Mitochondrial ATP inhibitors targeting the gamma subunit prevent metastasis

Abstract

High ATP production by mitochondrial ATP synthase is a novel therapeutic target for anticancer therapy, especially for preventing tumor progression. A mitochondrial-associated gene signature for metastasis, characterized by the gamma subunit of mitochondrial ATP synthase (ATP5F1C), is described. Knockdown of ATP5F1C expression significantly reduces ATP production, 3D anchorage-independent growth, and cell migration. Administration of bedaquiline, or a bedaquiline derivative moiety bearing a TPP moiety, downregulates ATP5F1C expression in vitro and prevents spontaneous metastasis in vivo. Mitochondrial ATP5F1C is a promising novel biomarker and molecular target for future drug development for the prevention of metastatic disease progression.

Description

[Technical field]

[0001] The present disclosure relates to the inhibition of mitochondrial ATP to prevent or reduce the likelihood of cancer stem cells (CSCs) metastasis. [Background technology]

[0002] Researchers are working to develop new anti-cancer treatments. Traditional cancer therapies (e.g., radiation, alkylating agents such as cyclophosphamide, and antimetabolites such as 5-fluorouracil) attempt to selectively detect and eradicate fast-growing cancer cells by interfering with cellular mechanisms involved in cell growth and DNA replication. Other cancer therapies use immunotherapy (e.g., monoclonal antibodies) that selectively bind mutant tumor antigens to fast-growing cancer cells. Unfortunately, these therapies often result in tumor recurrence at the same site or at one or more different sites, indicating that not all cancer cells have been eradicated. Recurrence may be due to insufficient chemotherapy doses and / or the emergence of cancer clones resistant to the therapy. Thus, new cancer treatment strategies are needed.

[0003] Advances in mutation analysis have enabled detailed study of genetic alterations that occur during cancer progression. Despite knowledge of the genomic landscape, modern oncology has had difficulty identifying primary driver mutations across cancer subtypes. This harsh reality appears to be due to the fact that each patient's tumor is unique and a single tumor may contain multiple distinct clonal cells. What is needed, therefore, are new approaches that focus on the commonalities between different cancer types. Targeting metabolic differences between tumor and normal cells is a promising novel cancer treatment strategy. Analysis of transcriptional profiling data from human breast cancer samples revealed the elevation of more than 95 mRNA transcripts associated with mitochondrial biogenesis and / or mitochondrial translation (Non-Patent Document 1). Furthermore, more than 35 of the 95 upregulated mRNAs encode mitochondrial ribosomal proteins (MRPs). Similarly, proteomic analysis of human breast cancer stem cells revealed significant overexpression of multiple mitoribosomal proteins and other proteins associated with mitochondrial biogenesis (Non-Patent Document 2).

[0004] Mitochondria are highly dynamic organelles that constantly divide, elongate, and connect with each other to form tubular networks or fragmented granules to meet cellular requirements and adapt to the cellular microenvironment. The balance between mitochondrial fusion and fission determines mitochondrial morphology, abundance, function, and spatial distribution, and thus influences many important mitochondria-dependent biological processes, such as adenosine triphosphate (ATP) production, mitophagy, apoptosis, and calcium homeostasis. And mitochondrial dynamics can be regulated by mitochondrial metabolism, respiration, and oxidative stress.

[0005] ATP is the universal bioenergetic "currency" of all living cells and tissues, including microorganisms such as prokaryotic bacteria and eukaryotic yeast. In eukaryotes, the mitochondrial organelle functions as the cellular "powerhouse." Mitochondria generate large amounts of ATP through the TCA cycle and oxidative phosphorylation (OXPHOS), while glycolysis contributes small amounts of ATP. Conversely, mitochondrial dysfunction induces ATP depletion, leading to mitochondrial-driven apoptosis (programmed cell death) and / or necrosis. Thus, we propose that ATP depletion therapy could be a viable strategy to target and eradicate even the "fittest" cancer cells.

[0006] In MCF7 breast cancer cells, under normoxic conditions, mitochondrial-driven OXPHOS contributes 80-90% of ATP production, while glycolysis contributes the remaining 10-20%. Thus, like normal cells, cancer cells are highly dependent on mitochondrial ATP production. However, it remains largely unknown whether ATP levels in cancer cells contribute to 3D anchorage-independent growth and cell migration, two hallmarks of metastatic spread. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sotgia et al., Cell Cycle, 11(23):4390‐4401 (2012) [Non-Patent Document 2] Lamb et al., Oncotarget, 5(22):11029‐11037 (2014) Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present disclosure is to identify novel therapeutic targets for anti-cancer therapy, in particular for preventing tumor progression. Another object of the present disclosure is to identify compounds with anti-cancer activity, in particular for preventing and / or reducing the likelihood of tumor recurrence and / or metastasis. Another object of the present disclosure is to provide companion diagnostics related to said therapeutic targets.

[0009] In view of the above background, it is an object of the present disclosure to describe therapeutic agents that can be used to eradicate CSCs, particularly CSCs that are responsible for tumor recurrence and / or metastasis. It is a further object of the present disclosure to describe compositions, such as pharmaceutical compositions, and methods for the treatment and prevention of cancer, including tumor recurrence and / or metastasis. [Means for solving the problem]

[0010] Bedaquiline (known as Sirturo) is an FDA-approved antibiotic in clinical use for the treatment of drug-resistant tuberculosis. Although it was initially thought to affect only mycobacterial ATP synthase, our studies have shown that it also potently inhibits yeast and human mitochondrial ATP synthase. High-resolution cryo-EM studies have shown that bedaquiline directly binds to the gamma subunit (ATP5F1C) that forms the axis of rotation of mitochondrial ATP synthase, which is critically involved in torque transmission and ultimately provides the mechanochemical energy required for ATP synthesis.

[0011] As shown herein, binding of bedaquiline to ATP5F1C leads to degradation of ATP5F1C in living cells. Bedaquiline induces downregulation of ATP5F1C protein expression accompanied by mitochondrial ATP depletion in both a time-dependent and concentration-dependent manner. Furthermore, ATP depletion induced by bedaquiline treatment effectively blocks spontaneous metastasis in vivo without significant toxicity in non-tumorigenic human cells (MCF10A) or chicken embryos.

[0012] In the present approach, the gamma subunit of mitochondrial ATP synthase (ATP5F1C) is identified as a novel therapeutic target for ameliorating aggressive cancer cell behavior, including tumor recurrence and / or metastasis.

[0013] Described herein is the use of the FDA-approved drug (1R,2S)-1-(6-bromo-2-methoxyquinolin-3-yl)-4-(dimethylamino)-2-naphthalen-1-yl-1-phenylbutan-2-ol, also known as bedaquiline, and its derivatives, to induce ATP depletion in cancer cells. In laboratory experiments, bedaquiline effectively induced ATP depletion in MDA-MB-231 breast cancer cells. For example, eight days of treatment with bedaquiline was sufficient to prevent the initiation of spontaneous metastasis in an in vivo xenograft model without affecting tumor growth. Bedaquiline and certain of its derivatives specifically target ATP5F1C, the gamma subunit of mitochondrial ATP synthase. This targeting is consistent with ATP5F1C being a functional biomarker and therapeutic target for the prevention of metastasis.

[0014] Also described herein are bedaquiline derivatives having a triphenylphosphonium (TPP) moiety. These derivative compounds induce ATP depletion in CSCs and effectively prevent and / or reduce the likelihood of tumor recurrence and / or metastasis. The bedaquiline derivatives are more potent than bedaquiline, selective for CSCs, and non-toxic to normal healthy cells.

[0015] The approach of the present invention can also be used to treat and / or prevent tumor recurrence and / or metastasis. Anti-cancer treatments often fail due to tumor recurrence or metastasis, especially after surgery. It is understood that the mitochondrial activity of CSCs is at least partially responsible for the cause of these treatment failures. Embodiments of the approach of the present invention can be used in situations where conventional cancer therapy fails and / or in conjunction with or prior to anti-cancer treatment to prevent or reduce the likelihood of treatment failure due to tumor recurrence and / or metastasis.

[0016] Some embodiments of the inventive approach may take the form of a method for treating or preventing at least one of tumor recurrence and metastasis in a subject, the method comprising administering to the subject a pharma- ceutical effective amount of bedaquiline or a bedaquiline derivative having a TPP moiety. Various bedaquiline derivatives are disclosed.

[0017] In some embodiments, the approach of the present invention can take the form of a method for preventing and / or reducing the likelihood of tumor metastasis and tumor recurrence in a patient. A cancer biological sample can be obtained from the patient. The levels of biomarkers of the ATP-associated metastasis gene signature consisting of ABCA2, ATP5F1C, COX20, NDUFA2 and UQCRB in the biological sample can be determined and compared to a threshold level. If the determined levels exceed the threshold level, a pharmacologic effective amount of a composition containing bedaquiline or a bedaquiline derivative having a TPP moiety can be administered. [Brief description of the drawings]

[0018] [Figure 1A] FIG. 1A shows a heatmap comparing the transcriptional profiles of ATP-related genes (OXPHOS and ATP-related transporters). [Figure 1B] 1B and 1C show the volcano plots of the GSE2034 GEO dataset and the GSE59000 GEO dataset, respectively. [Figure 1C]1B and 1C show the volcano plots of the GSE2034 GEO dataset and the GSE59000 GEO dataset, respectively. [Figure 1D] FIG. 1D is a Venn diagram showing the intersection of the two GEO datasets. [Figure 1E] FIG. 1E is a table of genes correlated with ATP5F1C. [Figure 2A] Figures 2A-2C are KM plots for ER(+) relapse-free survival (RFS), ER(+) distant metastasis-free survival (DMFS), and ER(+) LN-negative tamoxifen-treated RFS, respectively. [Figure 2B] Figures 2A-2C are KM plots for ER(+) relapse-free survival (RFS), ER(+) distant metastasis-free survival (DMFS), and ER(+) LN-negative tamoxifen-treated RFS, respectively. [Figure 2C] Figures 2A-2C are KM plots for ER(+) relapse-free survival (RFS), ER(+) distant metastasis-free survival (DMFS), and ER(+) LN-negative tamoxifen-treated RFS, respectively. [Diagram 3] FIG. 3 shows a Venn diagram of proteins in the MCF7 and T47D datasets and includes a table of upregulated ATP-related proteins in each dataset. [Figure 4] FIG. 4 shows Western blot analysis of MDA-MB-231 cells stably transduced with lentiviral vectors encoding shRNA targeting ATP5F1C in the Tet-On system. [Figure 5A] 5A-5D show the results of ATP5F1C knockdown on ATP production, cell migration, and 3D anchorage-independent growth. [Figure 5B]5A-5D show the results of ATP5F1C knockdown on ATP production, cell migration, and 3D anchorage-independent growth. [Figure 5C] 5A-5D show the results of ATP5F1C knockdown on ATP production, cell migration, and 3D anchorage-independent growth. [Figure 5D] 5A-5D show the results of ATP5F1C knockdown on ATP production, cell migration, and 3D anchorage-independent growth. [Figure 6] FIG. 6 shows Western blot analysis of MDA-MB-231 2D cell monolayers treated with various concentrations of bedaquiline over time. [Figure 7] FIG. 7 compares the fold change (mean signal) of BioTracker ATP-Red between control and bedaquiline-treated MDA-MB-231 cells. [Figure 8A] 8A and 8B show the time course of monolayer growth of MDA-MB-231 and MCF10A cells, respectively, treated with 10 μM bedaquiline. [Figure 8B] 8A and 8B show the time course of monolayer growth of MDA-MB-231 and MCF10A cells, respectively, treated with 10 μM bedaquiline. [Figure 9A] FIG. 9A shows the results of a tumor formation assay for different concentrations of bedaquiline (0.1, 1.0, and 10 μM). [Figure 9B] FIG. 9B shows the percentage of cells in each stage of the cell cycle in MDA-MB-231 cells treated with bedaquiline. [Figure 9C] Figures 9C and 9D show representative FACS traces for control and bedaquiline-treated cells, respectively. [Figure 9D] Figures 9C and 9D show representative FACS traces for control and bedaquiline-treated cells, respectively. [Figure 9E] FIG. 9E shows representative images of migration of MDA-MB-231 cells treated with bedaquiline, and FIG. 9F depicts said migration compared to controls. [Figure 9F] FIG. 9E shows representative images of migration of MDA-MB-231 cells treated with bedaquiline, and FIG. 9F depicts said migration compared to controls. [Figure 10A] 10A-10C show cell cycle populations for MDA-MB-231 cells treated with bedaquiline (1 and 10 μM) or vehicle alone after 48, 72, and 120 hours, respectively. [Figure 10B] 10A-10C show cell cycle populations for MDA-MB-231 cells treated with bedaquiline (1 and 10 μM) or vehicle alone after 48, 72, and 120 hours, respectively. [Figure 10C] 10A-10C show cell cycle populations for MDA-MB-231 cells treated with bedaquiline (1 and 10 μM) or vehicle alone after 48, 72, and 120 hours, respectively. [Figure 10D] 10D-10F show the results of live / dead cell analysis. MDA-MB-231 cells were treated with bedaquiline (1 and 10 μM) or vehicle alone for 48, 72, and 120 hours and then subjected to live / dead analysis by FACS. [Figure 10E] 10D-10F show the results of live / dead cell analysis. MDA-MB-231 cells were treated with bedaquiline (1 and 10 μM) or vehicle alone for 48, 72, and 120 hours and then subjected to live / dead analysis by FACS. [Figure 10F] 10D-10F show the results of live / dead cell analysis. MDA-MB-231 cells were treated with bedaquiline (1 and 10 μM) or vehicle alone for 48, 72, and 120 hours and then subjected to live / dead analysis by FACS. [Figure 10G] FIG. 10G shows the results of Western blot analysis of the effects of bedaquiline (0, 0.1, 1, and 10 μM) on PARP and p21 protein expression in MDA-MB after 120 hours of treatment. [Figure 11A] FIG. 11A shows the timeline of the CAM assay. [Figure 11B]FIG. 11B shows the number of viable eggs for each treatment in the CAM assay, and FIG. 11C shows the same data as survival rate. [Figure 11C] FIG. 11B shows the number of viable eggs for each treatment in the CAM assay, and FIG. 11C shows the same data as survival rate. [Figure 11D] FIG. 11D shows the average tumor weight after each treatment, and FIG. 11E shows the relative amount of metastases for each treatment compared to the control. [Figure 11E] FIG. 11D shows the average tumor weight after each treatment, and FIG. 11E shows the relative amount of metastases for each treatment compared to the control. [Figure 12] FIG. 12 shows the results of a tumor formation assay for bedaquiline and two bedaquiline derivatives bearing a TPP moiety. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The following description describes embodiments of the inventive approach in sufficient detail to enable one to practice the inventive approach. Although the inventive approach is described with reference to these specific embodiments, it is understood that the inventive approach can be embodied in different forms, and this description should not be construed as limiting any appended claims to the specific embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the inventive approach to those skilled in the art.

[0020] This description uses various terms that should be understood by a person of ordinary skill in the art. The following explanations are provided to avoid any misunderstanding.

[0021] The term "cancer" refers to a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes benign and malignant cancers. Examples of cancer include several types of carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial sarcoma), neuroendocrine tumors (carcinoid tumors, gastrinomas including but not limited to islet cell tumors), sarcomas, Schwannomas (including acoustic neuromas), medullary carcinomas, adenocarcinomas, melanomas, and leukemia or lymphocytic tumors. Specific examples of cancer include bladder cancer, squamous cell carcinoma, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous epithelial cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal or stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, reproductive tract cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, head and neck cancer, and multiple myelomas.

[0022] As used herein, the term "tumor" refers to the growth and proliferation of neoplastic cells, whether benign or malignant, including pre-cancerous and cancerous cells and tissues.

[0023] The term "metastasis" refers to the spread of cancer from its primary location to other parts of the body. Cancer cells can break away from the primary tumor, invade lymphatic and blood vessels, circulate in the bloodstream, and grow or "metastasize" as separate lesions in normal tissues elsewhere in the body. Multiple metastases can be local or distant. Metastasis is a series of processes that require tumor cells to break away from the primary tumor, travel through the bloodstream, and arrest at a distant site. At this new site, the cells establish a blood supply and grow, potentially forming a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cells control this behavior, and interactions between tumor cells and host cells at distant sites are also important.

[0024] The terms "treat", "treated", "treating" and "treatment" include reduction or amelioration of at least one symptom associated with or caused by the condition, disorder or disease being treated, particularly cancer. In certain embodiments, treatment involves reducing and / or ameliorating at least one symptom associated with or caused by the cancer being treated with the compounds of the invention. In some embodiments, treatment involves causing the death of a category of cells, e.g., CSCs, which may be involved in metastasis or recurrence, of a particular cancer in the host's body, which can be achieved by preventing further spread of cancer cells, e.g., by depriving these cells of energy production mechanisms, and / or by inhibiting CSC function. For example, treatment can include reduction of one or more symptoms of cancer, or complete eradication of cancer. As another example, the approach of the present invention can be used to inhibit mitochondrial metabolism in cancer, eradicate CSCs in cancer (killing at a rate faster than the rate of proliferation), eradicate TICs in cancer, eradicate circulating tumor cells in cancer, inhibit proliferation of cancer, target and inhibit CSCs, target and inhibit circulating tumor cells, prevent or reduce the likelihood of metastasis, prevent recurrence, sensitize cancer to chemotherapeutic agents, sensitize cancer to radiation therapy, sensitize cancer to phototherapy.

[0025] In the context of tumor recurrence and / or metastasis, the terms "prevent" and "reduce the likelihood of" refer to reducing the presence of CSCs, TICs, and circulating tumor cells that are likely involved in recurrence or metastasis in a subject to a level such that the likelihood of tumor recurrence and / or metastasis from the primary site is lower compared to a control (i.e., in the absence of a treatment to prevent or reduce the likelihood of tumor recurrence and / or metastasis). In practice, the treatments described herein for preventing and / or reducing the likelihood of tumor recurrence and / or metastasis target and inhibit or eradicate CSCs, TICs, and inhibit circulating tumor cells.

[0026] The terms "cancer stem cell" and "CSC" refer to a subpopulation of cancer cells within a tumor that have the capacity for self-renewal, differentiation, and tumor formation when transplanted into an animal host. Compared to "bulk" cancer cells, CSCs have a larger mitochondrial mass, enhanced mitochondrial biogenesis, and more active mitochondrial protein translation. As used herein, a "circulating tumor cell" is a cancer cell that sheds from the primary tumor into the vasculature or lymphatics and is carried throughout the body in the blood circulation. The CellSearch Circulating Tumor Cell Test can be used to detect circulating tumor cells.

[0027] As used herein, the phrase "pharmaceutical effective amount" refers to the amount that needs to be administered to a host or to a cell, tissue, or organ of a host to achieve a therapeutic result, such as regulating, modulating, or inhibiting protein kinase activity, e.g., inhibiting the activity of a protein kinase, or treating cancer. A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required for a given subject, using methods known and available in the art. For example, a physician or veterinarian can start the dose of the compound of the present invention contained in the pharmaceutical composition at a level below that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0028] As used herein, the phrase "active compound" refers to bedaquiline or a bedaquiline derivative compound described herein, which may include pharma- ceutically acceptable salts or isotopic analogs thereof. It is to be understood that one or more active compounds may be administered to a subject by any suitable approach, as known to one of ordinary skill in the art. It is also to be understood that the amount of active compound and the timing of its administration may depend on the individual subject being treated (e.g., age and weight, among other factors), the method of administration, the pharmacokinetic properties of the particular active compound or compounds, and the judgment of the prescribing physician. Thus, due to subject-to-subject variability, any dosages described herein are intended to be initial guidelines, and a physician may titrate the dose of the compound to achieve the treatment he or she deems appropriate for the subject. In considering the degree of treatment desired, a physician may balance a variety of factors, such as the age and weight of the subject, the presence of pre-existing disease, and the presence of other diseases. As described in more detail below, pharmaceutical formulations can be prepared for any desired route of administration, including but not limited to oral, intravenous, or aerosol administration.

[0029] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Some examples of materials that can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, sucrose, etc.; (2) starches, such as corn starch, potato starch, etc.; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, etc.; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter, suppository wax, etc.; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.; (10) glyceryl stearate, ... (11) glycols such as propylene glycol; (12) esters such as ethyl oleate, ethyl laurate, and the like; (13) agar; (14) buffers such as magnesium hydroxide, aluminum hydroxide, and the like; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffered saline; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0030] As used herein, the term "derivative" is a chemical moiety that is derived from or synthesized from a referenced chemical moiety. For example, compounds according to the inventive approach may be referred to as bedaquiline derivatives and have a TPP moiety conjugated at the 6-bromo position or at the dimethylamino.

[0031] As used herein, a "triphenylphosphonium" or "TPP" moiety refers to a phenylphosphonium (e.g., Cl - Or Br- The phosphorus quaternary lipophilic cation P, in its salt with + and three phenyl groups, which may be substituted or unsubstituted.

[0032] [ka]

[0033] Due to the ability of TPP cations to accumulate in mitochondria when conjugated to small molecules, they can be used to target small molecule therapeutics to mitochondria, it being understood that not all TPP conjugates behave in this manner, and some conjugates only increase mitochondrial uptake, if at all, compared to the unconjugated therapeutic.

[0034] Bedaquiline and certain bedaquiline derivatives bearing a TPP moiety can be used to selectively eradicate CSCs for the treatment and / or prevention of tumor recurrence and / or metastasis. Bedaquiline is a drug currently used to treat active tuberculosis, particularly multi-drug resistant tuberculosis, and is FDA approved. Mechanistically, bedaquiline blocks the proton pump of mycobacterial ATP synthase. Cellular energy production is dependent on ATP production, and loss of ATP production results in inhibition of mycobacterial growth.

[0035] As described herein, in vitro experiments have shown that bedaquiline also targets mitochondrial ATP synthase in malignant mammalian cells, reducing the rate of tumor recurrence and metastasis. In the present approach, tumor recurrence and / or metastasis can be treated and / or prevented by administering a pharmacologic effective amount of bedaquiline or a bedaquiline derivative having a TPP moiety as described herein to a subject with cancer. For example, in some embodiments for treating an adult subject, 400 mg of bedaquiline may be administered in tablet form once a day for 1-2 weeks, followed by 600 mg of bedaquiline, also in tablet form, three times a week for another 1-2 weeks. This exemplary dose is currently used to treat multi-drug resistant tuberculosis in adults. The pharmacologic effective amount of bedaquiline used to treat and / or prevent tumor recurrence and / or metastasis in the present approach may vary depending on the subject (e.g., age, weight, health, etc.) as is known in the art. In embodiments of the inventive approach involving bedaquiline derivatives having a TPP moiety, the pharmacologic effective amount used for the treatment and / or prevention of tumor recurrence and / or metastasis will be less than the pharmacologic effective amount of bedaquiline. Determination of the pharmacologic effective amount is considered to be within the ordinary level of skill of one of ordinary skill in the art upon review of this disclosure. It is understood that the treatment cycle may be the same or similar to the treatment cycle for the treatment of tuberculosis, or may differ depending on the particular embodiment, the bedaquiline derivative used, and other factors known in the art.

[0036] The present inventors developed the present approach by first exploring the role of ATP synthesis in cancer cells and then analyzing the impact of inhibition of ATP production in cancer cells. After identifying target genes for ATP inhibition, the present inventors evaluated various compounds for ATP inhibitory activity using various assays, such as tumorigenesis assay and chicken chorioallantoic membrane (CAM) assay. The following paragraphs describe the bioinformatics analysis of the role of ATP synthesis. This discussion highlights the importance of ATP5F1C, the gamma subunit of mitochondrial ATP synthase, in cancer metastasis. The ATP5F1C gene encodes a subunit of mitochondrial ATP synthase. Mitochondrial ATP synthase catalyzes ATP synthesis using the electrochemical gradient of protons across the inner membrane during oxidative phosphorylation. The catalytic portion of mitochondrial ATP synthase consists of five different subunits (α, β, γ, δ, ε). This gene encodes the gamma subunit of the catalytic core.

[0037] We have determined using a bioinformatics approach that mitochondrial ATP synthesis is a key determinant of 3D anchorage-independent growth and metastasis. As a first step, GEO transcriptional profiling datasets were analyzed to compare 2D, 3D, and in vivo tumor growth of MDA-MB-231 cells, a triple-negative breast cancer (TNBC) cell line. Heatmaps were generated that highlight that ATP-related genes were transcriptionally upregulated under both 3D growth conditions (anchor-independent and in vivo tumors), all compared to 2D adherent growth.

[0038] FIG. 1A shows a heatmap comparing the transcriptional profiles of ATP-related genes (OXPHOS and ATP-related transporters) using the GSE36953 GEO profiling dataset previously deposited in the NCBI database. Heatmaps are typically used in molecular biology to represent the levels of expression of a large number of genes across a large number of comparable samples (e.g., cells in different states, samples from different patients) obtained from DNA microarrays. Heatmaps are usually displayed in color, with green shading indicating negative log fold change (FC) values ​​and red shading indicating positive log FC values. The lighter the shading, the greater the log FC. Due to PCT rule 11.13, FIG. 1A is displayed in black and white. In the color version of FIG. 1A, green is displayed primarily in the first column, which is for 2D cells, and red shading is displayed primarily in the second and third columns, which are for 3D and xenograft cells. In Figure 1A, lighter shading represents a larger absolute value of log FC, and it can be seen that the 2D cells in the first row showed negative log FC. Total RNA was prepared from MDA-MB-231 cells, a TNBC cell line, under three different growth conditions: 2D adherent growth, 3D anchorage-independent growth, and in vivo tumor growth. Analysis was performed using Affymetrix Human Genome U133 Plus 2.0 Arrays. Heatmaps were generated using QIAGEN OmicSoft Suite Software. Heatmap scale bars of -4<log FC>+4 are shown. Note that under both 3D growth conditions (anchor-independent tumors and in vivo tumors), all ATP-related genes were transcriptionally upregulated compared to 2D adherent growth.

[0039] Transcriptional expression of ATP-related genes (OXPHOS and ATP-related transporters) was examined in two separate GEO datasets associated with human breast cancer metastasis, revealing ATP-related genes that serve as biomarkers for metastasis. Figures 1B, 1C show volcano plots for the GSE2034 GEO dataset and the GSE59000 GEO dataset, respectively. These types of plots are typically in color, similar to the heatmaps described above. In black and white, shading to the left of 0 represents green (indicating negative correlation) and shading to the right of 0 represents red (indicating positive correlation). GSE2034 compares breast cancer metastasis to no breast cancer metastasis. GSE59000 compares breast cancer metastasis to primary breast cancer tumors. These volcano plots were generated by examining the annotations present in OncoLand Metastatic Cancer (QIAGEN OmicSoft Suite) and performing a functional "core analysis" using Ingenuity Pathway Analysis Software (IPA; QIAGEN) on genes annotated with an uncorrected p-value cutoff of <0.05. Note that in both GEO datasets, the transcriptional profile of ATP-related genes (OXPHOS and ATP-related transporters) was increased and specifically associated with metastasis.

[0040] Figure 1D is a Venn diagram showing the intersection of the two GEO datasets, resulting in a five-member ATP-related translocation gene signature consisting of ABCA2, ATP5F1C, COX20, NDUFA2 and UQCRB. Figure 1E is a table of genes correlated with ATP5F1C. Most notably, ATP5F1C (also known as ATP5C1) encodes the gamma subunit of the soluble F1 catalytic core of complex V, the mitochondrial ATP synthase.

[0041] In bona-fide breast cancer metastatic lesions, transcriptional expression of ATP5F1C positively correlates with the co-expression of: i) five metastasis marker genes (EPCAM, MKI67, RRP1B, VCAM1, CXCR4); ii) four cell cycle regulatory genes (CDK1, CDK2, CDK4, CDK6); and iii) eleven cancer stem cell (CSC) marker genes (CDH1, ALDH2, ALDH1BA1, ALDH9A1, SOX2, VIM, CDH2, ALDH7A1, ALDH1B1, CD44, ALDH3B2, listed in ranked order of statistical significance). Tables 1-12 below show the expression data of various gene groups correlated with ATP5F1C. As can be seen, the transcriptional expression of ATP5F1C is also positively correlated with the co-expression of mitochondrial complexes I–V, transcripts encoded by mt‐DNA, as well as the other three members of the five-member translocation gene signature described above, namely UQCRB, COX20, and NDUFA2.

[0042] [Table 1] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 1. Complex I gene transcripts correlated with ATP5F1C

[0043] [Table 2] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 2. Complex II gene transcripts correlated with ATP5F1C

[0044] [Table 3] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 3. Complex III gene transcripts correlated with ATP5F1C

[0045] [Table 4] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 4. Complex IV gene transcripts correlated with ATP5F1C

[0046] [Table 5] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 5. Complex 5 gene transcripts correlated with ATP5F1C

[0047] [Table 6] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 6. Total ATP gene transcripts correlated with ATP5F1C

[0048] [Table 7] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 7. mt-DNA gene transcripts correlated with ATP5F1C

[0049] [Table 8] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 8. ABC gene transcripts correlated with ATP5F1C

[0050] [Table 9] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 9. Breast cancer stem cell marker gene transcripts correlated with ATP5F1C

[0051] [Table 10] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 10. Metastasis marker gene transcripts correlated with ATP5F1C

[0052] [Table 11] Correlated Gene Cytoband Spearman's... Spearman's correlation coefficient p-Value p-value q-Value Table 11. Cell cycle gene transcripts correlated with ATP5F1C

[0053] Similarly, expression of two members of this metastatic gene signature, namely ATP5F1C and UQCRB, was associated with increased maximal oxygen uptake (VO 2max ), and functionally correlated with a high percentage of (mitochondrial-rich) Type 1 fibers in human skeletal muscle tissue. ATP5F1C expression in skeletal muscle is also significantly increased after exercise training, reflecting improved muscle health in patients. Conversely, ATP5F1C levels decline with age and are reduced in progeria patients. These results strongly suggest that high ATP5F1C expression is a biomarker of increased mitochondrial ATP production at the cellular level.

[0054] Using Kaplan‐Meier (K‐M) analysis, ATP5F1C was determined to be a prognostic biomarker for distant metastasis and tumor recurrence, especially in ER(+) patients who were node‐negative at diagnosis and treated with tamoxifen (HR(RFS) = 2.77; P = 3.4E‐06; N = 471). Figures 2A–2C show KM plots for ER(+) recurrence‐free survival ("RFS"), ER(+) distant metastasis‐free survival ("DMFS"), and ER(+) LN‐negative tamoxifen‐treated RFS, respectively. Hazard ratios (HRs) are shown in these figures. To perform K‐M analysis for ATP5F1C, publicly available microarray data from breast cancer patients were interrogated using an open‐access online survival analysis tool. This approach allowed for direct in silico validation of ATP5F1C as a marker for tumor recurrence.

[0055] Furthermore, using existing GEO datasets, we determined that ATP-related genes and OXPHOS genes are transcriptional biomarkers for breast cancer circulating tumor cells (CTCs) in patients. Using a heatmap of ATP-ABC and OXPHOS genes from GSE55470, we determined that high ATP content in CTCs may be useful as a biomarker for identifying and tracking CTCs in whole blood, which may improve cancer diagnosis and prevent metastatic spread.

[0056] We also reviewed existing proteomic profiling data comparing 2D monolayers with 3D tumor-like masses of two distinct ER(+) breast cancer cell lines, namely MCF7 and T47D. Overall, 21 ATP-related proteins out of 1519 common proteins of these two cell lines were found to be upregulated in the 3D tumor-like masses in both data sets. Figure 3 shows a Venn diagram of proteins in both data sets, including a table of upregulated ATP-related proteins in each data set. From these 21 ATP-related proteins, seven subunits of mitochondrial ATP synthase were detected, including ATP5F1B, ATP5F1C, ATP5IF1, ATP5MG, ATP5PB, ATP5PD, and ATP5PO. Figure 3 shows a Venn diagram comparing the proteomic profiles of two ER(+) breast cancer cell lines (MCF7 and T47D) under 2D adherent and 3D anchorage-independent growth conditions. The shared ATP-related gene products (OXPHOS and ATP-related transporters) are listed below the Venn diagram. Of the 1519 common proteins, 21 ATP-related proteins were found to be upregulated in both data sets. The proteomic data were explored by performing a functional "core analysis" using Ingenuity Pathway Analysis Software (IPA; QIAGEN).

[0057] Taken together, our bioinformatic analysis confirms that increased mitochondrial ATP synthesis may be a key driver of 3D anchorage-independent growth and metastasis. Consistent with this, we determined that targeted knockdown of ATP5F1C protein expression with shRNA inhibited ATP production, cell migration, and 3D anchorage-independent growth.

[0058] We identified ATP5F1C as a clinical biomarker for cancer metastasis. To further validate its functional relevance as a potential therapeutic target, we used an shRNA approach to inducibly downregulate the expression of ATP5F1C by utilizing the Tet-On system integrated into a single lentiviral vector. Figure 4 shows a Western blot analysis of MDA-MB-231 cells stably transduced with lentiviral vectors encoding shRNAs targeting ATP5F1C in the Tet-On system. Three different shRNA constructs (a, b, c) were tested. In parallel, MDA-MB-231 cells were also transduced with an shRNA control vector. After 48 hours of shRNA induction by treatment with doxycycline (10 μM), the levels of ATP5F1C were assessed by Western blot assay. Note that only shRNA construct c showed inducible downregulation of ATP5F1C expression levels, as indicated by the light grey box around the results for +DOX. As a result, only cells transduced with construct c were used for further experiments compared to the shRNA control.

[0059] Using this approach, Figure 4 shows that expression of ATP5F1C is successfully downregulated in an inducible manner using low levels of doxycycline. Importantly, using this genetic approach to eliminate expression of ATP5F1C, we show that loss of ATP5F1C is sufficient to phenotypically inhibit i) ATP production, ii) cell migration, and iii) 3D anchorage-independent growth.

[0060] Figures 5A-5D show the results of ATP5F1C knockdown on ATP production, cell migration, and 3D anchorage-independent growth. First, Figure 5A compares BioTracker ATP-Red fold change (mean signal) between control and ATP5F1C knockdown. Induced downregulation of ATP5F1C reduces ATP levels by approximately 45% compared to shRNA control. Unpaired t-test, **p<0.005. Figure 5B shows representative images of cell migration after 16 hours for both control and ATP5F1C knockdown. Figure 5C shows data as a percentage of control. As can be seen, induced downregulation of ATP5F1C blocks cell migration by approximately 65% ​​compared to shRNA control. MDA-MB-231 cells were cultured in the presence of doxycycline (10 μM) for 32 hours and transferred to Transwell in the presence of doxycycline for 16 hours. Unpaired t-test, **p<0.005. Figure 5D shows the results of the tumor formation assay. As can be seen, knockdown of ATP5F1C inhibits 3D anchorage-independent growth. Induced downregulation of ATP5F1C blocks 3D tumor formation by approximately 50% compared to shRNA control. Unpaired t-test, ***p<0.0005.

[0061] The above data demonstrate that ATP5F1C is a suitable target for the treatment of tumor recurrence and metastasis. The inventors have determined that targeting ATP5F1C with bedaquiline or specific conjugates of bedaquiline prevents ATP production, cell migration, 3D anchorage-independent growth, and metastasis in vivo. Bedaquiline (structure [1] below) is an FDA-approved antibiotic conserved for the treatment of multidrug-resistant tuberculosis (TB). It is further described in U.S. Patent No. 7,498,343, issued March 3, 2009, and U.S. Patent No. 8,546,428, issued October 1, 2013, which are incorporated herein by reference in their entireties. Chemically, bedaquiline mechanistically inhibits mycobacterial ATP synthase. However, the studies described herein highlight that bedaquiline also specifically binds to and potently inhibits the activity of human mitochondrial ATP synthase. Ultrastructurally, detailed cryo-EM studies showed that the binding site of bedaquiline involves direct contact with the gamma subunit of mitochondrial ATP synthase, namely the C-ring (ATP5G1 / 2 / 3), which is in close contact with ATP5F1C.

[0062] [ka]

[0063] Bedaquiline binds to ATP5F1C in situ, inducing protein degradation. Figure 6 shows Western blot analysis of MDA-MB-231 2D cell monolayers treated with bedaquiline (0, 0.1, 1, and 10 μM) at four time points (24, 48, 72, and 120 hours of incubation). Note that the level of ATP5F1C protein expression was persistently reduced, especially at 10 μM bedaquiline, compared to the vehicle alone (DMSO) control. These results indicate that expression of ATP5F1C was downregulated in response to bedaquiline treatment. This effect was both time- and concentration-dependent.

[0064] Furthermore, the loss of expression of ATP5F1C induced by bedaquiline treatment resulted in up to 75% depletion of mitochondrial ATP. Figure 7 compares BioTracker ATP-Red fold change (mean signal) between control and bedaquiline-treated MDA-MB-231 cells. As can be seen, bedaquiline reduces mitochondrial ATP production. Note that bedaquiline significantly inhibited mitochondrial ATP production in MDA-MB-231 cells in a time-dependent manner at a concentration of 10 μM, as assessed using BioTracker ATP-Red 1 to specifically detect mitochondrial ATP levels. A maximum inhibition of 75% was observed at 120 hours of treatment. Data in Figure 7 are based on two-way ANOVA, Sidak multiple comparison test, *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

[0065] Bedaquiline-induced ATP depletion also inhibited the growth of MDA-MB-231 cells in 2D monolayers, but had no effect on MCF10A cells, a non-tumorigenic human breast epithelial cell line. Figures 8A and 8B show the monolayer growth of MDA-MB-231 and MCF10A cells, respectively, treated with 10 μM bedaquiline over time. At a concentration of 10 μM, bedaquiline effectively inhibits the 2D growth of MDA-MB-231 cells in a time-dependent manner. No effect was observed in MCF10A, which is considered a normal control human breast epithelial cell line. Unpaired t-test, *p<0.05, **p<0.005. This indicates that treatment with bedaquiline is selective for tumorigenic cells.

[0066] Furthermore, bedaquiline-induced ATP depletion also inhibited 3D anchorage-independent growth and cell migration of MDA-MB-231 cells, and bedaquiline treatment was sufficient to induce cell death, likely by blocking cell cycle progression, acting at the level of S-phase. Figure 9A shows the results of the tumor formation assay for different concentrations of bedaquiline (0.1, 1.0, and 10 μM). The inhibition of 3D tumor formation was concentration-dependent, and at a concentration of 10 μM, bedaquiline effectively blocked 3D tumor formation in MDA-MB-231 cells by approximately 65%. One-way ANOVA, Dunnett's multiple comparison test, **p<0.005, ***p<0.0005.

[0067] ATP depletion by bedaquiline also inhibits DNA synthesis and induces cell death. Figure 9B shows the percentage of cells in each phase of the cell cycle in MDA-MB-231 cells treated with bedaquiline. After 120 h of treatment, there was a 2-fold decrease in MDA-MB-231 cells in S phase, accompanied by a 2-fold increase in the Sub-G0 to G1 population. Two-way ANOVA, Sidak's multiple comparison test, ns = not significant, ***p < 0.0005. Figures 9C and 9D show representative FACS traces for control and bedaquiline-treated cells, respectively.

[0068] Treatment with bedaquiline also inhibits cell migration. Figure 9E shows a representative image of migration of MDA-MB-231 cells treated with bedaquiline, and Figure 9F represents the migration compared to the control. As can be seen, bedaquiline blocks MDA-MB-231 cell migration by approximately 50%. MDA-MB-231 cells were cultured in the presence of bedaquiline (10 μM) for 32 h and transferred to Transwell in the presence of bedaquiline (10 μM) for 16 h. Unpaired t-test, **p<0.005.

[0069] Further cell cycle analysis shows that bedaquiline treatment of MDA-MB-231 cells significantly reduces the population of cells in S phase and increases cell death in a dose- and time-dependent manner. Figures 10A-10C show the cell cycle populations for MDA-MB-231 cells treated with bedaquiline (1 and 10 μM) or vehicle alone after 48, 72, and 120 hours, respectively. Cell cycle analysis was performed using FACS. No effect was detected at 48 hours. However, at 72 and 120 hours, a decrease in the S phase population and a concomitant increase in the Sub-G0 to G1 population can be seen in Figures 10B, 10C. Two-way ANOVA, Sidak's multiple comparison test, ns=not significant, *p<0.01, **p<0.001, ***p<0.0005.

[0070] Figures 10D-10F show the results of live / dead cell analysis. MDA-MB-231 cells were treated with bedaquiline (1 and 10 μM) or vehicle alone for 48, 72, and 120 h and then subjected to live / dead analysis by FACS. No effect was detected at 48 h. However, at 72 and 120 h (Figures 10E, 10F), a decrease in the live cell population and an increase in the dead cell population can be seen. However, there was no increase in the apoptotic cell population (early or late), suggesting that cell death was due to necrosis. Two-way ANOVA, Sidak's multiple comparison test, ns=not significant, *p<0.01, **p<0.001, ***p<0.0005.

[0071] Figure 10G shows the results of Western blot analysis of the effect of bedaquiline (0, 0.1, 1, and 10 μM) on PARP and p21 protein expression in MDA-MBs after 120 hours of treatment. Note that PARP and p21 were reduced in a concentration-dependent manner. β-actin was used as a control for equal loading.

[0072] Next, to test the in vivo activity, the well-established CAM assay was used with MDA-MB-231 cells to measure the effects of bedaquiline on tumor growth, spontaneous metastasis, and embryotoxicity. Figure 11A shows the CAM assay timeline. 6 An inoculum of MDA-MB-231 cells was added to the CAM of each egg (day E9), after which the eggs were randomly divided into groups. Tumors were detectable on day E10 and were treated daily for 8 days with vehicle alone (1% DMSO in PBS) or bedaquiline. Eight days after drug administration, on day E18, all tumors were weighed and the lower part of the CAM was harvested to assess the number of metastatic cells analyzed by qPCR using primers specific for human Alu sequences. Treatment tolerability was assessed by scoring the number of live and dead chick embryos before each drug administration.

[0073] FIG. 11B shows the number of surviving eggs for each treatment, and FIG. 11C shows the same data as survival rate. The data show that bedaquiline treatment was not toxic to chick embryos at all concentrations tested in vivo. FIG. 11D shows the average tumor weight after each treatment. Bedaquiline had no statistically significant effect on tumor growth. In sharp contrast, however, bedaquiline dose-dependently inhibited spontaneous metastasis, with inhibition maximal at 84% at 120 μM treatment. FIG. 11E shows the relative amount of metastasis for each treatment compared to control. The above results show that pharmacological targeting of mitochondrial ATP synthase by bedaquiline can selectively prevent metastasis of tumor cells by inducing ATP depletion without causing toxicity.

[0074] It should be understood that bedaquiline is available in salt form (e.g., bedaquiline fumarate) and free base form, the latter of which may be more effective than the former in inducing mitochondrial ATP depletion in CSCs. Further evaluation of the relative efficacy and minimum inhibitory concentrations is ongoing.

[0075] The data discussed above are for unconjugated bedaquiline compounds. It should be noted that bedaquiline can be conjugated with a TPP moiety to increase mitochondrial uptake and, as a result, increase the inhibitory strength of the compound. This TPP moiety allows the therapeutic agent to more effectively penetrate the mitochondrial membrane and accumulate in cellular mitochondria. Furthermore, the increased metabolism of CSCs causes the bedaquiline-TPP conjugates to show preference for CSCs over normal, healthy cells. Initial evaluation indicates that the efficacy of the bedaquiline derivatives described herein is improved by 10% to over an order of magnitude when compared to unconjugated bedaquiline compounds. For example, initial evaluation of bedaquiline derivatives having structures [2B] and [3B] described below indicates that these bedaquiline derivatives are at least 2-5 times more potent than unconjugated bedaquiline in inducing ATP depletion, inhibiting the formation of tumor-like masses, and inhibiting spontaneous metastasis.

[0076] In general, the TPP moiety can be attached to the carboxylic acid ester via a linker and can be added as a substituent to any available position of the quinoline of the bedaquiline structure. Preferably, the TPP moiety is attached to the 6 carbon of the quinoline. For example, in some embodiments, the TPP moiety can be conjugated via a linking group by substitution of a bromine in the quinoline portion of the structure. The following general structure [2A] shows bedaquiline with a TPP moiety at the 6 carbon position, where m and n are each independently an integer between 0 and 16; A is O, N, or S; and X is a halogen or any other pharma- ceutically acceptable anion.

[0077] [ka]

[0078] In some preferred embodiments, n and m may be independently 3 to 16, more preferably 4 to 14, and even more preferably 4 to 10. Structure [2B] shown below illustrates one preferred embodiment where n is 4, m is 11, A is O, and X is Br, whereby the TPP moiety is conjugated to bedaquiline via a carboxylate linker. The name of structure [2B] in IUPAC nomenclature is 12-[4-[3-[(1R,2S)-4-(dimethylamino)-2-hydroxy-2-(1-naphthyl)-1-phenyl-butyl]-2-methoxy-6-quinolyl]butoxy]-12-oxo-dodecyl]-triphenyl-phosphonium bromide.

[0079] [ka]

[0080] In some embodiments, the TPP moiety can be conjugated to any available position of the quinoline moiety of the structure by an alkyl linker via a carbonate, carbamate, or urea. The general structure [3A] shown below shows bedaquiline with a TPP moiety attached to the 6 carbon position of the quinoline, where m and n are integers from 0 to 16, independently of each other; A and B are O, N, or S, or absent, independently of each other; and X is a halogen or any other pharma- ceutically acceptable anion.

[0081] [ka]

[0082] Structure [3B] shown below depicts one preferred embodiment where n is 4, m is 5, A is O, B is absent (and thus also resembles the product of general structure [2A]), and X is Cl, whereby the TPP moiety is conjugated to bedaquiline, also referred to herein as a "bedaquiline derivative having a TPP moiety."

[0083] [ka]

[0084] Structure [3B] exhibits significant improvements over unconjugated bedaquiline by at least 2-5 fold in the properties of ATP inhibition, inhibition of 3D tumor formation, and reduction in the relative amount of metastasis (i.e., as determined by the CAM assay). The results of a comparative tumor formation assay are shown in Figure 12 and discussed below.

[0085] It should be understood that the approach of the present invention is not limited to the linkers shown above. Some embodiments can take the form of a compound having the general structure [4] shown below, where R is any C aryl group terminating in a TPP moiety on one end and a carboxyl terminus on the other end, forming a covalent bond with a heteroatom on the linker as a carboxyl amide, ester, or thioester. 1 ~C 9 It may be alkyl, alkylcycloalkyl, cycloalkyl, alkylaryl, aryl, alkylheteroaryl, heteroaryl, alkylheterocyclo, heterocyclo.

[0086] [ka] LINKER

[0087] The linker may be as described above. Alternatively, the linker may be selected from the following non-exclusive examples:

[0088] [ka]

[0089] wherein m and n are, independently of each other, integers from 0 to 16. In such embodiments, the carboxyl connecting the TPP moiety and the alkyl chain can react with a heteroatom on the linker to form an ester, amide, or thioester, as the case may be.

[0090] It is understood that the conjugate compounds described herein can be synthesized from bedaquiline using common synthetic techniques known in the art. An exemplary synthetic scheme starting from bedaquiline is set forth below.

[0091] [ka] Dioxane Intermediate 1

[0092] The synthesis of intermediate 1, (1R,2S)-1-[6-[4-[tert-butyl(dimethyl)silyl]oxybutyl]-2-methoxy-3-quinolyl]-4-(dimethylamino)-2-(1-naphthyl)-1-phenyl-butan-2-ol was confirmed; LC-MS 663.7 [M+H] + , RT 5.57 minutes.

[0093] [ka] Intermediate 1 Intermediate 2

[0094] The synthesis of intermediate 2, ((1R,2S)-4-(dimethylamino)-1-[6-(4-hydroxybutyl)-2-methoxy-3-quinolyl]-2-(1-naphthyl)-1-phenyl-butan-2-ol, was confirmed; LC-MS 549.2 [M+H] + , RT 4.62 minutes.

[0095] [ka] Intermediate 2

[0096] The above exemplary synthesis scheme gives structure [3B], which has the IUPAC nomenclature name [6-[4-[3-[(1R,2S)-4-(dimethylamino)-2-hydroxy-2-(1-naphthyl)-1-phenyl-butyl]-2-methoxy-6-quinolyl]butoxy]-6-oxo-hexyl]-triphenyl-phosphonium; chloride. Synthesis of structure [3B] was confirmed; LC-MS 907.6 [M+H] + , RT 4.33 minutes.

[0097] Early evaluation of compounds of structures [2B] and [3B] has shown that these bedaquiline derivatives are at least five times more potent than unconjugated bedaquiline in ATP depletion, inhibition of tumor formation, and inhibition of spontaneous metastasis. FIG. 12 shows the results of a tumor formation assay comparing bedaquiline with bedaquiline derivatives of structures [2B] and [3B] at concentrations ranging from 0.5 μM to 10 μM. As can be seen, the bedaquiline derivatives with the TPP moiety are more potent than unconjugated bedaquiline at all concentrations tested. Both of these bedaquiline derivatives showed superior tumor formation inhibition at higher concentrations, with several-fold increases compared to unconjugated bedaquiline.

[0098] Other ATP synthase inhibitors that can be used in this approach include resveratrol, trans-resveratrol, quercetin, piceatannol, Bz423 (also known as 7-chloro-1,3-dihydro-5-(4-hydroxyphenyl)-1-methyl-3-(2-naphthalenylmethyl)-2H-1,4-benzodiazepin-2-one), and Gboxin (oxidative phosphorylation inhibitor, also known as 2-ethyl-1-methyl-3-[2-[[(1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl]oxy]-2-oxoethyl]-1H-benzimidazolium).

[0099] Although the data disclosed herein are primarily based on breast cancer cell lines, the compounds of the present approach have efficacy against other types of cancer. In previous studies, the inventors have demonstrated that mitochondrial biogenesis inhibitors successfully inhibit tumorsphere formation in a wide range of cell lines from multiple tumor types. Table 12 below lists cancer cell lines that have been shown to be sensitive to mitochondrial biogenesis inhibitors. This indicates that a wide range of cancer types are highly dependent on ATP for growth, including tumor recurrence and metastasis. Therefore, it is expected that inhibiting ATP production in CSCs of these types of cancers will inhibit the formation of tumor-like masses, thereby preventing and / or reducing tumor recurrence and / or metastasis. Therefore, based on these results, the present approach is effective against many types of cancer.

[0100] [Table 12] Table 12. Inhibition of mitochondrial biogenesis is effective against a wide range of cancer types.

[0101] The following paragraphs describe the materials and assays used to generate the data described herein. It is understood that one of ordinary skill in the art can perform the same assays described herein and / or use other assays known in the art to evaluate the physical, chemical, and pharmaceutical properties of the compounds described herein.

[0102] Bioinformatics Analysis: Unbiased label-free proteomics comparing 2D monolayers and 3D tumor-like masses was performed using MCF7 and T47D breast cancer cell lines. It is understood that other cell lines may be used without departing from the approach of the present invention. Informatics analysis was performed using a variety of publicly available GEO datasets (GSE36953; GSE2034; GSE59000; GSE55470) related to 3D growth, metastasis, and circulating tumor cells (CTCs) archived in the NCBI database. Gene expression profiling data was extracted from these GEO datasets. Heat maps were generated using QIAGEN OmicSoft Suite Software. Volcano plots were generated by inspecting annotations present in OncoLand Metastatic Cancer (QIAGEN OmicSoft Suite). Additionally, functional "core analysis" was performed on annotated genes using Ingenuity Pathway Analysis Software (IPA; QIAGEN). Gene co-expression profiles were extracted from The Metastatic Breast Cancer Project Provisional (2020) using cBioPortal (https: / / www.cbioportal.org / ) and mRNA expression profiling (RNA Seq V2 RSEM) was performed by RNA sequencing of metastatic breast cancer samples from 146 patients.

[0103] Kaplan-Meier (K-M) analysis was performed for ATP5F1C. Publicly available microarray data from up to 3,951 breast cancer patients were examined using an open access online survival analysis tool. For this purpose, data from ER(+) patients were analyzed. Biased array data were excluded from the analysis. This allowed identifying ATP5F1C (also known as ATP5C1) as an important prognostic marker. Hazard ratios were calculated with the best automatically selected cutoffs, p-values ​​were calculated using the log-rank test, and plotted in R. K-M curves were generated online using the K-M plotter (as high-resolution TIFF files) and univariate analysis: https: / / kmplot.com / analysis / index.php?p=service&cancer=breast. This approach allowed us to directly perform in silico validation of ATP5F1C as a marker of tumor recurrence (RFS, recurrence-free survival) and distant metastasis (DMFS, distant metastasis-free survival). For all these analyses, we utilized the latest 2020 version of the database.

[0104] Reagents and model cell lines: It should be clear that other cell lines may be used without departing from the approach of the present invention. BioTracker ATP-Red 1 (#SCT045) was purchased from Merck-Millipore, Inc. MDA-MB-231, a triple-negative human breast cancer cell line, was obtained from the American Type Culture Collection (ATCC). MCF10A cells, a non-tumorigenic human breast epithelial cell line, were also obtained from ATCC. MDA-MB-231 cells were cultured in DMEM (high glucose) supplemented with 10% Fetal Bovine Serum (FBS, Sigma Aldrich), 2 mM GlutaMAX (Gibco, Life Technologies, Waltham, MA, USA), and 1% penicillin / streptomycin (Gibco, Life Technologies). The MCF10A cell line was maintained in mammary epithelial cell growth medium (MEGM; Lonza, Basel, Switzerland) supplemented with 0.4% bovine pituitary extract (BPE), 0.1% insulin, 0.1% hEGF, 0.1% hydrocortisone, 0.1% GA-1000, and 100 ng / mL cholera toxin. All cell lines were cultured at 37°C and 5% CO2 in a humidified atmosphere.

[0105] ATP assay with BioTracker ATP-Red 1: After various treatments, cells were stained with BioTracker ATP-Red 1. After 30 min incubation, cells were washed twice with DPBS, harvested, and dissociated into a single cell suspension with a 40 μm cell strainer. Cells were analyzed using an Attune NxT flow cytometer. Mean signals (at 570 nm) were compared.

[0106] 3D Anchorage-Independent Growth Assay: This assay is also called the spheroid formation assay. Single cell suspensions were prepared using enzymatic and manual disintegration (25 g needle). Cells were then plated at a density of 500 cells / cm2 in spheroid medium (DMEM-F12+1X B-27+supplement+20 ng / ml EGF+Pen / Strep) under non-adherent conditions in culture dishes pre-coated with (2-hydroxyethyl methacrylate) (poly-HEMA, Sigma Aldrich Inc.), referred to as "spheroid plates". Cells were grown for 5 days and maintained at 37°C in a humidified incubator. After 5 days of culture, 3D spheroids larger than 50 μm were counted using an eyepiece ("graticule") and the percentage of plated cells that formed spheroids was calculated. This is referred to as percent spheroid formation. This was normalized to 1 (1–100% MFE). 3D mammosphere formation efficiency (MFE) was analyzed in both low and high ATP subpopulations of cells. All 3D mammosphere experiments were performed in triplicate and repeated at least three times independently.

[0107] shRNA lentiviral transduction: Lentiviral plasmids, packaging cells, and reagents were purchased from Genecopoeia. 48 hours after seeding, 293Ta packaging cells were transduced with lentiviral vectors encoding a set of shRNA clones of three constructs against all three variants of human ATP5F1C in the lentiviral psi-LVRInU6TGP vector with an inducible U6 promoter: CMV promoter-TetR-SV40 promoter-eGFP-IRES-puromycin. In parallel, a scrambled control psi-LVRInU6TGP vector (sh-Control) was transfected. Two days after transfection, the lentivirus-containing culture medium was passed through a 0.45 μm filter and added to target cells (MDA-MB-231) in the presence of 5 μg / ml polybrene. Infected cells were selected with a puromycin concentration of 1.5 μg / ml.

[0108] Western Blot: Cells were lysed in RIPA buffer (Sigma Aldrich, Inc.) containing 1 tablet of cOmplete TM inhibitor mix (Roche Applied Science, Indianapolis) and 1 tablet of PhosSTOP™ phosphate inhibitor per 10 mL of buffer and loaded onto SDS-polyacrylamide gels. The gels were transferred to 0.2 μm nitrocellulose membranes using a Trans-Blot Turbo Transfer System (Bio-Rad, Inc.). Membranes were incubated with each primary antibody diluted in Tris-buffered saline, 0.1% Tween 20 (Sigma Aldrich, Inc.) and 5% bovine serum albumin (BSA, Sigma Aldrich Inc.) (TBST) and incubated overnight at 4°C. Blots were then washed, incubated with the appropriate secondary antibodies, and detected with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Inc.) using a G-Box (Syngene, Inc). The antibodies and their dilutions used for Western blot analysis were as follows: mouse anti-ATP5F1C 1:500, mouse anti-β-actin 1:10,000, rabbit anti-PARP 1:1,000, rabbit anti-p21 1:1,000. The resulting immunoblot images were acquired using GeneSys Software (Syngene, Inc.).

[0109] Cell cycle analysis by FACS: Cell cycle analysis was performed on high and low ATP cell subpopulations by FACS analysis using an Attune NxT flow cytometer. Briefly, after trypsinization, resuspended cells were incubated with propidium iodide according to the manufacturer's recommendations (Merck Millipore, Inc.). At least 25,000 events were analyzed per condition. Gated cells were sorted into multiple cell cycle stages.

[0110] Cell migration assay: Briefly, 3.5 × 10 cells were cultured in 0.5 ml of serum-free DMEM containing 0.1% BSA. 4 Cells were added to the wells of a non-coated membrane modified Boyden chamber (Transwell) with a pore size of 8 μm. The lower chamber contained 10% fetal bovine serum in DMEM, which served as a chemoattractant. Cells were incubated at 37°C and allowed to migrate for 16 h. Non-invading cells were removed from the upper surface of the membrane by rubbing with a cotton swab. The chambers were stained with 0.5% crystal violet diluted in 100% methanol for 30-60 min, rinsed with water and examined under a bright-field microscope. Values ​​for invasion and migration were obtained by counting five fields per membrane (20x objective). This represents the average of three independent experiments.

[0111] Tumor growth, metastasis, and embryotoxicity assays: Xenograft assays were performed by INOVOTION (company identification code: 811310127) (La Tronche, France). Fertilized White Leghorn eggs were incubated for 9 days at 37.5 °C and 50% relative humidity. At this time point (E9), the chorioallantoic membrane (CAM) was dropped by puncturing a small hole in the air sac through the eggshell and placing a 1 cm eggshell above the CAM. 2 A window of 1 × 10 cells was opened. MDA-MB-231 tumor cell line was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. On day E9, cells were detached with trypsin, washed with complete medium, and suspended in transplantation medium. 6An inoculum of MDA-MB-231 cells was added to the CAM of each egg (E9), after which the eggs were randomly divided into groups. Tumors were detectable on day 10 (E10) and were subsequently treated once daily for 8 days with vehicle alone (1% DMSO in PBS) or with three different dosages of bedaquiline or bedaquiline conjugates described herein. On day 18 (E18), the upper part of the CAM was removed from each egg, washed with PBS, and then transferred directly into paraformaldehyde (fixed for 48 hours) and weighed. At least 14 tumor samples per group were collected and analyzed for tumor growth assays (n≧14). On day E18, 1 cm of the lower CAM was removed in at least 7 samples per group (n≧7). 2 A portion of the CAM was harvested and the number of metastatic cells was assessed. Genomic DNA was extracted from the CAM (commercially available kit) and analyzed by qPCR using primers specific for the human Alu sequence. Calculation of the Cq of each sample, the mean Cq, and the relative amount of metastasis in each group were managed directly by Bio-Rad® CFX Maestro software. One-way ANOVA with post-hoc tests was performed on all data. Tolerability of the treatment was evaluated by scoring the number of live and dead embryos before each dose.

[0112] Synthesis of compounds: LC column was Waters Sunfire C18 30x4.6mm. Gradient eluent: 5-100% methanol / water containing 0.05% formic acid. Time: 0-10 min. The following abbreviations were used in the above synthesis examples: tetrakis(dimethylamino)ethylene (TDAE), ethyl acetate (EtOAc), tetrahydrofuran (THF), pyridinium paratoluenesulfonate (PyTs), sodium bicarbonate (NaHCO 3 ), magnesium sulfate (MgSO 4 ), methanol (MeOH), 4-dimethylaminopyridine (DMAP), dichloromethane (DCM), ammonium hydroxide (NH 4 OH).

[0113] Intermediate 1, (1R,2S)-1-[6-[4-[tert-butyl(dimethyl)silyl]oxybutyl]-2-methoxy-3-quinolyl]-4-(dimethylamino)-2-(1-naphthyl)-1-phenyl-butan-2-ol, was synthesized as follows: bedaquiline (0.22 g, 0.40 mmol), cobalt(II) phthalocyanine (5.8 mg, 0.01 mmol), and (dibbpy)Ni(ii)Br in dry dioxane (4 ml). 2 To a stirred suspension of (14.1 mg, 0.028 mmol), (4-iodo-butoxy)-tert-butyldimethylsilane (260 μl, 1.00 mmol) and TDAE (187 μl, 0.80 mmol) were added at room temperature under nitrogen atmosphere. The mixture was stirred at +80° C. for 24 h, the solid residue was removed by filtration and the solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (10-15% EtOAc in isohexane) gave (1R,2S)-1-[6-[4-[tert-butyl(dimethyl)silyl]oxybutyl]-2-methoxy-3-quinolyl]-4-(dimethylamino)-2-(1-naphthyl)-1-phenyl-butan-2-ol (87.9 mg). LC-MS 667.7 [M+1] + , RT 5.75 minutes.

[0114] Intermediate 2, (1R,2S)-4-(dimethylamino)-1-[6-(4-hydroxybutyl)-2-methoxy-3-quinolyl]-2-(1-naphthyl)-1-phenyl-butan-2-ol, was synthesized as follows: To a stirred solution of (1R,2S)-1-[6-[4-[tert-butyl(dimethyl)silyl]oxybutyl]-2-methoxy-3-quinolyl]-4-(dimethylamino)-2-(1-naphthyl)-1-phenyl-butan-2-ol (85 mg, 0.128 mmol) in a mixture of THF (8 ml) and water (0.4 ml) under nitrogen atmosphere, PyTs (80 g, 0.318 mmol) was added and stirred at +50° C. for 4 h. The reaction mixture was concentrated and the residue was dissolved in EtOAc (20 ml) and saturated NaHCO 3 (10 ml), washed with saline (10 ml), and MgSO 4The mixture was dried over hexane, the solid residue was removed by filtration and the solvent was evaporated under reduced pressure. The crude product was purified on silica gel (20% EtOAc in isohexane and 1% MeOH in EtOAc) to give (1R,2S)-4-(dimethylamino)-1-[6-(4-hydroxybutyl)-2-methoxy-3-quinolyl]-2-(1-naphthyl)-1-phenyl-butan-2-ol (66.5 mg). LC-MS 549.4 [M+1] + , RT 4.62 minutes.

[0115] Compound [3B], [6-[4-[3-[(1R,2S)-4-(dimethylamino)-2-hydroxy-2-(1-naphthyl)-1-phenyl-butyl]-2-methoxy-6-quinolyl]butoxy]-6-oxo-hexyl]-triphenyl-phosphonium bromide, was synthesized as follows. To a stirred solution of intermediate 2, i.e. (1R,2S)-4-(dimethylamino)-1-[6-(4-hydroxybutyl)-2-methoxy-3-quinolyl]-2-(1-naphthyl)-1-phenyl-butan-2-ol (45 mg, 0.082 mmol), 5-carbopentyl(triphenyl)phosphonium bromide (37 mg, 0.082 mmol), and DMAP (11 mg, 0.082 mmol) in dry DCM (4 ml) under nitrogen atmosphere, DIC (13 μl, 0.082 mmol) was added and the mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure. The crude product was purified by filtration on silica gel (3% MeOH in DCM and 1% NH 4 Purification on hexane (MeOH:DCM) (3% MeOH:DCM) gave 53 mg of [6-[4-[3-[(1R,2S)-4-(dimethylamino)-2-hydroxy-2-(1-naphthyl)-1-phenyl-butyl]-2-methoxy-6-quinolyl]butoxy]-6-oxo-hexyl]-triphenyl-phosphonium bromide as a colourless solid. LC-MS 907.4 [M + ], RT 4.31 minutes.

[0116] Compound [2B], [12-[4-[3-[(1R,2S)-4-(dimethylamino)-2-hydroxy-2-(1-naphthyl)-1-phenyl-butyl]-2-methoxy-6-quinolyl]butoxy]-12-oxo-dodecyl]-triphenyl-phosphonium bromide, was synthesized from intermediate 2 and 11-carboxylundecyl(triphenyl)phosphonium bromide according to the method described above for compound [3B], to give 35 mg of 12-[4-[3-[(1R,2S)-4-(dimethylamino)-2-hydroxy-2-(1-naphthyl)-1-phenyl-butyl]-2-methoxy-6-quinolyl]butoxy]-12-oxo-dodecyl]-triphenyl-phosphonium bromide as a colorless solid. LC-MS 991.6 [M + ], RT 4.82 minutes.

[0117] Statistical analysis: All analyses were performed using GraphPad Prism 6. Data are presented as mean ± SD (or ± SEM where indicated). All experiments were performed independently at least three times, with 4 or more technical replicates for each experimental condition tested (unless otherwise stated, e.g., when one representative data is shown). Statistical significance was determined using Student's t-test or analysis of variance (ANOVA) test. For comparisons between multiple groups, statistical significance was determined using one-way ANOVA. P<0.05 was considered significant.

[0118] It should be understood that some embodiments of the approach of the present invention can have the form of a pharmaceutical composition, such as a composition for preventing metastasis and / or reducing the likelihood of metastasis. The pharmaceutical composition of the approach of the present invention may contain bedaquiline or a bedaquiline derivative having a TPP moiety as an active compound in any pharma- ceutically acceptable carrier. If a solution is desired, water can be the carrier of choice for water-soluble compounds or their salts. For water solubility, organic vehicles such as glycerol, propylene glycol, polyethylene glycol, or mixtures thereof can be suitable. Furthermore, methods for improving water solubility can be used without departing from the approach of the present invention. In the latter case, the organic vehicle can contain a significant amount of water. And in either case, the solution can be sterilized by suitable means known in the art, for example, filtration through a 0.22 micrometer filter. After sterilization, the solution can be dispensed into suitable containers, such as pyrogen-free glass vials. This dispensing is optionally performed in an aseptic manner. A sterile closure can then be placed on the vial, and the contents of the vial can be lyophilized, if desired. It is not intended that the present approach be limited to any particular form of administration, unless specifically stated.

[0119] In addition to the active compound, the pharmaceutical formulation of the present approach can contain other additives known in the art. For example, some embodiments can include pH adjusters, such as acids (e.g., hydrochloric acid) and bases or buffers (e.g., sodium acetate, sodium borate, sodium citrate, sodium gluconate, sodium lactate, sodium phosphate). Some embodiments can include antimicrobial preservatives, such as methylparaben, propylparaben, and benzyl alcohol. Antimicrobial preservatives are often included when the formulation is placed in a vial designed for multiple dose administration. The pharmaceutical formulations described herein can be lyophilized using techniques known in the art.

[0120] In embodiments involving oral administration of the active compound, the pharmaceutical composition may take the form of a capsule, tablet, pill, powder, solution, suspension, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate, calcium phosphate, and the like may be used with various disintegrants such as starch (e.g. potato or tapioca starch) and certain complex silicates, in combination with binders such as polyvinylpyrrolidone, sucrose, gelatin, acacia, and the like. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc may be included for tabletting purposes. Solid compositions of a similar type may be utilized as fillers in soft and hard-filled gelatin capsules. Materials related thereto also include lactose, and high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are desired for oral administration, the compounds of the subject of the present disclosure may be combined with various sweeteners, flavorings, colorings, emulsifying agents, and / or suspending agents, as well as diluents such as water, ethanol, propylene glycol, glycerin, and various similar combinations thereof.

[0121] Further embodiments provided herein include liposomal formulations of the active compounds disclosed herein. Techniques for forming liposomal suspensions are known in the art. If the compound is a water-soluble salt, it can be incorporated into lipid vesicles using conventional liposome technology. In such cases, the water solubility of the active compound allows the active compound to be substantially contained in the hydrophilic center or core of the liposome. The lipid layer utilized can be of any conventional composition and can contain or not contain cholesterol. If the active compound of interest is water-insoluble, again conventional liposome formation technology can be used to substantially contain the salt in the hydrophobic lipid bilayer that forms the structure of the liposome. In either case, the liposomes produced can be reduced in size, as by using standard sonication and homogenization techniques. The liposomal formulations containing the active compounds disclosed herein can be lyophilized to produce a lyophilizate, which can be reconstituted with a pharma-ceutically acceptable carrier, such as water, to regenerate the liposomal suspension.

[0122] With respect to pharmaceutical compositions, the pharma- ceutical effective amount of an active compound (e.g., bedaquiline or a bedaquiline derivative having a TPP moiety) described herein is determined by a medical professional and depends on the condition, size and age of the patient, and the route of delivery. In certain non-limiting embodiments, a dosage of about 0.1 to about 200 mg / kg has therapeutic efficacy, the weight ratio being the ratio of the weight of the active compound to the subject's body weight, including when a salt is used. In some embodiments, the dosage can be the amount of active compound required to provide a serum concentration of the active compound of up to about 1 to 5, 10, 20, 30, or 40 μM. In some embodiments, dosages of about 1 mg / kg to about 10 mg / kg, and in some embodiments, about 10 mg / kg to about 50 mg / kg can be used for oral administration. Typically, dosages of about 0.5 mg / kg to 5 mg / kg can be used for intramuscular injection. In some embodiments, dosages can be from about 1 μmol / kg to about 50 μmol / kg, or optionally from about 22 μmol / kg to about 33 μmol / kg of the compound for intravenous or oral administration. Oral dosage forms can contain any suitable amount of active compound, including, for example, 5 mg to 50, 100, 200, or 500 mg per tablet or other solid dosage form.

[0123] The pharmaceutical composition may use the active compound as a free base or as a salt. Common salts include monohydrates and hydrochloride hydrates, the latter of which may be useful for improving solubility. Exemplary pharmaceutical compositions are provided, but are intended to be non-limiting examples only. In capsule form, the composition may contain 50 mg or 100 mg of active compound per base. Other ingredients include gelatin, magnesium stearate, shellac glaze, sodium lauryl sulfate, starch, quinoline yellow (E104), erythrosine (E127), patent blue V (E131), titanium dioxide (E171), triiron tetroxide (E172), and propylene glycol. The delayed release tablet form may contain 60 mg or 120 mg of active compound, 3.6 mg or 7.2 mg of sodium, respectively, and inactive ingredients including lactose monohydrate, microcrystalline cellulose, sodium lauryl sulfate, sodium chloride, talc, lactose anhydrous, corn starch, crospovidone, magnesium stearate, and a cellulose polymer coating. It is understood that other pharmaceutical compositions may be used without departing from the approach of the present invention, and that the approach of the present invention is not intended to be limited to any particular formulation.

[0124] In some embodiments, the active compound may be present as a salt, such as a fumarate salt. The fumarate salt of the active compound is water insoluble. In some embodiments, the pharmaceutical composition may be in the form of a tablet, and the active compound may be present with inactive ingredients, such as colloidal silicon dioxide, crospovidone, hypromellose 2910, polysorbate 20, silicified microcrystalline cellulose, and sodium stearyl fumarate. In some embodiments, the pharmaceutical composition may be in the form of a tablet, and the active compound may be present with inactive ingredients, such as colloidal silicon dioxide, corn starch, croscarmellose sodium, hypromellose 219, lactose monohydrate, magnesium stearate, microcrystalline cellulose, and polysorbate 20. The exact formulation will depend on the particular embodiment, and those of ordinary skill in the art can use formulation methods known and available.

[0125] In some embodiments, the approaches of the present invention may take the form of a therapeutic method comprising administering to a patient in need thereof a pharma- ceutical effective amount of one or more pharmaceutical compositions and a pharma- ceutical acceptable carrier. For example, the approaches of the present invention may be used to eradicate a population of CSCs likely to cause metastasis, thereby preventing metastasis and recurrence from the original CSC population, or reducing the likelihood of metastasis and recurrence.

[0126] The approach of the present invention can be used to prevent and / or reduce the likelihood of tumor recurrence and metastasis. Anti-cancer treatments often fail due to tumor recurrence or metastasis, especially after surgery. The mitochondrial activity of CSCs is at least partially responsible for the failure of these treatments. The embodiment of the approach of the present invention can be used in situations where conventional cancer therapies fail and / or in conjunction with anti-cancer treatments to prevent or reduce the likelihood of failure due to tumor recurrence and / or metastasis. A pharmacologic effective amount of a pharmaceutical composition containing bedaquiline or a bedaquiline derivative having a TPP moiety as described herein as an active compound may be administered to a patient. The patient may be suffering from cancer or at risk of suffering from cancer or at risk of tumor recurrence and / or metastasis.

[0127] As described in the applicant's co-pending U.S. Provisional Patent Application No. 62 / 686,881, filed June 19, 2018, and U.S. Provisional Patent Application No. 62 / 731,561, filed September 14, 2018, both of which are incorporated herein by reference in their entireties, e-CSCs represent a proliferation-associated CSC phenotype. In addition to bulk cancer cells and CSCs, it should be understood that the approach of the present invention may be used to target a hyperproliferative cell subpopulation that the inventors call e-CSCs. The e-CSCs exhibit a gradual increase in stemness markers (ALDH activity and tumor-like formation activity), a strong increase in mitochondrial mass, and an increase in glycolytic and mitochondrial activity.

[0128] In view of the above, it should be understood that the inventive approach can take a variety of forms depending on the embodiment. For example, an embodiment of the inventive approach can take the form of a composition, particularly a pharmaceutical composition. The therapeutic compound can be the active ingredient and can be present in a pharma- ceutical effective amount.

[0129] An embodiment of the approach of the present invention may also take the form of a method for preventing or reducing the likelihood of tumor recurrence and / or metastasis. In some embodiments, an effective amount of a composition having bedaquiline as an active compound may be administered. In some embodiments, an effective amount of a composition having a bedaquiline derivative having a TPP moiety as described herein as an active compound may be administered.

[0130] Some embodiments of the approach of the present invention can take the form of a companion diagnostic using the ATP-related gene signature described herein. By using the gene signature as a companion diagnostic, cancer patients who can benefit from ATP inhibition therapy using bedaquiline or a bedaquiline derivative having a TPP moiety, as described above, can be identified. After identification, the candidate can be administered a pharmacologic effective amount of a composition comprising an active compound, bedaquiline or a bedaquiline derivative having a TPP moiety, as the active compound.

[0131] A biological epithelial sample of cancer can be obtained, and the level of each biomarker of the selected gene signature can be determined from the biological sample using a method for measuring biomarker expression that is known and available in the art. The determined level is compared to a threshold level for each biomarker of the signature, and a pharmacologic effective amount of an ATP inhibitor is administered when the determined level of the biomarker of the gene signature exceeds the threshold level. Preferably, the ATP inhibitor is administered when the determined levels of all biomarkers of the gene signature exceed the threshold level for the biomarker. The threshold level for each biomarker can be determined using a non-cancerous epithelial sample from the same subject.

[0132] The terminology used in the description of the present invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. When used in the description of the present invention and the appended claims, the terms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. The present invention encompasses numerous alternatives, modifications, and equivalents, as will become apparent from a consideration of the following Detailed Description of the Invention.

[0133] It is to be understood that the terms "first", "second", "third", "a)", "b)" and "c)" etc. may be used herein to describe various elements of the invention, and the claims are not limited by these terms. These terms are used only to distinguish one element of the invention from another element. Thus, a "first element" described below can be referred to as an aspect of an element, and can also be referred to as a third element, without departing from the teachings of the invention. Thus, the terms "first", "second", "third", "a)", "b)" and "c)" etc. are not necessarily intended to impose any order or other hierarchy on the associated elements, but are used merely for identification purposes. The order of operations (or steps) is not limited to the order presented in the claims.

[0134] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Furthermore, terms as defined in commonly used dictionaries shall be interpreted as having a meaning that is not inconsistent with the meaning of said terms in the context of this application and related art, and shall not be interpreted in an ideal or overly formal sense unless so clearly defined herein. The terminology used in describing the invention herein is for the purpose of describing particular embodiments only, and is not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety into this application. In the event of a conflict of terminology, the present specification shall control.

[0135] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0136] The phrase "treatment cycle" refers to a course of treatment, such as a dosing schedule, that is repeated periodically or on a predefined basis. A treatment cycle may include several days of treatment followed by several days of rest. By way of example only, an agent may be administered once daily for two weeks, followed by two weeks without treatment, over a four-week treatment cycle. It is understood that treatment cycles may depend on a number of factors, such as the individual's medical condition, age, sex, and weight, as well as one or more specific agents and / or methodologies, to elicit a desired response for each individual.

[0137] Unless otherwise clearly indicated by the context, it is specifically contemplated that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. By way of illustration, if the specification states that "a composite comprises components A, B, and C," it is specifically contemplated that any of A, B, or C, or any combination thereof, can be omitted and discarded.

[0138] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations thereof) should be interpreted to include the materials or steps recited and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, as used herein, the term "consisting essentially of" should not be interpreted as the equivalent of "comprising."

[0139] The term "about" as used herein in reference to a measurable numerical value, e.g., amount or concentration, is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even no more than ±0.1% of the specified amount. Ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​within that range.

[0140] Thus, while specific embodiments of the present invention have been described, it should be understood that the present invention, as defined by the appended claims, is not limited to the specific details set forth in the above description, since many obvious variations of the invention are possible without departing from the spirit or scope of the invention as hereinafter claimed.

Claims

1. 1. A method for treating or preventing at least one of tumor recurrence and metastasis in a subject, the method comprising: A method comprising the step of administering to the subject a pharma- ceutical effective amount of a bedaquiline derivative having a TPP moiety.

2. The bedaquiline derivatives having a TPP moiety include: 【Chemistry 1】 2. The method of claim 1, wherein m and n are each independently an integer from 0 to 16; A is O, N, or S; and X is a halogen or a pharma- ceutically acceptable anion.

3. The bedaquiline derivatives having a TPP moiety include: 【Chemistry 2】 2. The method of claim 1, wherein m and n are, independently of each other, integers from 0 to 16; A and B are, independently of each other, O, N, S, or absent; and X is a halogen or a pharma- ceutically acceptable anion.

4. The bedaquiline derivatives having a TPP moiety include: 【Chemistry 3】 The method of claim 1, wherein

5. The bedaquiline derivatives having a TPP moiety include: 【Chemistry 4】 The method of claim 1, wherein

6. structure: 【Chemistry 5】 A compound of the formula: A compound wherein m and n are, independently of each other, integers from 0 to 16; A is O, N, or S; and X is a halogen or a pharma- ceutically acceptable anion.

7. The compound is: 【Chemistry 6】 6. The compound of claim 5,

8. The compound is: 【Chemistry 7】 6. The compound of claim 5,

9. structure: 【Chemistry 8】 A compound of the formula: A compound wherein m and n are, independently of each other, integers from 0 to 16; A and B are O, N, S, or absent; and X is a halogen or a pharma- ceutically acceptable anion.

10. 1. A method for preventing tumor metastasis and tumor recurrence in a patient's body and / or reducing the likelihood of tumor metastasis and tumor recurrence, the method comprising: obtaining a cancer biological sample from said patient; determining, or having determined, in said biological sample the levels of biomarkers of an ATP-related metastasis gene signature consisting of ABCA2, ATP5F1C, COX20, NDUFA2 and UQCRB; comparing said determined level with a threshold level for said biomarker; and If the determined level exceeds the threshold level, the method comprises administering a pharma- ceutical effective amount of a composition containing an active compound selected from bedaquiline and a bedaquiline derivative having a TPP moiety.

11. The method of claim 10, wherein the bedaquiline derivative having a TPP moiety is a compound according to any one of claims 6 to 9.

12. 1. A method of treating an individual suffering from cancer, comprising: The method comprises: administering to said individual a pharma- ceutical effective amount of a bedaquiline derivative having a TPP moiety. A method comprising:

13. The method of claim 12, wherein the bedaquiline derivative having a TPP moiety is a compound according to any one of claims 6 to 9.

14. The method of claim 12, wherein the cancer is breast cancer.

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