of RAD51 spares BRCA1 function and is a common feature of BRCA1-deficient breast tumors, breast cancer patients harboring BRCA1 loss-of-function mutations may also benefit from inhibition of RAD51.

[0156] Although functional HR is important for maintaining genomic stability, enhanced homologous recombination DNA repair activity impedes chemotherapy and ionizing radiation treatments for cancer. Elevated expression levels of RAD51 positively correlate with breast cancer tumor malignancy and have been identified in several TNBC cell lines and metastatic patient samples. Several studies have attempted to exploit RAD51 inhibition to promote lethality in cancer cells. Inhibition of RAD51 with small molecule inhibitors can sensitize cancer cells to chemotherapy or IR [e.g., DIDS, B02, RI-1, and IBR2]. For example, RI-1 was identified in a high-throughput screen to potentiate RAD51 filament formation and HR activity, which serendipitously adds to Cys319. Unfortunately, RI-1 has multiple electrophilic centers in the complex biphenol morpholino structure, thus inducing irreversible Michael addition, and incompatibility for in vivo applications due to toxicity.

[0157] RAD51 Cys319 represents a key functional site within the RAD51 protein. Homomultimeric RAD51 filaments coordinate around the Cys319 residue, which is located within the Src homology 3 (SH3) domain and near the ATPase domain (PDB:1N0W). Thus, post-translational thiol modification or pharmacological targeting of Cys319 can disrupt RAD51 function through multiple mechanisms.

[0158] We now identify herein Cys319 of RAD51 as a specific target for electrophilic nitroalkenes that, upon alkylation, inhibit RAD51 binding to ssDNA (Figures 3A-3I). Thus, administration of synthetic homologues of endogenously occurring fatty acid nitroalkenes provides a viable option for inactivating RAD51. Clinical administration of intravenous and oral formulations of NO2-OA (IV IND, 122583; oral IND, 124524) has been safe and passed multiple Phase I drug-drug interaction studies, and the oral formulation is currently in multicenter Phase II trials for treating chronic inflammation-related diseases.

[0159] Nitroalkene fatty acids have anticancer pleiotropic effects that affect other DNA DSB repair pathways in addition to HDR. Comparison of all four pathways that repair DNA DSBs showed that in addition to HDR, NO2-OA also inhibits single-strand annealing (SSA), a conserved DNA DSB repair pathway specific to homologous repeat sequences, resulting in deletion of sequences between repeat sequences (Figures 14A and 14B). This supports Cys in other proteins as a target of nitrated fatty acids (NFAs) at or upstream of SSA. In vivo combination therapy of NO2-OA with the PARPi talazuparib shows significantly greater tumor shrinkage compared to talazoparib or NO2-OA alone in a TNBC xenograft model (MDA-MB-231 cells) (Figure 15).

[0160] The results disclosed herein indicate that exogenously administered nitroalkene fatty acids play a role in regulating DNA repair and other signaling responses that can improve the treatment of drug-resistant cancers.

[0161] In certain embodiments, the compounds disclosed herein inhibit cell migration and invasion, which is necessary for cancer cell spreading and metastasis.

[0162] In certain embodiments, inhibition of Rad51-mediated DNA repair by electrophilic nitroalkene fatty acids renders TNBC cells more sensitive to PARP inhibition, leading to death and potentially inhibiting invasion and metastasis. Approximately 15% of all breast cancers (BC) are triple negative, lacking the three receptors that classify and define the treatment strategies for the majority of BC: estrogen receptor (ER), progesterone receptor (PR), and ERB2 (also known as HER2). Thus, TNBC is an aggressive type of BC that does not have targeted therapy and disproportionately affects young women and women of African descent. Up to 20% of TNBC patients also carry germline BRCA1 or BRCA2 mutations (gBRCAm), resulting in defects in homologous recombination DNA repair (HDR). Olaparib and talazoparib are FDA approved for metastatic gBRCAm-positive TNBC patients due to successful phase 3 clinical trials showing the benefits of PARPi monotherapy over chemotherapy for germline BRCA-mutated TNBC patients. These findings reinforce the idea that genetic defects in HDR pave the way for cancer cell killing by PARPi by inhibiting single-stranded DNA repair and highlight the significance of the co-administration approach disclosed herein to chemically induce HDR deficiency by inhibiting Rad51 to amplify PARPi efficacy. Thus, the 80% of TNBC patients who are negative for gBRCAm and have inconsistent responses to PARPi may benefit from PARPi therapy in this case, despite their BRCA-like phenotype (BRCAness). In particular: i) By chemically inducing Rad51 inhibition to limit TNBC cell HDR, the patient cohort eligible for PARPi treatment can now also include patients who are wild-type for BRCA or harbor mutations in other HDR-associated genes; ii) Several cancers, including TNBC, have increased Rad51 expression. Preclinical models show that depletion of Rad51 by siRNA and shRNA sensitizes to a) PARPi, b) radiation treatment in pancreatic cancer and multiple myeloma, c) chemotherapy in non-small cell lung cancer (NSCLC) and glioma, and d) reduces primary tumor growth and brain metastases in mouse TNBC models. Drug targeting of Rad51 is a coveted anti-cancer therapy, but to date no small molecule Rad51 inhibitor is safe enough to initiate phase 1 / 2 development; iii) PARPi resistance is a problem for patients and is a result of restoration of BRCA1 or BRCA2 open reading frames by cancer cells, increased use of non-homologous end joining (NHEJ) for repair, and Rad51 overexpression. The low toxicity of PARPi, and encouraging its success, together with data showing that co-treatment of PARPi with nitroalkene fatty acids greatly enhances PARPi efficacy, provide new opportunities to limit drug resistance and enhance TNBC cell killing.

[0163] In certain embodiments, cancer cell growth and / or migration and / or invasion are suppressed through the regulation of NF-κB signaling by treatment with at least one nitroalkene fatty acid disclosed herein.One embodiment contemplates administering a therapeutically effective amount of nitroalkene fatty acid to a subject who has, is suspected of having, is at risk of developing, is undergoing treatment, or is in remission from triple-negative breast cancer.For example, triple-negative breast cancer cell growth, migration, and invasion are suppressed through the regulation of NF-κB signaling by treatment with nitroalkene fatty acid, particularly electrophilic nitroalkene fatty acid (10-nitro-octadec-9-enoic acid, referred to herein as "NO2-OA"), while non-tumorigenic breast epithelial cells are resistant to the effects of NO2-OA due to their more intact mechanism for maintaining redox homeostasis.

[0164] Compared to other breast cancer phenotypes, TNBC is an aggressive subtype with poor prognosis. Patients are four times more likely to present with visceral metastases to the lungs, liver, and brain within five years of diagnosis. DNA damage-inducing strategies such as ionizing radiation, cisplatin, and doxorubicin remain the primary treatment, as TNBC does not respond to endocrine therapy or other more targeted chemotherapeutic agents. Systemic adverse responses to DNA-directed chemotherapeutic agents, including cardiotoxicity and nephrotoxicity, limit chemotherapy options due to cytotoxic effects on noncancerous cells.

[0165] NO2-OA inhibited cultured TNBC cell survival, motility, and tumor cell proliferation-related signaling responses to the extent that in vivo tumor growth in MDA-MB-231 xenografted mice was attenuated by oral administration of NO2-OA. This observation also motivates more detailed timing and dose-response studies of NO2-OA effects on tumor growth and metastasis of multiple breast cancer phenotypes in preclinical animal models.

[0166] At lower concentrations, there was selective cytotoxicity of NO2-OA to TNBC cells compared to nontumorigenic MCF-10A breast ductal epithelial cells. One meaningful explanation for this selectivity of action arose from the analysis of both basal GSH levels and the formation and fate of NO2-OA-SG adducts in control and TNBC cells. Due to the abundance and reactivity of the GSH thiol, GSH is the most important intracellular reaction target of endogenously generated and exogenously administered oxidants and electrophilic species. The rate of MRP1-mediated efflux of GSH-adducted electrophiles from cells contributes to defining the net intracellular concentration, half-life, target protein reactions, and cellular and tissue responses to lipid electrophiles. MRP1 is highly expressed in MCF-10A cells compared to TNBC cells, motivating the LC-MS / MS determination of extracellular NO2-OA-SG levels in the medium of NO2-OA-treated MCF-10A cells versus NO2-OA-treated MDA-MB-231 and MDA-MB-468 cells. Consistent with the relative degree of MRP1 expression, MCF-10A cells formed and transported 4-5 times more NO2-OA-SG adducts to the extracellular compartment compared to TNBC cells (Figure 6A). This transport of larger amounts of NO2-OA-SG by MCF-10A versus MDA-MB-231 and MDA-MB-468 cells was notable also because the basal GSH concentration and GSH:GSSG ratio in MCF-10A cells were more stable after treatment with NO2-OA. In contrast, the GSH concentration and GSH:GSSG ratio in MDA-MB-231 and MDA-MB-468 cells decreased within a short time after treatment with NO2-OA. These results indicate that the transport of NO2-OA-SG by MRP1, and the more sufficient antioxidant capacity of the MCF10A cell line in contrast to TNBC cells, play a role in defining the vulnerability of TNBC cells to NO2-OA signaling action. Another electrophile, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), exerts antitumor activity by inducing apoptosis in various cancers. CDDO rapidly depletes mitochondrial GSH and induces increased ROS generation in pancreatic cancer cells.In contrast, NO2-OA did not significantly affect the rate of cellular H2O2 production after both short-term and long-term (6 h) treatment of TNBC cells, indicating that inhibition of TNBC cell growth and survival by NO2-OA was not due to the induction of ROS.

[0167] When MCF-10A cells' MRP1 transport activity was inhibited by the organic acid probenecid, a more TNBC-like phenotype was conferred in terms of sensitivity to NO2-OA. For example, the effects of NO2-OA on cell growth arrest and death (Fig. 6C-D), cell cycle arrest (cyclin D1, p21), and apoptosis control mediators (PARP-1, caspase-3) all supported the idea that NO2-OA signaling action is enhanced in MRP1-depleted cells due to more favorable pharmacokinetics in the intracellular compartment. This affirms that the cellular concentration of GSH, the reaction of GSH with NO2-OA, and the subsequent transport of NO2-OA-SG by MRP1 influence the downstream response to NO2-OA. Other mechanisms, yet to be described, may also be responsible for this differentiation of breast epithelial cell responses.

[0168] Although the antiproliferative effects of NO2-OA on macrophages, vascular smooth muscle cells, and fibroblasts have been observed in models of chronic vascular and pulmonary disease, the effect of fatty acid nitroalkenes on cancer cell growth was not examined. This motivated experimental investigation because there are a limited number of reports suggesting that upregulation of Nrf2 signaling may result in intrinsic or acquired chemoresistance. In contrast, we observed inhibition of TNBC growth by NO2-OA in vitro and in vivo (Figure 4B-E). This growth inhibition of TNBC cells was the result of altered signaling responses specific to TNBC cells but not to non-transformed MCF-10A cells. Increased p21 expression and decreased cyclin D1 expression (Figure 5D) were observed along with an increase in the sub-G1 population of TNBC cells (Figure 5A-C). Two distinct pathways of apoptotic signaling were employed by NO2-OA in TNBC cells, initiated by both mitochondria (caspase-9 activation) and death receptor (caspase-8 activation; Figure 5F)-regulated mechanisms. Collectively, these data reveal that NO2-OA exerts pleiotropic anticancer properties through inhibition of cell proliferation and induction of apoptosis in TNBC. At this stage, the more detailed mechanisms of NO2-OA-induced apoptotic cell death remain unclear, but the electrophilic thiocyanate sulforaphane also reduces Bcl-2 expression, activates cytochrome c release from mitochondria, and increases FasL expression in TNBC cells. These actions imply that electrophilic fatty acid nitroalkene derivatives may mediate similar actions in controlling apoptosis. Inhibition of NF-κB signaling by NO2-OA also limits TNBC cell migration and invasion. Proinflammatory cytokines such as TNFα enhance the metastatic potential of TNBC, as upregulation of TNFα expression and activity in TNBC patients is strongly associated with the tumor metastatic phenotype. TNFα stimulates epithelial-mesenchymal transition (EMT) and chemokine gene expression through activation of AP-1 and NF-κB signaling in TNBC cells.Herein, NO2-OA significantly inhibited TNFα-induced TNBC cell migration and invasion (Figure 6). Decreased expression of pro-metastatic genes uPA and ICAM-1 via decreased NF-κB transcriptional activity was also induced by NO2-OA (Figure 9D). Consistent with this, electrophilic 15-deoxy-Δ. 12,14 -Prostaglandin J2, dithiolthiones, and dimethyl fumarate also inhibit breast cancer cell migration. NO2-OA also reduced the migration of MDA-MB-231 cells in the absence of TNFα induction (Figure 8B). The electrophilic cyclopentenone 15-deoxy-Δ 12,14 - Because prostaglandin J2 also interferes with breast cancer cell migration through inhibition of F-actin reorganization and focal adhesion disassembly, it is likely that NO2-OA inhibits cell migration by interacting with molecular targets in addition to NF-κB.

[0169] Proteolysis of NF-κB subunits contributes to the termination of NF-κB activation. RelA protein is controlled by ubiquitin- and proteasome-dependent degradation signals that terminate NF-κB activation. Thiol alkylating and S-nitrosating agents also promote the degradation of NF-κB subunit p50 via posttranslational modification of Cys62 in HT29 and HCT116 tumor cell lines. Thus, alkylation of NF-κB RelA by NO2-OA induces functional responses similar to other alkylating agents. Notably, alkylation of RelA by NO2-OA induced an increase in RelA ubiquitination in TNBC cells, an effect that was not observed with the non-electrophilic NO2-SA (Figure 10D). Peroxisome proliferator-activated receptor γ (PPARγ) acts as an E3 ubiquitin ligase and induces RelA protein ubiquitination and degradation through physical interaction with RelA protein. The PPARγ ligands troglitazone and pioglitazone increase PPARγ E3 ligase activity by promoting its interaction with RelA protein, which shortens RelA half-life. Because NO2-OA is a partial agonist of PPARγ, it can be speculated that NO2-OA also activates PPARγ E3 ligase activity, thus further destabilizing RelA protein in TNBC.

[0170] Inhibition of NF-κB signaling represents a viable anticancer strategy, especially since aberrant activation of NF-κB is closely associated with the development of various human cancers. Dimethyl fumarate, an immunomodulatory electrophile that is FDA-approved as an oral drug to treat multiple sclerosis, also inhibits NF-κB activity in breast cancer cells and inhibits TNBC cell proliferation. The present results that NO2-OA inhibited multiple TNBC cell functions: proliferation, survival, migration, and invasion imply that electrophilic lipid nitroalkene species may also have utility as chemotherapeutic agents.

[0171] The lipid electrophile NO2-OA affects NF-κB signaling in TNBC at multiple levels, including suppression of IKKβ phosphorylation, inhibition of IκBα degradation, and enhanced ubiquitination and proteasomal degradation of RelA. These actions contribute to the inhibition of TNBC cell migration and invasion in vitro. TNBC cells are more sensitive to NO2-OA in part due to lower GSH concentrations, and inhibition of NO2-OA transport as NO2-OA-SG adducts, a consequence of lower MRP1 expression. This GSH-deficient redox vulnerability of TNBC cells promotes more extensive protein thiol alkylation and oxidation reactions, eliciting chemotherapeutic signaling responses at lower electrophile concentrations. The concentration of non-protein-adducted endogenous free NO2-FA in healthy human plasma and urine is typically 1-5 nM. Oral administration of NO2-OA increased mouse tumor NO2-OA levels sufficiently to induce a pharmacological response, as evidenced by inhibition of MDA-MB-231 xenograft tumor growth.

[0172] In some embodiments, the method disclosed herein comprises administering to a subject in need of treatment a pharmaceutical composition, for example a composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of one or more compounds disclosed herein. The compound can be administered orally, parenterally (including subcutaneous injection (SC or depo-SC), intravenous (IV), intramuscular (IM or depo-IM), intrasternal injection or infusion techniques), sublingually, intranasally (inhalation), intrathecally, topically, ophthalmically, or rectally. The pharmaceutical composition can be administered in a dosage unit formulation containing conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, and / or vehicles. The compound is preferably formulated into pharmaceutical preparations such as tablets, capsules, or elixirs suitable for oral administration, or pharmaceutical preparations in sterile solutions, emulsions, or suspensions suitable for parenteral or topical administration or inhalation. Typically, the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art.

[0173] In some embodiments, one or more of the disclosed compounds (including compounds linked to detectable labels or cargo moieties) are mixed or combined with a suitable pharma- ceutically acceptable carrier to prepare a pharmaceutical composition. Suitable pharmaceutical carriers or vehicles for administration of the compounds provided herein include any such carriers known to be suitable for a particular method of administration. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes exemplary compositions and formulations suitable for pharmaceutical delivery of the compounds disclosed herein. In addition, the compounds may be formulated as the only pharmacoactive ingredient in the composition or may be combined with other active ingredients.

[0174] After mixing or adding the compound to the pharma- ceutically acceptable carrier, the resulting mixture may be a solution, suspension, emulsion, etc. Liposomal suspensions may also be suitable as pharma- ceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends on several factors, including the intended method of administration and the solubility of the compound in the selected carrier or vehicle. If the compound exhibits insufficient solubility, methods for solubilization may be used. Such methods are known and include, but are not limited to, using cosolvents such as dimethyl sulfoxide (DMSO), using surfactants such as Tween®, and dissolving in aqueous sodium bicarbonate. Derivatives of the compound, such as salts or prodrugs, may also be used in formulating effective pharmaceutical compositions. The disclosed compounds may also be prepared with carriers that protect the compound against rapid elimination from the body, such as sustained release formulations or coatings. Such carriers include controlled release formulations, such as, but are not limited to, microencapsulated delivery systems. Formulations may also be obtained by dissolution with medium and long chain natural oils.

[0175] The disclosed compounds and / or compositions can be packaged in multi- or single-dose containers. The compounds and / or compositions can also be provided in kits that include components that can be assembled for use, for example. For example, one or more disclosed compounds can be provided in lyophilized form, and a suitable diluent can be provided as separate components for combination before use. In some examples, the kit can include the disclosed compounds and a second therapeutic agent (e.g., an antiretroviral agent) for co-administration. The compound and the second therapeutic agent can be provided as separate components. The kit can include multiple containers, each container holding one or more unit doses of the compound. The containers are preferably adapted for the desired method of administration, including, but not limited to, tablets, gel capsules, sustained release capsules, etc. for oral administration; depots, pre-filled syringes, ampoules, vials, etc. for parenteral administration; and patches, medipads, creams, etc. for topical administration.

[0176] The pharmaceutical composition may be in dosage unit form, for example, an injectable fluid, an oral delivery fluid (e.g., a solution or suspension), an intranasal delivery fluid (e.g., for delivery as an aerosol or vapor), a semi-solid form (e.g., a topical cream), or a solid form, such as a powder, pill, tablet, or capsule form.

[0177] The active compound is contained in a pharma- ceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject being treated. The therapeutically effective concentration can be empirically determined by testing the compound in known in vitro and in vivo model systems for the disorder being treated. In some cases, the therapeutically effective amount of a compound is an amount that relieves or improves at least one symptom of the disorder for which the compound is administered. Typically, the composition is formulated for single dose administration. The concentration of the active compound in the drug composition can depend on the absorption, inactivation, and excretion rates of the active compound, the administration schedule, and the amount administered, as well as other factors known to those skilled in the art.

[0178] In some examples, about 0.1 mg to 1000 mg of the disclosed compounds, mixtures of such compounds, or physiologically acceptable salts or esters thereof are formulated into unit dosage forms with physiologically acceptable vehicles, carriers, excipients, binders, preservatives, stabilizers, flavorings, and the like. The amount of active substance in these compositions or preparations is such that a suitable dosage in the indicated range will be obtained. The term "unit dosage form" refers to physically discrete units suitable as single dosages for human subjects and other mammals, each unit containing a predetermined quantity of active substance calculated to produce a desired therapeutic effect in association with a suitable pharmaceutical excipient. In some examples, the compositions are formulated into unit dosage forms, each dosage containing about 1 mg to about 1000 mg (e.g., about 2 mg to about 500 mg, about 5 mg to 50 mg, about 10 mg to 100 mg, or about 25 mg to 75 mg) of one or more compounds. In other examples, the unit dosage form contains about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, or more of a disclosed compound.

[0179] The disclosed compound or composition may be administered as a single dose or divided into several smaller doses administered at intervals. The therapeutic composition may be administered in a single dose delivery by continuous delivery over an extended period of time in a repeated administration protocol (e.g., multiple times a day, once a day, once a week, or once a month). It is understood that the exact dosage, timing, and duration of treatment are a function of the disease being treated and can be empirically determined using known testing protocols or by extrapolation from in vivo or in vitro test data. It is noted that concentration and dosage values ​​may also vary with the severity of the condition being alleviated. In addition, it is understood that for a specific subject, dosage regimens may be adjusted over time according to individual need and the professional judgment of the person administering or supervising the administration of the composition, and that the concentration ranges described herein are merely exemplary.

[0180] When administered orally as a suspension, these compositions are prepared according to techniques well known in the art of pharmaceutical formulations and may contain microcrystalline cellulose to provide volume, alginic acid or sodium alginate as a suspending agent, methylcellulose as a thickening agent, and sweeteners / flavoring agents.As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, and lactose, and / or other excipients, binders, fillers, tablet disintegrants, diluents, and lubricants.When oral administration is desired, the compound is typically provided in a composition that protects the compound from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.The composition may also be formulated in combination with an antacid or other such ingredients.

[0181] Oral compositions generally can contain inert diluents or edible carriers, and can be compressed into tablets or can be enclosed in gelatin capsules.For the purpose of oral therapeutic administration, one or more active compounds can be combined with excipients and used in the form of tablets, capsules, or lozenges.Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, etc. may contain any of the following ingredients or compounds of similar nature: binders, such as, but not limited to, gum tragacanth, gum arabic, corn starch, or gelatin; excipients, such as, but not limited to, microcrystalline cellulose, starch, or lactose; disintegrants, such as, but not limited to, alginic acid and corn starch; lubricants, such as, but not limited to, magnesium stearate; gildants, such as, but not limited to, colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; and flavoring agents, such as peppermint, methyl salicylate, or fruit flavors.

[0182] When the dosage unit form is a capsule, it can contain liquid carriers such as fatty oils in addition to the above-mentioned types of materials.In addition, dosage unit form can contain various other materials that modify the physical form of dosage unit, such as sugar and other enteric coatings.The compound can also be administered as a component of elixirs, suspensions, syrups, cachets, chewing gums, etc.Syrups can contain sucrose as a sweetener, as well as certain preservatives, dyes and colorings, and flavorings in addition to the active compound.

[0183] When administered orally, the compound can be administered in a conventional dosage form for oral administration. These dosage forms include conventional solid unit dosage forms of tablets and capsules, as well as liquid dosage forms such as solutions, suspensions, and elixirs. When solid dosage forms are used, the dosage form is preferably of a sustained release type, such that the compound need only be administered once or twice daily. In some examples, the oral dosage form is administered to a subject 1, 2, 3, 4, or more times daily. In additional examples, the compound can be administered orally to a human in a dosage range of 1-1000 mg / kg body weight in single or divided doses. One illustrative dosage range is 0.1-200 mg / kg body weight orally (e.g., 0.5-100 mg / kg body weight orally) in single or divided doses. For oral administration, the composition may be provided in the form of a tablet containing about 1 to 1000 milligrams of active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 milligrams of active ingredient. It will be understood, however, that the specific dose level and frequency of administration for any particular patient may vary and will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, age, body weight, general health, sex, diet, method and time of administration, rate of excretion, drug combination, severity of the particular condition, and the host receiving the therapy.

[0184] Injectable solutions or suspensions can also be formulated using suitable non-toxic parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, nonirritating fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application can contain any of the following components: sterile diluents, such as water for injection, saline solution, fixed oils, naturally occurring vegetable oils, such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fatty vehicles, such as ethyl oleate, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol and methylparaben; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, and phosphates; and agents for adjusting tonicity, such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable materials. Buffers, preservatives, antioxidants, etc. can be incorporated where necessary.

[0185] For intravenous administration, suitable carriers include physiological saline, phosphate buffered saline (PBS), and solutions containing viscosity agents and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.Liposomal suspensions, including tissue-targeted liposomes, can also be suitable as pharma-ceutically acceptable carriers.

[0186] The compound can be administered parenterally, for example, by IV, IM, depo-IM, SC, or depo-SC. When administered parenterally, a therapeutically effective amount of about 0.1 to about 500 mg / day (e.g., about 1 mg / day to about 100 mg / day or about 5 mg / day to about 50 mg / day) can be delivered. When the depot formulation is used for injection once a month or once every two weeks, the dose can be about 0.1 mg / day to about 100 mg / day, or a monthly dose of about 3 mg to about 3000 mg.

[0187] The compounds may also be administered sublingually. When administered sublingually, the compounds should be administered 1 to 4 times daily in the amounts described above for IM administration.

[0188] Compound can also be administered intranasally.When administered by this route, suitable dosage form is nasal spray or dry powder.The dosage of compound for intranasal administration is the amount described above for IM administration.When administered by nasal aerosol or inhalation, these compositions can be prepared according to the techniques well known in the art of pharmaceutical preparations, and can be prepared as a solution in saline solution using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents.

[0189] The compound can be administered intrathecally.When administered by this route, the appropriate dosage form can be a parenteral dosage form.The dosage of the compound for intrathecal administration is the amount described above for IM administration.

[0190] The compound can be administered externally. When administered by this route, the appropriate dosage form is cream, ointment, or patch. When administered externally, an exemplary dosage is about 0.5 mg / day to about 200 mg / day. Since the amount that can be delivered by a patch is limited, two or more patches may be used.

[0191] The compound can be administered rectally by suppository.When administered by suppository, an exemplary therapeutically effective amount can be in the range of about 0.5mg to about 500mg.When administered rectally in the form of suppository, these compositions can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, a synthetic glyceride ester of polyethylene glycol, which is solid at room temperature but liquefies and / or dissolves in the rectal cavity to release the drug.

[0192] It should be apparent to one of ordinary skill in the art that the exact dosage and frequency of administration will depend on the particular compound being administered, the particular condition being treated, the severity of the condition being treated, the age, weight, and general health of the particular subject, as well as other drug therapies the individual may be receiving, as will be known to the administering physician or other clinician skilled in the therapy of retroviral infections, diseases, and related disorders. EXAMPLES

[0193] Experimental Procedure - Nitroalkene fatty acids impair RAD51 function and potentiate the effects of DNA damaging agents on the growth of triple-negative breast cells

[0194] Cell culture and reagents. HEK293T, MDA-MB-231, MDA-MB-468, Hs578T, and BT-549 cells (American Type Culture Collection) were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 5% FBS (HyClone), 100 units / ml penicillin, 100 mg / ml streptomycin (Gibco), non-essential amino acids (Gibco), and 2 mM l-glutamine (Gibco). Doxorubicin (Selleckchem), cisplatin (Sigma), or olaparib (Selleckchem) were dissolved in DMSO or DMF (cisplatin). Nitro-oleic acid (10-octadec-9-enoic acid) (NO2-OA) and biotinylated NO2-OA were synthesized as previously described. Pure NO2-OA was diluted in DMSO and added to cells after solvation in assay medium. Relative cell numbers were compared by measuring the luminescence signal generated by ATP using the CellTiter-Glo (Promega) assay. Cells were plated in 96-well plates at 5,000 (MDA-MB-231) or 6,600 (BT549 or Hs578T) cells per well. Cells were treated with the indicated concentrations of doxorubicin, cisplatin, or olaparib for 72 hours in the presence or absence of 2 μM NO2-OA supplemented every 24 hours.

[0195] NO2-OA in vivo.

[0196] Animals used for this study were approved by and maintained in accordance with the University of Pittsburgh IACUC guidelines. MDA-MB-231 cells (0.5 × 106) were injected into the mammary fat pad (fourth gland on the left) of 6-week-old female nude mice in a volume of 20 μL of sterile saline. When tumors reached 100 mm 3When tumor volumes reached a mean volume of 100 mg / kg, mice were treated with OA + vehicle (n = 8), OA-NO2 + vehicle (n = 7), OA + talazoparib (0.3 mg / kg) (n = 10), or OA-NO2 (15 mg / kg) + talazoparib (n = 11). Tumor volumes were compared using P values: * <0.01, *** <0.001, **** The surgical procedure was previously described.

[0197] Plasmid.

[0198] Direct repeat green fluorescent protein (DR-GFP) reporter and I-SceI pCAGGS plasmids were kindly donated by Prof. Maria Jasin. pLVX Neo-RAD51 was cloned by PCR amplification of RAD51 using PCR primers incorporating SpeI (5') and BamHI (3') restriction sites. The PCR product was then ligated into the corresponding restriction sites of pLVX-Neo (Clonetech) and transformed into DH5α maximum efficiency cells (Invitrogen). Cysteine-to-serine mutated pLVX Neo RAD51 plasmids (137, 312, or 319) were generated using the QuikChange II site-directed mutagenesis kit (Agilent) and pLVX Neo-RAD51 as template.

[0199] DSB repair assay.

[0200] Measurements of NHEJ, HR, Alt-EJ, and SSA assays were performed as previously described. HR activity was measured by counting GFP-positive cells by flow cytometry using a BD LSRII (BD Biosciences) at the MWRI Flow Cytometry Core. RAD51-overexpressing cells were generated by stable transfection of pLVX RAD51 IRES Neo and selection with Geneticin (Invitrogen).

[0201] Kinetic DSB repair assay.

[0202] U2OS cells were prepared as above, except that 5 hours after compound treatment, cells were transferred to an Incucyte Zoom (Essen) automated live-cell imaging fluorescence microscope at 37°C and 5% CO2. Cell confluence and the number of green objects per mm2 were determined using Incucyte Zoom software. The number of green objects per field was normalized to cell confluence to correct for NO2-OA-induced effects on cell proliferation.

[0203] Immunostaining and imaging.

[0204] To analyze RAD51 foci formation, 10,000 cells were plated on CultureWell 16-well chambered coverglass (MIDSCI) coated with poly-L-lysine (Sigma) and incubated overnight in 5% FBS medium. Cells were then treated with NO2-OA, irradiated with 5Gy (Gammacell 40Exactor γ-irradiator, Best Medical), and incubated for 6 hours. Cells were fixed with 10% formalin for 20 minutes at 4°C, and immunostained with RAD51 (Santa Cruz) or γH2AX (EMD Biosciences) antibodies. Z-stack images were acquired using a Nikon A1R confocal microscope with a 60x oil immersion objective, and acquisition was performed using NIS elements software. Quantification of z-stacks and foci was completed using ImageJ software.

[0205] Cell cycle analysis.

[0206] Cell cycle analysis was performed using propidium iodide on MDA-MB-231 cells treated with DMSO or 5 μM NO2-OA. Samples were analyzed at the MWRI Flow Cytometry Core using a BD LSRII (BD).

[0207] Western blotting and immunoprecipitation.

[0208] Cell lysates and immunoprecipitates were prepared. For immunoprecipitation analysis, one million HEK293T cells were transiently transfected with Fugene6 (Promega) and 2 μg of pQCXIP(EV) or FLAG-RAD51 pQCXIP plasmid and precipitated with anti-FLAG M2 affinity gel (Sigma).

[0209] Affinity capture of RAD51 by biotinylated OA-NO2.

[0210] HEK293T were transiently transfected with Fugene6 (Promega) and 5 μg of RAD51 expression vector (wild type, C312S, or C319S). After 24 h, cells were treated with 5 μM biotin-NO2-OA or biotin-SA-NO2 in 5% FBS medium for 1 h. Cells were prepared as described above. Precipitation of biotinylated NO2-OA was accomplished by incubating 1 mg of total cell lysate with 8 μl of streptavidin agarose beads for 16 h at 4°C. Detection of RAD51 was accomplished by immunoblotting with RAD51 antibody (1:2000) probed with actin antibody (1:3000) as a loading control.

[0211] Protein purification for in vitro RAD51-ABL binding assay.

[0212] Recombinant His-tagged RAD51 in pET21a vector was transformed into E. coli BL21(DE3)pLysS cells (EMD Millipore) and purified.

[0213] DNA binding assay.

[0214] Reactions were carried out in black 96-well plates (Greiner) in 20 mM, pH 7.5 HEPES, 10 mM MgCl2, 0.25 μM BSA, 2% glycerol, 30 mM NaCl, and 4% DMSO in a reaction volume of 50 μL. Purified RAD51 protein (Abcam) and OA (negative control) or NO2-OA were preincubated for 5 min at 25°C. 2 mM ATP and 100 nM 5'-Alexa Fluor488ssDNA poly-dT (Integrated DNA technologies) were added to the reaction and incubated for 90 min at 37°C. DNA binding was measured using fluorescence polarization (FP) on a Tecan Spark20M (ex / em 480 nm / 535 nm). Quenching of compound fluorescence was detected as above in the absence of RAD51 protein.

[0215] Molecular modeling.

[0216] The structures of RAD51 (PDB: 1N0W (30) and NO2-OA were aligned using PyMOL 1.7.1. The structure of NO2-OA2 was generated using ChemDraw 15 (PerkinElmer) and converted to a 3D structure using Open Babel version 2.3.1.

[0217] Statistical analysis.

[0218] Data represent the mean ± SEM of three independent experiments unless otherwise indicated. A p value < 0.05 was considered statistically significant. Nonlinear curves were generated in GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA) for statistical analysis. EC50 values ​​and standard errors were calculated using a nonlinear dose-response variable slope model from three independent experiments. Significance was tested by one-way ANOVA with Tukey post-hoc test for multiple groups or by t-test if there were less than three groups. The number of RAD51 foci was analyzed using ImageJ. Nuclear borders were identified individually in more than 50 cells per treatment group from three independent experiments.

[0219] Results: Nitroalkene fatty acids impair RAD51 function and potentiate the effects of DNA damaging agents on triple-negative breast cell growth

[0220] NO2-OA inhibits TNBC cell growth, RAD51 foci formation, and sensitivity to ionizing radiation.

[0221] The data show that NO2-OA inhibits multiple aspects of NF-κB signaling in TNBC epithelial cells, but not in non-tumorigenic breast epithelial cells, by a) alkylating functionally critical thiols in the inhibitor of NF-κB subunit kinase b (IKKb), thus limiting downstream IκKa phosphorylation, and (b) alkylating functionally critical thiols in the NF-κB RelA protein, thus preventing DNA binding and promoting RelA polyubiquitination and proteasomal degradation. We assessed whether NO2-OA could also enhance TNBC DNA damage in vivo. MDA-MD-231 cells were implanted into the mammary glands of mice, and tumors were grown to 100 mm 3 When tumor volume reached 1000 mg / kg, mice were treated with 15 mg / kg of the non-electrophilic fatty acid oleic acid (OA) or NO2-OA by gavage for 4 weeks. Mice treated with NO2-OA had a significantly reduced tumor growth rate when compared to OA-treated controls (Figure 1A). Probing tumor levels of the DNA damage biomarker γH2AX by immunoblotting showed that OA-NO2-treated mice expressed higher levels of γH2AX (Figure 1B). Densitometric quantification of γH2AX / β-actin protein levels of tumors in OA and NO2-OA-treated mice increased γH2AX in NO2-OA-treated mice. This response became statistically significant after Grubbs outlier detection and exclusion of OA-treated mouse #2. The orthotopic tumor in OA mouse #2 was the largest tumor in this study, potentially causing necrosis to cause enhanced γH2AX levels.

[0222] The TNBC growth inhibitory effect of NO2-OA was then evaluated in combination with DNA damaging agents. Cell lines MDA-MB-231, BT-549, and Hs578T were treated with NO2-OA daily for 3 days, and the relative cell numbers were quantified by measuring the ATP-dependent luminescence signal generated using Ultra-Glo luciferase with the substrate luciferin. The EC of TNBC cell growth inhibition was 0.01 to 0.01. 50The values ​​ranged from 1.98 ± 0.52 μM (Hs578T) to 3.78 ± 0.48 μM (BT-549), with MDA-MB-231 cells exhibiting an EC50 value of 3.66 ± 0.14 μM (Figure 1C). Next, we tested the DNA damaging agents doxorubicin and cisplatin in combination with daily treatment with 2 μM NO2-OA. NO2-OA enhanced the growth inhibition of doxorubicin 7-fold and 5-fold in MDA-MB-231 and Hs578T cells, respectively (Figure 1D). Growth of BT-549 cells was unaffected. Co-treatment of NO2-OA with cisplatin showed a similar trend for MDA-MB-231 and Hs578T cells, exhibiting a 6-fold and 3-fold increased growth inhibition, respectively, whereas growth inhibition of BT-549 cells was suppressed 1.4-fold (Figure 1E). Because a subset of TNBC cells are sensitive to PARP inhibition and display a BRCAness phenotype in the presence of wild-type BRCA1, the PARP-1 inhibitor olaparib was evaluated to determine whether combination treatment with NO2-OA enhanced efficacy. MDA-MB-231, Hs578T, and BT-549 cells all displayed 5-fold, 17-fold, and 3-fold enhanced growth inhibition, respectively, when olaparib was combined with NO2-OA (Figure 1F). To specifically demonstrate that combination treatment with olaparib and NO2-OA, but not with OA, affected proliferation, and to evaluate the effect of daily olaparib medium replacement, MDAMB-231 cell proliferation was quantified in a dose-response assay. Daily administration of olaparib in combination with OA did not significantly alter the EC50 of olaparib in MDA-MB-231 cells. In contrast, olaparib in combination with NO2-OA significantly inhibited growth (Figure 1G). Next, talazoparib, another PARPi, was tested in combination with NO2-OA for growth inhibition and for determination of combination index using the Chou-Talalay method. MDA-MB-231 cells were plated in 96-well plates and treated daily for 3 days with talazoparib (0.01-75 μM) and OA-NO2 or NDA-NO2 (0.2-12 μM).Growth inhibition (relative cell number) of MDA-MB-231 cells was determined by luminescent detection of ATP (CellTiter-Glo) (Figure 1K). Growth inhibition was nearly 100% in wells treated with 12 or 6.0 μM NO-OA and 0.01–75 μM talazoparib (Figure 1K, top half). This translated into a robust combination index showing drug synergy (CI<1) between NO2-OA and talazoparib (Figure 1K, bottom half). Thus, not only the targeted PARP-1 inhibition presented, but also standard TNBC chemotherapeutic drugs enhanced antiproliferative effects in a synergistic rather than additive manner when co-administered with NO2-OA in TNBC cells.

[0223] The increased tumor γH2AX levels in vivo and sensitization of TNBC cells to DNA damaging agents led us to further explore DNA damage repair modulation by NO2-OA, especially in the context of Olaparib-induced responses. Because Olaparib sensitivity is a hallmark of HR-deficient cells, we assessed whether NO2-OA affects DNA repair by HR. To specifically explore DNA double-strand break repair, MDA-MB-231 cells were exposed to 5 Gy of IR and RAD51 foci were quantified. Treatment of breast cancer cells with NO2-OA inhibited RAD51 foci formation, as reflected by a) the number of cells with more than five foci, and b) the response of vehicle-treated cells after IR (Figure 1I). Cell cycle analysis of MDA-MB-231 cells confirmed that no significant changes to the cell cycle occurred that could indirectly alter RAD51 foci formation. Evaluation of nuclear γH2AX staining probing DNA damage in MDA-MB-231 cells in the presence or absence of 5 Gy IR found that NO2-OA significantly increased nuclear γH2AX localization in irradiated MDAMB-231 cells compared to vehicle controls, indicating increased DSBs and overall DNA damage (Figure 1I). Increasing concentrations of NO2-OA also enhanced breast cancer cell death in clonogenic assays following irradiation with 2 Gy IR. Evaluation of the DNA damaging effects of OA and NO2-OA on non-transformed MCF10A and MDA-MB-231 cells following 5 Gy IR found that nuclear γH2AX staining was only increased in TNBC cells treated with NO2-OA2, but not in MCF10A cells.

[0224] NO2-OA reduces HR efficiency but not NHEJ efficiency. The NO2-OA-dependent effect on HR DNA repair was further investigated by utilizing the DRGFP reporter assay. This analysis quantifies intracellular recombination of the integrated cDNA cassette of two tandem non-fluorescent GFP constructs after the introduction of an I-SceI break into the system by measuring the fluorescent GFP protein produced after successful recombination. Daily NO2-OA treatment of U2OS cells carrying the DR-GFP construct revealed that the number of GFP-positive cells was significantly reduced by 2-fold after 48 hours after I-SceI transfection compared to native OA or vehicle controls (Figure 2A). Rather than performing static measurements of detached cells using flow cytometry, a novel strategy was used to measure the kinetics of HR changes in live cells by following the appearance of GFP-positive cells over time in monolayers using automated fluorescence microscopy. To account for changes in cell density over time, cell confluency was measured after 4-hourly treatment of DR-GFP U2OS cells with vehicle, 5 μM OA, or NO2-OA for 3 days. The appearance of GFP-positive cells following I-SceI-induced cleavage was quantified over time, normalized to cell confluency, and NO2-OA-treated cells were compared to OA-treated and untreated cells. Daily administration of 5 μM NO2-OA reduced the number of GFP-positive cells by 2-fold over 68 h when compared to controls (Figure 2B). The effect of NO2-OA on suppression of DSB repair by both HR and NHEJ pathways was examined by utilizing an EJ5-GFP NHEJ reporter assay in which GFP cDNA is separated from a transcription promoter with the puro gene flanked by two I-SceI cleavage sites. In contrast to the effect seen by DR-GFP-mediated HR measurements, EJ5-GFP U2OS cells showed no effect of OA-NO2 on NHEJ. This was shown by the absence of change in the number of GFP-positive cells by flow cytometric analysis at 48 h or by live cell fluorescence microscopy over 68 h after I-SceI cleavage (Figures 2C, 2D).

[0225] NO2-OA targets RAD51 Cys319 and reduces RAD51 phosphorylation.

[0226] The inhibition of IR-induced RAD51 foci formation and DR-GFP HR reporter functionality by NO2-OA was further studied by verifying whether overexpression of RAD51, a crucial HR protein, in HR reporter cells could rescue the effects of NO2-OA. HR activity, as measured by the percentage of GFP-positive cells relative to OA control treatment, was significantly increased in U2OS DR-GFP reporter cells stably overexpressing RAD51 treated with 5 μM NO2-OA compared to control reporter cells (Figure 3A). Protein structural data (PDB:1N0W) indicate that Cys319 is a solvent-exposed nucleophile at the C-terminus of RAD51 that is susceptible to reaction with RI-1, a reagent that also has Michael acceptor properties. Furthermore, fluorophore addition to Cys319 disrupts RAD51 filament formation in vitro. We hypothesized that NO2-OA reacts with RAD51 Cys319. Indeed, biotin-NO2-OA supported affinity precipitation of RAD51 from cell lysates using streptavidin-labeled beads, whereas the non-electrophilic biotin-oleate (OA) and biotin-10-nitro-octadecanoate (SA-NO2) did not (Figure 3B). Comparing the reaction of RAD51 Cys312Ser or Cys319Ser mutants with biotin-OA-NO2 revealed preferential reaction of NO2-OA with Cys319 (Figure 3C). RAD51 Cys312Ser and RAD51 WT control were readily affinity precipitated by biotin-NO2-OA, unlike when RAD51 Cys319Ser was expressed in mutant cells. Of note, the RAD51 Cys312Ser mutant exhibited increased precipitation of NO2-OA, which may reflect disruption of the disulfide bond between RAD51 Cys312 and Cys319, or another intracellular protein that obscures Cys319. The ability of NO2-OA to specifically disrupt RAD51 binding from DNA was explored in vitro by quantifying changes in fluorescence polarization of Alexa Fluor488-conjugated single-stranded oligonucleotides.NO2-OA, but not OA, reduced the relative polarization of RAD51 in the presence of ATP and DNA (Figure 3D). Control experiments found that OA and NO2-OA did not cause nonspecific effects via fluorophore quenching that reduced fluorescence polarization. Computational analysis revealed that alkylation of RAD51 Cys319 by NO2-OA was further stabilized by hydrophobic interactions with Pro318 and hydrogen bonding with Glu322 of RAD51 (Figure 3E).

[0227] Cys319 maps to the C-terminus of RAD51 in one of two ABL-SH3 binding domains (amino acids 283–286 and 318–321). In addition to disrupting RAD51 filaments, NO2-OA inhibited the heterodimerization of RAD51 with ABL. IP analysis revealed that purified RAD51 and catalytic ABL core (only SH2, SH3, and kinase domains) complex formation was abolished by NO2-OA (Figure 3F). ABL controls RAD51 activity through sequential phosphorylation of RAD51 Tyr54 and then Tyr315. We examined the effects of NO2-OA on RAD51-ABL complex formation and RAD51 Tyr315 phosphorylation by transfecting 293T cells with FLAGRAD51 and ABL core. After cells were treated with 0–5 μM NO2-OA for 1 h, FLAG IP analysis revealed that NO2-OA reduced the amount of ABL bound to RAD51 (Figure 3G). Along with the inhibition of RAD51-ABL complex formation, RAD51 Tyr315 phosphorylation was also inhibited by NO2-OA in FLAG-RAD51- and ABL-expressing cells. To clarify whether NO2-OA alkylates endogenous RAD51 in TNBC cells, biotin-NO2-OA was added to MDA-MB-231 and MDA-MB-468 cells. Biotin-NO2-OA-RAD51 complex formation in streptavidin precipitation was observed in lysates of both cell lines (Figure 3H). Overall, these data reveal that NO2-OA inhibited HR by forming adducts with RAD51 and possibly additional HR-associated target proteins to enhance sensitivity to DNA-directed cancer therapy (Figure 3I).

[0228] Experimental Procedure – Inhibition of Triple-Negative Breast Cancer Cell Survival, Migration, Invasion, and Tumor Growth by Nitroalkene Fatty Acids

[0229] Cell culture and reagents.

[0230] Cell lines were purchased from ATCC. MDA-MB-231 and MCF7 cells were cultured in Dulbecco's modified Eagle's medium, and MDA-MD-468 cells were cultured in modified minimum essential medium (Gibco, Gaithersburg, MD), each supplemented with 5% fetal bovine serum (Hyclone, Logan, UT). MCF-10A cells were cultured in growth medium consisting of DMEM:F12 (1:1) in 5% horse serum (Hyclone) and supplemented with 0.5 μg / mL hydrocortisone, 0.1 μg / mL cholera toxin, 20 ng / mL EGF, and 10 μg / mL insulin (Sigma-Aldrich, St. Louis, MO). Cells were incubated at 37° C. in a 5% CO2 atmosphere. siRNA against human RelA (L-003533-00-0005), human MRP1 / ABCC1 siRNA (L-007308-00-0005), and non-targeting control siRNA (D-001810-10-05) were purchased from Dharmacon RNAi Technologies. Lipofectamine 2000 or 3000 (Life Technologies) were used for cell transfection. The MRP1 inhibitor probenecid (4-[(dipropylamino)sulfonyl]benzoic acid) was purchased from Enzo Life Sciences and dissolved in 1 M sodium hydroxide. The NF-κB inhibitor JSH-23 (4-methyl-N1-(3-phenyl-propyl)-benzene-1,2-diamine) and the proteasome inhibitor MG-132 (Z-LLeu-D-Leu-L-Leu-al) were purchased from Sigma-Aldrich. The IKKβ inhibitor Bay11-7082 (3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile) was purchased from Calbiochem, and TNFα was purchased from BD Biosciences.

[0231] Cell treatment for IKKβ phosphorylation, IκBα phosphorylation, and IκBα degradation.

[0232] All studies used two TNBC cell lines, namely MDA-MB-231 and MDA-MB-468. To determine the effect of NO2-OA on TNFα-induced IKKβ phosphorylation in TNBC cells, cells were pretreated with NO2-OA (5 μM), NO2-SA (5 μM), or BAY11-7082 (10 μM) in serum-free medium (DMEM containing 0.1% fatty acid-free BSA) for 2 h before TNFα (20 ng / mL) stimulation for 5 min. For IκBα degradation, cells were treated as described above and stimulated with 20 ng / mL TNFα for 10 min. For IκBα phosphorylation, cells were pretreated with NO2-OA (5 μM), NO2-SA (5 μM), or MG-132 (10 μM) in combination with BAY11-7082 (10 μM) in serum-free medium for 2 h followed by TNFα (20 ng / mL) stimulation for 10 min.

[0233] NO2-FA synthesis and use.

[0234] Oleic acid (OA; octadec-9-enoic acid) was purchased from Nu-Chek Prep (Elysian, MN). Nitro-stearic acid (NO2-SA; 10-nitro-octadecanoic acid) was obtained by reduction of 10-nitro-oleic acid. Specifically, NO2-OA was dissolved in THF / methanol and cooled, then sodium borohydride was added. The flask was stirred and an aliquot was monitored by UV analysis until complete loss of nitroalkene occurred, then the reaction was stopped with acetic acid. NO2-SA was purified by first adding additional cysteine ​​to any remaining NO2-OA, and then NO2-SA was fractionated on silica gel using an ethyl acetate / hexane gradient in a chromatography. OA, NO2-OA, and NO2-SA were dissolved in anhydrous methanol and diluted into culture medium with a maximum methanol concentration of 0.1 vol / vol% immediately before use in all experiments. Biotinylated NO2-FA (Bt-NO2-OA, Bt-NO2-SA, and Bt-OA) were synthesized from the corresponding free fatty acids and biotin-(polyethylene glycol)-amine.

[0235] Cell growth assay.

[0236] Cells were plated in 96-well plates at a density of 5000 cells per well. After overnight attachment, the medium was replaced and cells were treated with 0–15 μM NO2-OA, NO2-SA, or 0.1% methanol (vehicle) for 48 h. In MRP inhibition studies, MCF-10A cells were pretreated with 0.25 mM probenecid for 1 h followed by 0–25 μM NO2-OA for 48 h. Cells were counted using the FluoReporter dsDNA quantification kit (Molecular Probes) according to the manufacturer's instructions. Fluorescence was measured using a SpectraMax M2 plate reader (Molecular Devices). The half-maximal inhibitory concentration (IC50) of NO2-OA was determined by using CalcuSyn software from Biosoft. Three individual experiments were performed (n=5 / each) and statistical comparisons between doses of the two cell lines were determined by two-way ANOVA followed by Tukey's post-hoc test.

[0237] Fluorescence-activated cell sorting (FACS).

[0238] MCF-10A, MDA-MB-231, and MDA-MB-468 cells were plated at a density of 2.5×105 cells in 6-well plates for 24 h, followed by treatment with 0.1% methanol (vehicle), 5 μM NO2-OA, NO2-SA, or OA for 24 h. Adherent and nonadherent cells were collected, centrifuged at 2000×rpm for 10 min, washed with ice-cold phosphate-buffered saline, fixed with cold 70% ethanol for 30 min at 4°C, and stained with 50 μg / mL propidium iodide (Sigma-Aldrich). FACS analysis was performed at the University of Pittsburgh Department of Immunology Unified Flow Core Facility. Three individual experiments were performed, and statistical comparisons between phases (G0 / G1, S, and G2 / M) were determined by one-way ANOVA followed by Tukey post-hoc test.

[0239] Cell migration analysis.

[0240] MDA-MB-231 and MDA-MB-468 cells were subjected to cell migration analysis in Boyden chambers. Each experiment was performed after coating the bottom surface of a 12-well membrane filter (BD Biosciences) with 10 μg / mL fibronectin for 12 hours. Cells were pretreated with 5 μM NO2-OA or NO2-SA for 1 hour and then in culture medium containing 1% FBS in the absence or presence of TNFα (20 ng / mL) for an additional 2 hours. Cells were trypsinized and washed with migration medium (DMEM containing 0.1% fatty acid-free BSA) to remove serum. Then, 10 5Cells at a density of 1000 / well were placed in the upper chamber containing migration medium with the same pretreatment conditions. Cells were allowed to migrate for 5 hours against 5% FBS chemoattractant. Non-migrating cells from the upper surface were removed using a cotton swab. Migrating cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and then stained with 0.5% crystal violet (Sigma-Aldrich) for 15 minutes. The migrated cell density of the filter was observed by microscopy. Crystal violet on the migrated cells was destained with 10% acetic acid and the absorbance of each filter was determined at A573 nm. Images represent three individual experiments and statistical comparison between treatments was determined by one-way ANOVA followed by Tukey post-hoc test.

[0241] Cell invasion assay.

[0242] MDA-MB-468 cells were pretreated with NO2-OA (5 μM), NO2-SA (5 μM), or the NF-κB inhibitor JSH-23 (10 μM) for 1 h, then in the absence or presence of TNFα (20 ng / mL) for an additional 2 h in culture medium containing 1% FBS. The cells were then suspended in migration medium and placed in the upper well of an invasion chamber (EMD Millipore). A chemoattractant (5% FBS) was placed in the lower chamber for 24 h at 37°C to attract the invasive cells. The cells were then harvested and the invasion rate was determined according to the manufacturer's protocol. Three individual experiments were performed and statistical comparisons between treatments were determined by one-way ANOVA followed by Tukey's post-hoc test.

[0243] Luciferase analysis of NF-κB activity.

[0244] Luciferase chemiluminescence-based analysis of NF-κB transcriptional activity was performed. MDA-MB-231 and MDA-MB-468 cells (approximately 70% confluent) in 12-well plates were transiently transfected with NF-κB-luciferase reporter plasmid (Stratagene, La Jolla, CA) using Lipofectamine 3000. After transfection (24 h), cells were pretreated with NO2-OA (5 μM), NO2-SA (5 μM), OA (5 μM), or JSH-23 (20 μM) for 2 h, followed by 20 ng / mL TNFα for an additional 4 h. Each transfection was performed in triplicate. Luciferase activity was measured using a dual luciferase assay kit (Promega). Relative light units (RLU) were measured using a 96-well plate luminometer according to the manufacturer's instructions (Victor II, PerkinElmer Life Sciences). Protein concentrations were determined using the BCA assay (Thermo Fisher Scientific). Data represent the ratio of treated samples to control in the context of RLU / protein content mean + / - SD. Three individual experiments were performed and statistical significance was determined by Kruskal-Wallis test followed by Dunn's post-hoc test with Bonferroni correction for multiple comparisons.

[0245] NO2-FA protein alkylation reaction.

[0246] To determine whether NO2-FA binds to RelA(p65) or IKKβ in TNBC cells, MDA-MB-231 or MDA-MB-468 cells were treated with 5 μM biotinylated (Bt) lipids Bt-NO2-OA, Bt-NO2-SA, or Bt-OA in DMEM containing 5% FBS. After 2 h, cells were harvested in lysis buffer containing 1% Triton-X, 10% glycerol, 150 mM NaCl, 10 mM HEPES, 1 mM EDTA, 1 mM EGTA, and supplemented with a mixture of protease and phosphatase inhibitors (Roche Applied Science). Total cell lysates (0.5–1 mg) were mixed and incubated with streptavidin agarose beads (Sigma-Aldrich) overnight at 4 °C. Beads were washed three times using lysis buffer. After SDS-PAGE, immunoblotting was performed using anti-RelA mouse monoclonal antibody (Santa Cruz Biotechnology) or anti-IKKβ rabbit polyclonal antibody (Cell Signaling). Proteomic analysis of RelA alkylation by NO2-OA was also performed using recombinant RelA protein and LC-MS / MS analysis.

[0247] Immunoprecipitation and NO2-OA-induced RelA protein polyubiquitination.

[0248] To determine the induction level of RelA protein polyubiquitination by NO2-FA, MDA-MB-231 and MDA-MB-468 cells were treated with 0.1% methanol (vehicle), NO2-OA (5 μM), or NO2-SA (5 μM) for 6 h, and then cell lysates were collected in lysis buffer supplemented with a mixture of protease and phosphatase inhibitors. The lysates were clarified by centrifugation at 14,000×g for 10 min. Protein lysates (1 mg) were incubated overnight at 4°C with anti-RelA antibody and protein G / A conjugated agarose beads (EMD Millipore, Bedford, MA). Immunoprecipitated fractions were obtained by centrifugation at 14,000×g for 1 min at room temperature and washed three times with lysis buffer. Immunoprecipitated RelA was resolved by 8% SDS-PAGE gel and transferred to nitrocellulose membranes (Bio-Rad) for immunoblotting probed with anti-ubiquitin antibody (Santa Cruz Biotechnology). Blots were then stripped and probed with anti-RelA antibody to assess the amount of RelA protein pull-down.

[0249] Western blotting.

[0250] Western blotting was performed. 20–60 μg of total lysate per lane was loaded onto 7%, 10%, or 12% SDS-PAGE and transferred to nitrocellulose or polyvinylidene difluoride membranes (Bio-Rad). Membranes were probed with primary antibodies against caspase-3, multidrug resistance protein 1 (MRP1), poly[ADP-ribose] polymerase-1 (PARP-1), ubiquitin, RelA; cyclin D1, p21, caspase-9, MRP4, IKKβ, pIKKβ, IκBα, or pIκBα (Cell Signaling); caspase-8 (R&D Systems). Samples were normalized to β-actin (Sigma-Aldrich) or GAPDH (Trevigen).

[0251] Protein bands were visualized and digitized images were quantified using Image Lab software (Bio-Rad). Immunoblots represent at least three individual experiments. Quantitative results are the average of three or more individual experiments, and statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

[0252] RNA extraction, qPCR, and RT2 Profiler PCR arrays.

[0253] To determine the effect of NO2-OA on the expression of NF-κB target genes in TNFα-induced MDA-MB-231 and MDA-MB-468 cells, cells were pretreated with NO2-OA (5 μM) for 2 h and then stimulated with TNFα (20 ng / mL) for 6 h. Total RNA samples of tissues or cells were extracted using TRIZOL reagent according to the manufacturer's instructions (Invitrogen). Total RNA (1 μg) was reverse transcribed using iScript cDNA kit (Bio-Rad) according to the manufacturer's instructions. cDNA (25 ng) was used for each subsequent quantitative real-time PCR (qRT-PCR) reaction. All qRT-PCR was performed in a StepOne PLUS PCR system (Thermo Fisher Scientific) using TaqMan gene expression assays. Fold changes were calculated using the ΔΔCt method with 18S ribosomal RNA or human β-actin RNA serving as internal controls. Three independent experiments were performed and statistical significance was determined by one-way analysis of variance followed by Tukey's post-hoc test. 2 For the Profiler PCR array, MDA-MB-468 cells were treated or not with NO2-OA (5 μM) for 24 h. Expression of 84 human NF-κB target genes was analyzed using a 96-well plate format as instructed in the manufacturer's handbook (Qiagen). PCR amplification was performed by the StepOne PLUS PCR system, and fold changes in gene expression were calculated according to the manufacturer's instructions.

[0254] Analysis of NO2-OA GSH conjugate (NO2-OA-SG) and NO2-OA in cell culture medium.

[0255] MCF-10A, MDA-MB-231, or MDA-MB-468 cells were cultured in 6-well plates (1 × 10 per well). 6 MCF-10A cells were cultured in 1000 mM NaCl (100 mM NaCl) for 24 h. Prior to treatment, the cell medium was replaced with DMEM containing 5% FBS. NO2-OA (5 μM) was added to the medium and cells were incubated at 37°C for 60 min before collecting the cell culture medium. For MRP1 inhibition studies, MCF-10A cells were pretreated with 1 mM probenecid for 1 h and then co-treated with 5 μM NO2-OA for an additional 1 h. For MRP1 siRNA knockdown studies, MCF-10A cells were transiently transfected with non-targeting siRNA (scrambled) or MRP1 siRNA for 48 h followed by treatment with 5 μM NO2-OA for 1 h. Cells were washed with PBS and then gently scraped off the plate in 1 mL of PBS. 100 μL of the cell suspension was lysed by sonication and used for protein concentration measurement via BCA protein assay. The remaining 0.9 mL of cell suspension was used to determine the amount of intracellular NO2-OA-SG. NO2-OA-SG and free NO2-OA were extracted using a modified Bligh-Dyer method, with NO2-OA-SG partitioning into the polar phase and NO2-OA partitioning into the organic phase. 15Extraction was performed after adding NO2-d4-OA (5 nM) as an internal standard for free NO2-OA. Samples were centrifuged at 2,800 rpm at room temperature for 5 min. The lower (organic) layer was transferred to a clean vial, dried, and reconstituted in methanol before MS analysis. The upper (aqueous) layer containing NO2-OA-SG was desalted and concentrated using a 3 mL C18 SPE column (Thermo Fisher Scientific). The column was preconditioned with one column volume of 100% methanol followed by two column volumes of 5% methanol before sample loading. The sample was vortexed and equilibrated at 4 °C for 5 min before extraction. The sample was washed with two column volumes of 5% methanol and elution was performed with 3 mL of 100% methanol after the column was dried under vacuum for 30 min. The solvent was then evaporated under N2 and the sample was reconstituted in methanol for further analysis.

[0256] GSH and GSSG extraction and analysis.

[0257] MCF-10A, MDA-MB-231, and MDA-MB-468 cells were cultured at 3 × 10 5 Cells were seeded at a density of 1000 cells / well. After overnight culture, cells were treated with 5 μM NO2-OA for the indicated times. At each time point, cell medium was aspirated and washed twice with sterile PBS. Cells were then incubated with PBS containing 25 mM N-ethylmaleimide (NEM) for 15 min at 37°C. Induction solution (50 μL of 15% MeOH, 40 mM HEPES, 50 mM NaCl, 1 mM EDTA, 2 μM [ 13 C2 15 N]-GSH, 2 µM 13 C4 15N2]-GSSG, and 25 mM NEM) were added to each well and incubated at room temperature for 15 min. Then, 50 μL of 10% (w / v) sulfosalicylic acid solution was immediately added to each well to stabilize GSH and GSSG. Supernatants were collected by centrifugation at 15,000 RPM for 10 min at 4°C. Samples were diluted 1:5 in 5% sulfosalicylic acid and 20 μL was injected for HPLC-MS / MS analysis. The number of cells at time 0 was quantified by Hoechst33258 DNA staining assay and expressed as nmol / cell (×10 6 ) were used for normalized GSH or GSSG levels, expressed as

[0258] Liquid chromatography-mass spectrometry (LC-MS / MS). NO2-OA, NO2-OA-SG, GSH, and GSSG were analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS / MS) using a Shimadzu / CTC PAL HPLC coupled to a Sciex5000 triple quadrupole mass spectrometer (Sciex, San Jose, CA). The NO2-OA, NO2-OA-SG gradient solvent system consisted of water + 0.1% acetic acid (solvent A) and acetonitrile + 0.1% acetic acid (solvent B). NO2-OA and its metabolites were resolved using a Luna C18 reversed-phase column (2 mm × 100 mm, Phenomenex, Torrence, CA) at a flow rate of 0.65 mL / min. The sample was applied to the column at 30% B and eluted with a linear increase of solvent B (30 to 100% in 9.7 min). The column was washed with 100% B for 3 min and then returned to the initial conditions for equilibration (2 min). The NO2-OA-SG conjugates were resolved using a Luna C18 reversed-phase column (2 mm x 150 mm, Phenomenex) at a flow rate of 0.25 mL / min. Samples were applied to the column at 20% B, held for 5 min, and eluted with a linear increase in solvent B (20 to 98% solvent B in 20 min), followed by a 4.5 min wash step with 98% B and 4 min restoration to the initial condition. MS analysis for NO2-FA used electrospray ionization in negative ion mode with collision gas set at 5 units, curtain gas at 40 units, ion source gas #1 at 55 units and #2 at 60 units, ion spray voltage at -4500 V, and temperature at 600 °C. The declustering potential was -80 eV, entrance potential at -5, collision energy at -35, and collision exit potential at -3. Multiple reaction monitoring (MRM) was used for the analysis of lipids that exhibit loss of a nitro group (m / z 46) upon collision-induced dissociation. 15MRM: 326.2 / 46 and 331 / 47 for NO2-d4-OA, respectively. The following parameters on the mass spectrometer were used in positive ion mode for the NO2-OA-SG conjugate: Gas #1 was 50 and Gas #2 was 55, ion spray voltage was 5000V, and source temperature was set at 550°C, declustering potential was 70 eV, entrance potential was 5, collision energy was 17, and collision exit potential was 5. The following MRM transitions were used: NO2-OA-SG and 15 635.2 / 506.2 and 640.2 / 511.2 for NO2-d4-OA-SG, respectively. The method for the simultaneous determination of GSH and GSSG involved sample (20 μL) separation on a Phenomenex C18 (2.1×150 mm, 3.5 μ pore size) column. The solvent system used was aqueous 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B) at a net flow rate of 0.6 mL / min. A linear gradient from 2% B to 75% B over 0.1-6.2 min, followed by a 2 min wash with 100% B and a 6 min re-equilibration with 2% B was used for the separation. Unlabeled and 13 C4 15 N2GSSG elutes at 2 min, and unlabeled and 13 C4 15 N GS-NEM eluted at approximately 2.7 min. The Sciex5000 mass spectrometer settings were as follows: CAD 4 units, curtain gas 40 units, GS1 45 units, GS2 50 units, ion spray voltage 5500 V, source temperature 550° C., EP 5 V, and CXP 10 V. Multiple reaction monitoring was performed in positive ion mode. The transitions for each species were as follows: GSH (Q1 308.3→Q3 179.1; declustering potential (DP) 60 V, collision energy (CE) 18.5 V). 13 C4 15 N GSH (Q1 311.3→Q3 182.1; DP 60V, CE 18.5V). GS-NEM(Q1 433.0→Q3 304.2;DP 65V, CE 38V); 13 C4 15N GS-NEM(Q1 436.0→Q3 307.2;DP 65V, CE 38V);GSSG(Q1 613.2→Q3 355.2;DP 60V, CE 24V); 13 C4 15 N GSSG (Q1 619.2 → Q3 361.2; DP 60V, CE 24V). Calibration curves were generated using known GSH and GSSG standards and an isotopic internal standard, showing linearity over 100,000-fold with a quantification limit of 1 nM for both GS-NEM and GSSG. Sample [GSH] and [GSSG] were determined from the analyte:IS area ratio, and intracellular GSH and GSSG were determined as a function of the number of cells (10 6 ) and normalize the result to 10 6 Expressed as nmol of GSH or GSSG per cell.

[0259] Statistical analysis.

[0260] Data analysis was performed using Prism6 software (GraphPad Software). Results are presented as mean ± SD, except for tumor volume, which is presented as mean ± SEM, in Figure 4E. Statistical analysis was performed using Student's t-test, one-way ANOVA, or two-way ANOVA, as appropriate. Statistical significance was achieved at p<0.05.

[0261] Results - Inhibition of triple-negative breast cancer cell survival, migration, invasion, and tumor growth by nitroalkene fatty acids

[0262] NO2-OA inhibits TNBC cell growth and survival.

[0263] The endogenously occurring lipid electrophile NO2-OA and its non-electrophilic control fatty acids (NO2-SA and OA) were evaluated for their effects on normal and cancerous breast ductal epithelial cell growth and signaling responses (Figure 4A). To examine whether NO2-OA preferentially inhibits TNBC cell growth, Hoechst33258 was used to count non-tumorigenic breast epithelial cells (MCF-10A), an ER+ breast cancer cell line (MCF7), and two TNBC cell lines (MDA-MB-231 and MDA-MB-468). Each cell line was treated for 48 h with a range of NO2-OA concentrations (0-15 μM). NO2-OA significantly inhibited the growth of both TNBC cell lines, but did not significantly inhibit the growth of ER+ cells or the growth of MCF-10A cells (Figure 4B, 4C, 4D). The IC50 of NO2-OA was significantly greater in noncancerous MCF-10A (7.7 ± 1.93 μM) and MCF7 (11.61 ± 3.59 μM) cells in contrast to TNBC MDA-MB-231 (2.7 ± 0.11 μM) and MDAMB-468 (1.6 ± 0.11 μM) cells (Figure 4E). In addition to preferential TNBC cell growth inhibition, MTT detection of intact cellular electron transfer mechanisms revealed that NO2-OA also significantly reduced the viability of both MDA-MB-231 and MDA-MB-468 cells, but not MCF7 or MCF-10A cells. Cytotoxicity was not detected in any cell line for up to 24 h at a NO2-OA concentration of 5 μM, typically used for subsequent cell signaling and functional studies whose durations range from 1 to 8 h. The non-electrophilic NO2-SA (Figure 4A), which is structurally related to NO2-OA, had no effect on TNBC cell growth, supporting the attribution of NO2-OA-mediated TNBC cell growth inhibition to the electrophilic nitroalkene moiety.

[0264] NO2-OA reduces MDA-MB-231 xenograft tumor growth.

[0265] Considering that TNBC cell growth and survival are inhibited by NO2-OA, the efficacy of NO2-OA on tumor growth was investigated in a mouse xenograft model of TNBC. MDA-MB-231 cells were injected into the fourth mammary fat pad in the inguinal region of 6-week-old female athymic nude mice. Oral gavage of NO2-OA (7.5 mg / kg / day), NO2-SA (7.5 mg / kg / day), or sesame oil (vehicle control) was initiated, and the mean tumor size was increased to 50–100 mm. 3 The tumor growth rate was continued for 4 weeks after reaching 10 days. In mice treated with NO2-OA, there was significantly reduced tumor growth versus vehicle control and NO2-SA-treated mice at 27 days after treatment (Figure 4F). There was no weight loss in either NO2-OA-treated or control mice over the course of treatment.

[0266] These results support that NO2-OA mediates in vivo growth inhibition of MDA-MB-231 cells without overt toxic effects.

[0267] NO2-OA induces cell cycle arrest and apoptotic cell death in TNBC cells.

[0268] To determine whether the decreased number of cells was due to NO2-OA-induced cell cycle alterations, fluorescence-activated cell sorting analysis (FACS) was performed. NO2-OA significantly increased the percentage of cells in G2 / M phase and decreased the percentage of cells in G0 / G1 phase upon 24 h treatment in MDA-MB-231 and MDA-MB-468 cells (Figure 5A, 5B). Notably, all cell cycle populations (G0 / G1, S, and G2 / M) in MCF-10A cells were unaffected by NO2-OA (Figure 5C). Cell cycle inhibition by NO2-OA was accompanied by increased p21 protein expression and decreased cyclin D1 protein expression in both MDA-MB-231 and MDA-MB-468 cells, but not in MCF-10A cells (Figure 5D). Consistent with the lack of effects on cell growth and survival, NO2-SA did not affect cell cycle populations or the expression levels of cell cycle control proteins in MCF-10A, MDA-MB-231, and MDA-MB-468 cells (Figure 5D). Gene expression levels of cyclin D1 and p21 were also determined by quantitative RT-PCR. NO2-OA downregulated cyclin D1 gene expression and upregulated p21 gene expression after 24 h of treatment in MDA-MB-231 and MDA-MB-468 cells, but not in MCF-10A cells. These results indicate that NO2-OA selectively induced cell cycle arrest in TNBC cells. An increased sub-G1 cell population was evident in both MDA-MB-231 and MDA-MB-468 cells after 24 h of NO2-OA treatment. To determine whether the effect of NO2-OA on sub-G1 cells in TNBC cells was apoptosis-mediated, cleavage of PARP-1 was examined by Western blotting. Treatment with NO2-OA for 24 h promoted caspase-3-mediated cleavage of PARP-1 in MDA-MB-231 and MDA-MB-468 cells (Figure 5E), but not in MCF-10A cells, indicating that NO2-OA preferentially induced TNBC apoptosis via caspase-3 activation. It is also possible that the increase in p21 blocks cell cycle entry into S phase, resulting in an increase in sub-G1 cells.To further explore the apoptotic signaling response to NO2-OA in TNBC cells, activation of initiator caspases (caspase-8 for the extrinsic pathway and caspase-9 for the intrinsic pathway) was analyzed using antibodies that detect both the procaspase and activated (cleaved) forms of these initiator caspases. NO2-OA treatment increased the cleavage of caspase-8 and caspase-9 in both MDA-MB-231 and MDA-MB-468 cells, suggesting that NO2-OA induced apoptosis via both intrinsic (mitochondria-dependent) and extrinsic (death receptor-dependent) apoptotic signaling mechanisms in TNBC cells (Figure 5F). Collectively, these results confirm that NO2-OA selectively regulates cell cycle arrest and apoptosis in TNBC cells versus MCF-10A cells.

[0269] Extracellular NO2-OA-glutathione adduct excretion is associated with MRP1 expression.

[0270] In the intracellular compartment, GSH and its reactive Cys moiety are more abundant than protein thiols, and thus GSH and other low molecular weight thiols are the most important targets for oxidation and alkylation by free radicals, oxidants, and electrophiles. In the case of NO2-OA, which easily diffuses and reaches intracellular compartments and organelle protein targets, a GSH conjugate (NO2-OA-SG) is formed that can be actively transported out of the cell by the GSH conjugate efflux pump MRP1. This phenomenon was further investigated by measuring the concentration of extracellular NO2-OA-SG in the medium of MCF-10A, MDA-MB-231, and MDA-MB-468 cells after 1 h treatment with 5 μM NO2-OA. In contrast to NO2-OA-SG released by MCF-10A cells, there was a significantly lower level of NO2-OA-SG transported into the medium of both MDA-MB-231 and MDA-MB-468 cells (Figure 6A). This 4-5-fold difference in extracellular NO2-OA-SG levels produced by MCF-10A and TNBC cells prompted us to compare the relative extent of MRP1 protein expression and GSH and GSSG content in TNBC and noncancerous cell lines. Western blot analysis detected MRP1 protein expression in MCF-10A cells, whereas MRP1 was undetectable in both TNBC cell lines (Figure 6B). MRP4 mRNA was detected at low levels in all three cell types, but protein expression was not evident by Western blotting.

[0271] MRP1 affects NO2-OA bioactivity in MCF-10A cells.

[0272] Two strategies, namely, the use of the organic anion transport inhibitor probenecid, which is often used as an MRP inhibitor, and knockdown of MRP1 by siRNA, facilitated the investigation of the role of MRP1 in the cellular response to NO2-OA. Both probenecid and MRP1 siRNA knockdown (knockdown efficiency of about 70%) enhanced the intracellular levels of NO2-OASG adducts in MCF-10A cells (Figure 6C-D). Notably, probenecid also significantly enhanced MCF-10A cell growth inhibition by NO2-OA (Figure 6E). The IC 50 (7.23 ± 0.15 μM) was reduced two-fold in MCF-10A cells pretreated with probenecid compared to treatment with NO2-OA alone (14.23 ± 1.05 μM) (Figure 6F). Furthermore, probenecid increased the extent of NO2-OA-induced cell cycle arrest in MCF-10A cells, as reflected by increased p21 levels and a concomitant decrease in cyclin D1 expression (Figure 6G). Probenecid also enhanced NO2-OA-induced apoptosis in MCF-10A cells in the context of increased caspase-3 activation and PARP-1 cleavage (Figure 6H). These observations are consistent with both the intracellular concentration and cell growth / survival signaling actions of NO2-OA being influenced by the extent of NO2-OA's reaction with GSH and the subsequent transport of NO2-OA-SG by MRP1.

[0273] GSH and GSSG respond to NO2-OA in MCF-10A cells versus TNBC cells.

[0274] LC-MS quantification of GSH and GSSG after 0–12 h of treatment with 5 μM NO2-OA revealed that basal GSH levels (19.3 ± 1.9 nmol / 10 6 cells) was found to be significantly lower than the basal GSH level (8.3 ± 0.8 nmol / 10 6 cells) and MDA-MB-468 cells (12.9 ± 0.5 nmol / 10 6The results revealed that the GSSG levels at time 0 (Fig. 7A) were approximately 1.5-fold greater in MCF-10A cells than in MDA-MB-231 cells, resulting in an initial GSH:GSSG ratio of 82 ± 16 compared to 653 ± 68 in MDA-MB-231 cells and 2003 ± 163 in MDA-MB-468 cells. MCF-10A cells maintained their GSH:GSSG ratio over the first 6 h after NO2-OA treatment, whereas the GSH:GSSG ratio decreased rapidly in MDA-MB-231 and MDA-MB-468 cells due to the decreased GSH concentration. Collectively, the data in Figures 6 and 7 support that there may be an expected greater interaction between NO2-OA and cellular protein targets in TNBC cells due to the more favorable pharmacokinetics (greater intracellular concentrations and longer t0.5) conferred by lower GSH concentrations and the inhibition of NO2-OA-SG transport by the MRP1-deficient TNBC cell phenotype. In MCF-10A cells, NO2-OA can be more readily glutathionylated and transported, thus limiting the interaction with signaling pathway proteins.

[0275] NO2-OA inhibits TNFα-induced TNBC cell migration and invasion.

[0276] Inflammatory stimuli such as TNFα induce responses in the tumor microenvironment that promote TNBC tumor metastasis and invasion. Because electrophilic NO2-FA mediates anti-inflammatory signaling effects, we evaluated the effect of NO2-OA on TNFα-induced TNBC cell migration. Boyden chamber migration analysis showed that TNFα increased the migration of both MDA-MB-231 and MDA-MB-468 cells (Figure 8A, images 3 and 8) compared to basal conditions (Figure 8A, images 2 and 7). NO2-OA significantly inhibited both MDA-MB-231 and MDA-MB-468 cell migration induced by TNFα (Figure 8A, images 4 and 9; Figure 5, B and C). NO2-OA moderately inhibited basal, i.e., unstimulated, migration of MDA-MB-231 and MDA-MB-468 cells (Figure 8, B and C). Next, cells were placed into transwell permeable supports coated with Matrigel for invasion assays to evaluate the potential effect of NO2-OA on the invasive phenotype of TNBC cells. TNFα-induced invasion was significantly inhibited by NO2-OA treatment of MDA-MB-468 cells, whereas a non-electrophilic control fatty acid (NO2-SA) exerted a minor effect on tumor cell invasion (Figure 8D). The inhibitory effect of NO2-OA on MDA-MB-468 invasion was compared to the cellular response to the NF-κB inhibitor JSH-23, which inhibits the nuclear translocation of RelA subunits. Similar to JSH-23, NO2-OA inhibited TNFα-induced invasion in MDA-MB-468 cells (Figure 8D).

[0277] NO2-OA inhibits TNFα-induced NF-κB transcriptional activity in TNBC cells.

[0278] The inhibition of MDA-MB-468 cell invasion by JSH-23 (Figure 8D) suggests that NO2-OA may also inhibit TNFα-induced breast cancer cell migration due to its ability to inhibit NF-κB signaling. To test this idea, we examined the effect of NO2-OA on TNFα-activated NF-κB transcriptional activity in TNBC cells. MDA-MB-231 and MDA-MB-468 cells were transiently transfected with an NF-κB luciferase reporter plasmid and treated with 5 μM NO2-OA for 2 h, followed by activation with 20 ng / mL TNFα for 4 h. In addition to NO2-OA, the non-electrophilic lipid controls NO2-SA (5 μM) and OA (5 μM) were also examined. NO2-OA significantly inhibited NF-κB-dependent transcription of luciferase in both TNBC cell lines compared to TNFα alone, whereas NO2-SA and OA had no effect. Furthermore, the degree of inhibition of NF-κB-dependent luciferase expression by NO2-OA was comparable to that induced by the NF-κB inhibitor JSH-23 (20 μM) (Fig. 9, A and B). These data indicate that the electrophilic reactivity of NO2-OA is responsible for the inhibition of TNFα-induced NF-κB transcriptional activity in TNBC cells.

[0279] NO2-OA inhibits NF-κB-regulated gene expression associated with TNBC tumor metastasis.

[0280] Inhibition of NF-κB transcriptional activity by NO2-OA suggested that the expression of metastasis-related downstream target genes may be decreased. To investigate this, important NF-κB target genes were assessed via RT2 Profiler PCR array analysis in MDA-MB-468 cells treated with NO2-OA (5 μM) for 24 h. The expression levels of NF-κB target genes regulated by NO2-OA were compared to MDA-MB-468 untreated control cells. The data revealed that treatment with NO2-OA reduced the mRNA expression of numerous NF-κB target genes, including ICAM-1 and uPA, two critical mediators of tumor progression and metastasis (Figure 9C). TNFα induces the expression of both ICAM-1 and uPA in MDAMB-231 cells (37, 38). To more directly examine whether NO2-OA suppresses TNFα-induced expression of ICAM-1 and uPA in TNBC cells, MDA-MD-231 or MDA-MD-468 cells were treated with 5 μM NO2-OA and 20 ng / mL TNFα. Simultaneous treatment with either NO2-OA or RelA siRNA resulted in suppression of TNFα-induced expression of ICAM-1 and uPA genes in TNBC cells (Figures 9D, 9E; 9G, 9H). The effects of NO2-OA and RelA siRNA on RelA-dependent target gene expression were further evaluated by qRT-PCR (Figures 9F; 9I). RelA mRNA levels were suppressed by RelA siRNA treatment but not by NO2-OA. Both NO2-OA and RelA siRNA inhibited gene expression of TNFα-induced ICAM-1 and uPA gene expression via an NF-κB-dependent mechanism. To determine whether NO2-OA suppresses TNFα-induced metastasis-induced ICAM-1 and uPA gene expression during cell migration, transcript levels of ICAM-1 and uPA genes were assessed in MDA-MB-468 cells studied in Boyden chamber migration assays (Figure 8C).Under these conditions, NO2-OA significantly inhibited TNFα-induced expression of ICAM-1 and uPA in migrating tumor cells, again supporting that NO2-OA inhibited the expression of NF-κB-regulated genes involved in metastasis.

[0281] NO2-OA suppresses TNFα-induced IKKβ / IκBβ signaling in TNBC.

[0282] To better define the mechanism responsible for the inhibition of TNFα-activated NF-κB signaling by NO2-OA, MDA-MB-231 or MDA-MB-468 cells were pretreated with NO2-OA (5 μM) or the IKK inhibitor BAY11-7082 (10 μM) for 2 h prior to TNFα stimulation (20 ng / mL, 5 min).

[0283] TNFα-induced IKKβ phosphorylation was reduced by both NO2-OA and BAY11-7082 (Figure 10A). Both NO2-OA and BAY11-7082 also inhibited the degradation of IκBβ after TNFα stimulation (20 ng / mL, 10 min) (Figure 10B). Furthermore, decreased IκBβ phosphorylation occurred in cells pretreated with NO2-OA or BAY11-7082 and the proteasome inhibitor MG-132 (10 μM) (Figure 7C). This indicates that NO2-OA suppresses TNFα-induced IKKβ phosphorylation and IκBβ degradation, and these actions inhibit downstream NF-κB signaling in TNBC cells.

[0284] NO2-OA alkylates IKKβ and RelA proteins.

[0285] Cys179, located in the activation loop of IKKβ, is a target for oxidation and electrophile alkylation reactions. Because NO2-OA suppresses TNFα-induced phosphorylation of IKKβ and IκBβ in TNBC cells (Figure 10A, 10C), we investigated the possibility that NO2-OA directly modifies IKKβ. Biotinylated lipids (Bt-NO2-OA, Bt-NO2-SA, and Bt-OA) were synthesized to facilitate affinity capture-mediated measurements of NO2-OA and control fatty acid addition to IKKβ. MDA-MB-231 or MDA-MB-468 cells were treated with 5 μM Bt-NO2-OA, Bt-NO2-SA, or Bt-OA for 2 h, and then all alkylated proteins were pulled down from whole-cell lysates using streptavidin-conjugated beads. Western blotting revealed that IKKβ was pulled down by Bt-NO2-OA, but not by a nonelectrophilic control fatty acid (Fig. S10D). Similarly, Bt-NO2-OA, but not the control fatty acid, promoted the pull-down of NF-κB RelA (Fig. S11A). NO2-OA inhibited LPS-induced NF-κB transcriptional activity, which was partially the result of alkylation of RelA Cys38 and inhibition of RelA DNA binding. LC-MS / MS proteomic analysis showed that RelA Cys105 was also alkylated by NO2-OA, but the functional significance of the alkylation of RelA Cys105 by NO2-OA was unclear. Overall, Bt-NO2-OA promoted the pull-down of IKKβ and RelA, and direct proteomic analysis revealed alkylation of RelA by NO2-OA. These observations highlight that NO2-OA mediates PTMs that inhibit multiple aspects of proinflammatory NF-κB signaling.

[0286] NO2-OA stimulates proteasomal degradation of RelA protein.

[0287] NF-κB proteolysis contributes to the termination of NF-κB signaling. Thiol alkylating and nitrosating agents induce the degradation of NF-κB subunit p50 via PTM of Cys62 in both HT29 and HCT116 tumor cell lines. Because NO2-OA covalently attaches to RelA in both MDA-MB-231 and MDA-MB-468 cells (Figure 11A), we investigated the consequences of PTM by NO2-OA on RelA protein stability. To validate this putative mechanism, we first examined whether endogenous RelA protein expression responds to NO2-OA. MDA-MB-231, MDA-MB-468, and MCF-10A cells were treated with 5 μM NO2-OA or control lipids (NO2-SA and OA) for 24 h. NO2-OA decreased the abundance of RelA in TNBC cells, whereas NO2-SA and OA had no effect (Figure 11B). In contrast, RelA protein levels in MCF-10A cells were not altered by NO2-OA (Figure 11B). In all three cell lines, RelA mRNA levels were not altered by NO2-OA. These data support that NO2-OA affects RelA protein stability through alkylation of RelA in TNBC cells. RelA is controlled by ubiquitin- and proteasome-dependent degradation signals that govern NF-κB activation. To determine whether RelA modification by NO2-OA induces ubiquitination of endogenous RelA in TNBC cells, MDA-MB-231 or MDA-MB-468 cells were treated with 5 μM NO2-OA or NO2-SA for 5 h. RelA protein was immunoprecipitated, and its polyubiquitination was detected by anti-ubiquitin. NO2-OA, but not NO2-SA, promoted polyubiquitination of RelA in both TNBC cell lines (Fig. S11C), indicating that NO2-OA interacts with RelA in TNBC cells and destabilizes the RelA protein by promoting its ubiquitination and proteasomal degradation.

[0288] Structure-function relationship of electrophilic nitro fatty acid regioisomers in inhibition of HDR and lethality against TNBC cell lines.

[0289] Computational analysis of the crystal structure of Rad51 suggests that changing the position of the nitroalkene substituent in the nitroalkene fatty acid closer to the C-terminus enhances Rad51 protein binding via hydrogen bond stabilization of the carboxyl group adjacent to the Cys319 residue. Based on this analysis, we designed 7-NDA (7-NO2-nonadec-7-enoic acid). 7-NDA has a lower IC compared to NO2-OA. 50 , and shows a better combination index when combined with talazoparib (Figure 12).

[0290] The data show significantly reduced tumor volume in TNBC xenografted mice treated with nitroalkene fatty acids in combination with PARPi compared to either NFA or PARPi alone. 6 MDA-MB-231 cells were orthotopically injected into the mammary fat pad of nude mice. Tumor volumes were measured over time by caliper in mice treated with OA+vehicle (n=8), NO2-OA+vehicle (n=7), OA+talazoparib (0.3 mg / kg) (n=10), or NO2-OA (15 mg / kg)+talazoparib (Figure 14).

[0291] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be considered as limiting the scope of the invention. The present invention provides, for example, the following items. (Item 1) Subjects who have, are suspected of having, or are at risk for developing cancer, or are in remission from cancer, (a) a therapeutically effective amount of at least one compound (a) selected from: (a)(i) a nitroalkene fatty acid; (a)(ii) an unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between said electron-withdrawing group and said leaving group; (a)(iii) a thiolated nitro fatty acid; or (a)(iv) a dicarboxylic acid compound containing an electron-withdrawing group; a therapeutically effective amount of at least one anti-neoplastic agent (b); and The cancer is a cancer with a genetic etiology of defects in DNA repair genes, a cancer with a high frequency of natural genomic instability, a cancer that is treated with a DNA damaging agent, or a cancer that is treated with a combination of a DNA damaging agent and immunotherapy. method. (Item 2) 2. The method of claim 1, wherein the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, ovarian cancer, brain cancer, or skin cancer. (Item 3) The method of item 1, wherein the cancer is triple-negative breast cancer. (Item 4) The cancer may be fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, or bronchogenic carcinoma. 2. The method of claim 1, wherein the cancer is renal cell carcinoma, hepatocarcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, or lymphoma. (Item 5) 5. The method according to any one of items 1 to 4, wherein the anti-neoplastic agent is a poly(ADP-ribose) polymerase inhibitor. (Item 6) 6. The method of claim 5, wherein the cancer is triple negative breast cancer and the subject is negative for gBRCAm. (Item 7) 6. The method of claim 5, wherein the subject is resistant to single-agent treatment with a poly(ADP-ribose) polymerase inhibitor. (Item 8) 5. The method according to any one of items 1 to 4, wherein the anti-neoplastic agent is a DNA damaging agent or a DNA damaging treatment. (Item 9) 5. The method according to any of items 1 to 4, wherein the anti-neoplastic agent is doxorubicin, cisplatin, olaparib, rucaparib, niraparib, talazoparib, veliparib, camptothecin, or irradiation treatment. (Item 10) 1. A method comprising administering to a subject having, suspected of having, or at risk for developing triple-negative breast cancer, or in remission of triple-negative breast cancer, a therapeutically effective amount of at least one compound (a) selected from (a)(i) a nitroalkene fatty acid, (a)(ii) an unsaturated fatty acid having an electron-withdrawing group, a leaving group, and a carbon-carbon double bond disposed between the electron-withdrawing group and the leaving group, (a)(iii) a thiolated nitro fatty acid, or (a)(iv) a dicarboxylic acid compound containing an electron-withdrawing group. (Item 11) 11. The method according to any one of items 1 to 10, wherein the compound (a) is a RAD51 inhibitor. (Item 12) 12. The method according to any one of items 1 to 11, wherein the compound (a) is the nitroalkene fatty acid. (Item 13) Item 13. The method according to item 12, wherein the nitroalkene fatty acid is a compound containing at least one carbon-carbon double bond and at least one nitro group. (Item 14) The nitroalkene fatty acid has Formula I:

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Claims

1. A composition for use in treating a subject having, suspected of having, at risk of developing, or in remission from cancer, said composition comprising at least one anti-neoplastic agent (b); the composition is co-administered to the subject with 7-NO 2 -nonadec-7-enoic acid, 5-NO 2 -eicosa-5-enoic acid, (6-NO 2 -octadec-6-enoic acid) or 8-NO 2 -nonadec-7-enoic acid; The composition, wherein the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, ovarian cancer, brain cancer, or skin cancer.

2. The composition for use according to claim 1, wherein the cancer is triple-negative breast cancer.

3. The composition for use according to claim 1 or 2, wherein the anti-neoplastic agent is a poly(ADP-ribose) polymerase inhibitor.

4. The composition for use according to claim 1 or 2, wherein the anti-neoplastic agent is a DNA damaging agent.

5. 3. The composition for use according to claim 1 or 2, wherein the anti-neoplastic agent is doxorubicin, cisplatin, olaparib, rucaparib, niraparib, talazoparib, veliparib, camptothecin, or an irradiation treatment agent.

6. The compound is 7-NO ２ The composition for use according to any one of claims 1 to 4, wherein the hydroxyl group is -nonadeca-7-enoic acid.

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