|Year : 2020 | Volume
| Issue : 3 | Page : 110-118
In silico drug repurposing: An antifungal drug, itraconazole, repurposed as an anticancer agent using molecular docking
Sanika Dhorje, Poonam Lavhate, Amrita Srivastav
Department of Biotechnology, Modern College of Arts, Science and Commerce, Savitribai Phule Pune University, Pune, Maharashtra, India
|Date of Submission||11-May-2020|
|Date of Acceptance||12-May-2020|
|Date of Web Publication||18-Aug-2020|
Dr. Amrita Srivastav
Department of Biotechnology, Modern College of Arts, Science and Commerce, Savitribai Phule Pune University, Pune, Maharashtra.
Source of Support: None, Conflict of Interest: None
Introduction: In recent years, increased cancer incidences and death rates due to it, have turned cancer to be a major problem worldwide. Approximately more than 7 million people globally die from cancer. Among the various types of cancer, breast cancer is the most prevalent type of malignant neoplasms among the women. Owing to the increasing triple-negative breast cancer (TNBC) cases per year, there is a high demand for the development of new potential drugs within a short period. Objectives: The objectives of this study were to overcome the traditional drug discovery challenges and to deal with hazardous diseases with potential drugs within a less time using molecular docking as the most important bioinformatics tool used for computer-aided drug designing (CADD). Materials and Methods: For designing drug against TNBC, Smoothened (SMO) protein involved in the hedgehog pathway is selected, and an antifungal agent itraconazole is taken as a drug, which already exists but is repurposed using bioinformatics tools such as National Centre for Biotechnology Information (NCBI), Protein Data Bank (PDB), KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database, Computed Atlas of Surface Topography of proteins (CASTp)/metaPocket, PubChem, DrugBank, MarvinView, Discovery Studio, and AutoDock tool. Similarly, the effect of the drug was tested in vitro on TNBC cell line (MDA-MB-231) using 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Results: It was observed that cell viability decreased when different drug concentrations were used against TNBC cell lines in vitro as compared with the control sample, which lacked the drug sample. The cell viability observed was 100% in the control sample, 91% in 15.625 µM drug concentration, 71.5% in 31.25 µM drug concentration, 65.25% in 62.5 µM drug concentration, 54.75% in 125 µM drug concentration, 40.5% in 250 µM drug concentration, and 43% in 500 µM drug concentration. Conclusion: Repurposing of drug with the help of molecular docking is an effective method of drug development, which reduces time and cost of development of drug, and as it has already been approved, its safety measures are already known to make them safe to use. It is concluded that itraconazole shows an inhibitory effect on SMO, and thus it can be used as an anticancer agent.
Keywords: Docking, hedgehog, itraconazole, neoplasm, repurposing, smoothened
|How to cite this article:|
Dhorje S, Lavhate P, Srivastav A. In silico drug repurposing: An antifungal drug, itraconazole, repurposed as an anticancer agent using molecular docking. MGM J Med Sci 2020;7:110-8
|How to cite this URL:|
Dhorje S, Lavhate P, Srivastav A. In silico drug repurposing: An antifungal drug, itraconazole, repurposed as an anticancer agent using molecular docking. MGM J Med Sci [serial online] 2020 [cited 2022 Dec 2];7:110-8. Available from: http://www.mgmjms.com/text.asp?2020/7/3/110/292374
| Introduction|| |
Increasing incidences of cancer in recent years have turned it to be a major problem worldwide. Approximately more than 7 million people globally die from cancer. Among the various types of cancer, breast cancer is one of the most hazardous malignant neoplasms among women. Per year more than 1 million new cases of breast cancer are diagnosed, and this leads to the development of new potential drugs within a short period. Among all the other types of cancer, breast cancer is ranked number one in Indian females at a rate as high as 25.8 per 100,000 women and mortality rate 12.7 per 100,000 women. The age-adjusted incidence rate of breast carcinoma is as high as 41 per 100,000 women for Delhi, followed by Chennai (37.9), Bengaluru (34.4), and Thiruvananthapuram (33.7). The poor clinical prognosis, the limited long-term efficacy of chemotherapy, and the absence of targeted therapies support the research to identify new targets and to develop novel therapies against this cancer. A statistical data of age-adjusted rate over time during 1982–2014, in some places were observed, which are as follows: Bengaluru (annual percentage change: 2.84%), Bhopal (2.00%), Chennai (2.44%), Delhi (1.44%), and Mumbai (1.42%). Increase in mortality as well as morbidity rate in Indian subcontinent was reported as described in global and Indian studies. The traditional method (de novo method) of drug discovery is a challenging task as it is time consuming, which requires large investment. On average, approximately 12–15 years are required to test drug in a clinical trial, and then the drug is released in market. Cervical cancer was one of the most hazardous types of cancers, but breast cancer has surpassed the cervical cancer in case of significant incidence, mortality, and morbidity rate. Breast cancer globally is most common in females with around 1.7 million cases in 2012. It represents around 25.2% of all other cancers in women globally. There is a very low success rate in traditional method of drug discovery; safety of the new drug is not predictable. Hence, computer-aided drug designing (CADD) is used as an alternative method for drug development, which has accelerated the process of drug discovery and reduced the cost and time.
Drug repositioning (drug repurposing) is the process of identifying new therapeutic uses for already existing drugs. This process is used to overcome the traditional drug discovery challenges and to face new arising diseases with potential drugs within less time. During the development of a certain drug, its potential for an interested disease is only focused, this can lead to miss out other therapeutic uses of a drug. Repositioned drugs used are mostly marketed drugs or failed drugs, which are safe but discarded due to other reason. Hence, the initial 6–9 years are not required for the development of new drugs, but instead directly preclinical testing and clinical trials are carried out, thus reducing costs, time, and risk, as its safety is already known.
As cancer is a major disease worldwide, there is a constant need for the development of a new potential drug within less time to face new arising cases of cancer. Drug repurposing saves time and money, making drug treatment affordable to all kinds of patients, and there is no problem of drug availability, as these repurposed drugs are already in the market.
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with approximately 15% of the total cases. Treatment of TNBC remains challenging due to the absence of common breast cancer receptors such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Hence, neither endocrine therapies nor HER2-targeted agents are effective in treating TNBC. This leaves chemotherapy as the only main therapeutic option for the treatment of TNBC. But the lack of ER and other growth factor receptors in TNBC limit drug treatment options to chemotherapy. Hence, it is necessary to identify new pathways important for growth and survival of TNBC and to develop a targeted therapy. One such pathway is Hedgehog (Hh) pathway, which is upregulated in TNBC.
The Hh signaling pathway plays an important role in early embryonic development by controlling cell proliferation and differentiation. By adulthood, this pathway is largely inactive other than in stem cell populations and in maintaining tissue homeostasis. Activation of the pathway has also been heavily implicated in cancer, and is especially prevalent in basal cell carcinoma, medulloblastoma, and pancreatic cancer.
The Hh pathway activity is regulated by Patched (PTCH), which is a 12-pass transmembrane protein that suppresses the activity of Smoothened (SMO), a 7-pass transmembrane protein, which activates the GLI transcription factors. On Hh ligand (Sonic-SHH or Desert-DHH or Indian Hedgehog-IHH), binding to PTCH causes release of its inhibitory action on SMO, resulting in SMO ciliary translocation and activation. Activated SMO promotes trafficking of SUFU-GLI complexes to the cilium, followed by the dissociation of GLI proteins from SUFU. Activated GLI1 transcription factors enter nucleus and lead to upregulation of Hh target genes and induce proliferation, epithelial mesenchymal transition, angiogenesis, metastasis, and activation of other cancer stem cell pathways.
SMO transmembrane is a central regulator of Hh pathway, and hence has been the primary focus for the development of small molecule Hh pathway inhibitors. Some of the SMO inhibitors are cyclopamine, vismodegib, Novartis, IPI-926 and Exelixis. These inhibitors showed positive response in inhibiting Hh pathway, which decreased the cell proliferation of basal cell carcinoma. Hence, these SMO inhibitors alone or in combination show promising treatment against TNBC. A drug can be screened and repurposed as SMO inhibitor with the help of molecular docking by predicting its binding affinity with SMO protein. SMO inhibitors can act as an effective drug as they haves more binding energy and are therefore more stable.
Itraconazole is a synthetic triazole antifungal agent used in the treatment of systemic and superficial fungal infections. It is a Food and Drug Administration (FDA)-approved drug, which inhibits fungal cytochrome P450 enzymes, resulting in a decrease in fungal ergosterol synthesis, required for the membrane integrity of fungal cells. Because of its low toxicity profile, this agent can be used for long-term maintenance treatment of chronic fungal infections. It has been used against histoplasmosis, blastomycosis, cryptococcal meningitis, candidiasis, aspergillosis, and in some dermatological and nail infections.
Recently, itraconazole was known to show inhibitory action on Hh pathway. Itraconazole-binding affinity against SMO can be predicted with the help of molecular docking, and hence, its potency as an anticancer agent can be measured. This will predict whether an itraconazole can be a promising agent in treating TNBC. Further, it can be confirmed by using different concentrations of itraconazole on TNBC cell line and performing different cell viability assays to measure the toxicity of drug on cancerous cell.
| Materials and methods|| |
The proposed research work was carried out at the biotechnology laboratory, Modern College of Arts, Science and Commerce, Ganeshkhind, Pune, Maharashtra, India. The work took 8 months to complete in all aspects, which included in silico studies, in which results were verified using different tools as mentioned further. Biological triplicates had confirmed biochemical results.
With the help of NCBI (National Center for Biotechnology Information), leading cancer-causing deaths all over the world was discovered and studied. Breast cancer showed the highest death rate worldwide. Further study of breast cancer and its subtypes was done. The study leads to TNBC, which contributes in majority to most breast cancer types. TNBC is the deadliest and the most aggressive breast cancer, lacking all the three prevalent breast cancer receptors (ER, PR, and HER2). This makes TNBC hard to target through endocrine therapies or HER2-targeted agents. Hence, to design effective drugs, the pathways were studied, which are present in TNBC; Hh pathway was selected for the drug-designing protocol as this pathway is heavily active in basal cell carcinoma, medulloblastoma, and pancreatic cancer. Hh pathway induces proliferation, epithelial–mesenchymal transition, angiogenesis, metastasis, and activation of other cancer stem cell. Blocking this pathway could help stop the proliferation of cancer cells and lead them to apoptosis. With the help of NCBI and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database, SMO protein [Figure 1] was studied to be the central protein for the activation of the Hh pathway. Targeting this protein could help in the inhibition of Hh pathway and further proliferation of stem cells. To develop a targeted therapy for TNBC faster, already marketed drugs that somehow show the effect on Hh pathway by targeting SMO were studied. Recently, itraconazole was known to show inhibitory action on Hh pathway. For this, literature related to drug repurposing—already repurposed drugs and current research on repurposing from NCBI were studied. This study helped in selecting the drug “itraconazole” [Figure 2], which is present in the market as an antifungal drug. Its properties and structure were studied from DrugBank and PubChem.
|Figure 1: Top five binding sites of Smoothened protein predicted by Computed Atlas of Surface Topography of proteins|
Click here to view
The crystal structure of the targeted SMO transmembrane protein (PDB: 4JKV) was selected as a receptor and retrieved from the RCSB PDB. The elimination of water molecules and the selection of the single peptide chain of the receptor were performed using “Pymol”. All ligand structures were extracted from the NCBI PubChem database in SDF format and converted into PDB format using an online tool “Pymol.” The graphical user interface program “Auto-Dock Tool 4.2” was used for docking ligand with the selected smoothened protein. DrugBank was also used to view the three-dimensional (3D) and two-dimensional (2D) structure of itraconazole. Also, the Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties of itraconazole and its target information were studied through DrugBank. Binding sites of SMO were predicted with the help of Computed Atlas of Surface Topography of proteins (CASTp) software and Discovery Studio., Among the predicted binding sites, top five binding sites predicted by Discovery Studio and CASTp were selected.
The ligand energy was also optimized by optimizing the rotatable bonds, torsion angle, and submerging the nonpolar hydrogen bonds. AutoDock Tool grid box, which includes the target protein and ligand for docking, was set at 60, 60, and 60 and (x, y, and z) center with −18.015, 7.905, and −8.516, respectively, to include all the present amino acid residues of ligand-binding pockets of the receptor [Figure 1]. Final docked conformations were obtained using the AutoDock Tool, and the results were analyzed. The results were visualized using “AutoDock Tool 4.2” [Figure 3].
|Figure 3: AutoDock Tool grid box showing all the coordinates and dimensions|
Click here to view
In vitro drug toxicity
TNBC cell line MDA-MB-231 was brought from NCCS (National Centre of Cell Science), Savitribai Phule Pune University. The cell line received was cultured in L-15 medium. It was first made adaptable to DMEM (Dulbecco’s Modified Eagle Medium) by culturing them in 50% L-15 medium and 50% DMEM. The next subculture was done in 100% DMEM, and henceforth the cell line was grown and maintained in DMEM. The cells were subcultured after 24h.
Cell viability assay—MTT assay
MTT assay is a colorimetric assay that measures the reduction of yellow 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase (SDH). The MTT enters the cells and in mitochondria gets reduced to an insoluble, colored (dark purple) formazan product because of the action of SDH. The cells are then solubilized with an organic solvent (e.g., isopropanol/dimethyl sulfoxide [DMSO]), and the released, solubilized formazan reagent is measured using a spectrophotometer. As the reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells. More the absorbance more is the product formed, which is directly proportional to the number of viable cells.
| Preparation of drug stock|| |
To develop a targeted therapy for TNBC faster, already marketed drugs, which somehow show effect on Hh pathway by targeting SMO, were studied. For this, literature related to drug repurposing—already repurposed drugs and current research on repurposing from NCBI were studied. This study helped in selecting the drug “itraconazole,” which is present in the market as an antifungal drug. Its ADMET properties and structure were studied from DrugBank and PubChem.
Drug: Itraconazole (Candituf) capsule of concentration 100 mg/capsule was the drug used.
Molecular weight: The molecular weight of the drug was 705.641g.
Different drug concentrations were prepared for MTT assay in 2mL Eppendorf tubes.
Drug concentration range studied was as follows: 15.625, 31.25, 62.5, 125, 250, and 500 µM.
Cell counting is done just before the cells are plated into 96-well microtiter plate. This gives the idea of cell population per milliliter of cell culture, and also it helps in deciding how much volume of media should be taken from the cultured flask such that per well (100 µL) of 96 micro-well plates contains 20,000 cells. Cell counting was done using hemocytometer and trypan blue as staining dye.
A total of 20,000 cells were plated per well in a 96 micro-well plate, along with control and six test samples. The cells were incubated for 24h so that they will get adhered to the wells. Later, exhausted media was removed from the wells, and different concentrations of the drug along with fresh complete media were added into the wells. In control, instead of adding drug, only media was added. The microtiter plate was incubated for 24h. A total of 10 µL MTT reagent was added to each well. The cells were incubated for 3h. After incubation, purple color was visible due to the formation of crystals of formazan. The media was removed from the well, leaving the purple-colored crystals in the well. A total of 100 µL DMSO was added to each well and mixed well to dissolve the crystals. Blank was prepared by adding DMSO in an empty well. The cells were again kept for incubation for half an hour. After incubation, absorbance was recorded at 570nm using a microtiter plate reader [Figure 4].
|Figure 4: 96 micro-well plate with result 1 of MTT assay. Control—extreme left, drug concentration from 500 to 15.625 µM—left to right|
Click here to view
Absorbance values that are lower than the absorbance of control cells indicate a reduction in the rate of cell proliferation.
| Results|| |
Final docked conformations for the best ligand–receptor structure from the docked structures were opted based on the binding energy and the number of H bonds formed between the target and the ligand [Figure 5].
|Figure 5: Docking result of itraconazole with Smoothened protein showing one atom in hydrogen bond|
Click here to view
After completing docking, the binding sites were analyzed for its lowest binding energies, as lower the binding energy, higher will be the affinity of the ligand to the target protein [Figure 6], and the same conformation was checked for the formation of the hydrogen bond and interaction of itraconazole with “LYS395,” the amino acid of binding site number 4 of SMO [Figure 7]. In MTT assay, the cell viability observed for each concentration of the drug is as noted in [Table 1].
|Figure 7: Interaction of itraconazole with “LYS395” amino acid of binding site number 4 of SMO|
Click here to view
|Table 1: Cell viability with respect to drug concentration according to MTT assay|
Click here to view
On the basis of the results of cell toxicity assay, the 250 µM concentration was found to inhibit the growth of cancer cells, and the viability of the cells was found to be the least, that is, up to 40% of cell viability was observed in 250 µM drug concentration as compared to the control showing 100% cell viability [Figure 8].
| Discussion|| |
TNBC accounts for acute breast cancers, prevalent in large numbers in young women and those from African and Hispanic descendants. Owing to the lack of druggable known targets, most patients with TNBC are treated with chemotherapy. The poor clinical prognosis, the limited long-term efficacy of chemotherapy, and the absence of targeted therapies support the research to identify new targets and to develop novel therapies against this cancer. Thus, drug repurposing is an alternative method through which such diseases can be cured. Recurrence of breast cancer after chemotherapy is thought to arise from resistant breast cancer stem cells, which are eventually able to repopulate the tumor. The Hh signaling pathway has been shown to regulate the proliferation and survival of breast cancer stem cells, and to promote the resistance to chemotherapy through the activation of multidrug resistance and pro-survival pathways. Extensive preclinical data highlight the key contribution of Hh signaling in cancer stem cell reprogramming in TNBC. Furthermore, the expression of some Hh effectors, such as SMO and GLI1, is significantly increased in TNBC in comparison to non-TNBC. SMO, as a central regulator of the pathway and an accessible cell membrane component, has been the primary focus for the development of small molecule Hh pathway inhibitors.
The development of anticancer drugs is a lengthy and expensive process. After a novel compound is identified or designed, preclinical and clinical data from phase I, II, and III clinical trials are generated before approval. CADD has become an important method for drug development, which has greatly accelerated the efficiency of drug discovery and also reduced costs. Molecular docking, as the core technology, is a computational method in which small molecule ligands are docked to the active pockets of the receptor (the target protein) to predict candidate drugs. This method can be used to predict if a given drug is potentially able to bind other targets. Docking studies have been successfully exploited in drug repurposing. Drug repurposing/repositioning has been suggested as a strategy to minimize time and cost expenses until the drug reaches the market, compared to traditional drug design.
The recent determination of SMO crystal structures now offers the possibility to perform large structure-based screens for new antagonists. Itraconazole is a triazole antifungal treatment widely used in the prevention and systemic treatment of a broad range of fungal infections. The mechanism of action for this antifungal activity is through the decrease of ergosterol synthesis, required for membrane integrity of fungal cells, via inhibition of the lanosterol 14 α-demethylase (14DM) catalyst. There is evidence that at the clinically relevant doses, itraconazole has potent antiangiogenic activity, and that it can inhibit the Hh signaling pathway and may also induce autophagic growth arrest. Itraconazole inhibits the Hh pathway by acting directly on SMO but, unlike other drugs, it binds to a different site on the SMO protein. Itraconazole can therefore be used in combination with, or in cases of drug resistance, as an alternative to other Hh pathway inhibitors. Chemotherapy with itraconazole is promising for heavily pretreated patients with TNBC.
| Conclusion|| |
With the help of in silico tools, superior interactions of itraconazole at two binding sites of SMO were shown with minimum binding energy. Therefore, it is concluded that itraconazole shows an inhibitory effect on SMO, and thus can be used as an anticancer agent. These in silico results were confirmed in vitro by performing MTT assay, where different drug concentrations were used. As the concentration of drugs increased, the number of viable cells decreased in the well. This proved that itraconazole shows an anticancer effect. Repurposing of drugs with the help of molecular docking is an effective method of drug development, which reduces the time and cost of development of drugs, and as it has already been approved, its safety measures are already known to make them safe to use. Hence, another targeted therapy using itraconazole can be considered for the treatment of TNBC.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Malvia S, Bagadi SA, Dubey US, Saxena S Epidemiology of breast cancer in Indian women. Asia Pac J Clin Oncol 2017;13:289-95.
Ocana A, Pandiella A Targeting oncogenic vulnerabilities in triple negative breast cancer: Biological bases and ongoing clinical studies. Oncotarget 2017;8:22218-34.
Ghoncheh M, Pournamdar Z, Salehiniya H Incidence and mortality and epidemiology of breast cancer in the world. Asian Pac J Cancer Prev 2016;17:43-6.
Hodos RA, Kidd BA, Shameer K, Readhead BP, Dudley JT In silico
methods for drug repurposing and pharmacology. Wiley Interdiscip Rev Syst Biol Med 2016;8:186-210.
Dong C, Yang R, Li H, Ke K, Luo C, Yang F, et al
. Econazole nitrate inhibits PI3K activity and promotes apoptosis in lung cancer cells. Sci Rep 2017;7:17987.
Jin G, Wong ST Toward better drug repositioning: Prioritizing and integrating existing methods into efficient pipelines. Drug Discov Today 2014;19:637-44.
Jamdade VS, Sethi N, Mundhe NA, Kumar P, Lahkar M, Sinha N Therapeutic targets of triple-negative breast cancer: A review. Br J Pharmacol 2015;172:4228-37.
Habib JG, O’Shaughnessy JA The hedgehog pathway in triple‐negative breast cancer. Cancer Med 2016;5:2989-3006.
Lacroix C, Fish I, Torosyan H, Parathaman P, Irwin JJ, Shoichet BK, et al
. Identification of novel smoothened ligands using structure-based docking. PLoS One 2016;11:e0160365.
Pantziarka P, Sukhatme V, Bouche G, Meheus L, Sukhatme VP Repurposing Drugs in Oncology (ReDO)—Itraconazole as an anti-cancer agent. Ecancermedicalscience 2015;9:521.
Kim J, Tang JY, Gong R, Kim J, Lee JJ, Clemons KV, et al
. Itraconazole, a commonly used antifungal that inhibits hedgehog pathway activity and cancer growth. Cancer Cell 2010;17:388-99.
Foulkes WD, Smith IE, Reis-Filho JS Triple-negative breast cancer. N Engl J Med 2010;363:1938-48.
Tomao F, Papa A, Zaccarelli E, Rossi L, Caruso D, Minozzi M, et al
. Triple-negative breast cancer: New perspectives for targeted therapies. Onco Targets Ther 2015;8:177-93.
Di Mauro C, Rosa R, D’Amato V, Ciciola P, Servetto A, Marciano R, et al
. Hedgehog signalling pathway orchestrates angiogenesis in triple-negative breast cancers. Br J Cancer 2017;116:1425-35.
Chacón RD, Costanzo MV Triple-negative breast cancer. Breast Cancer Res 2010;12(Suppl 2):S3.
Liu TL, Liu MN, Xu XL, Liu WX, Shang PJ, Zhai XH, et al
. Differential gene expression profiles between two subtypes of ischemic stroke with blood stasis syndromes. Oncotarget 2017;8:111608-22.
Pounds R, Leonard S, Dawson C, Kehoe S Repurposing itraconazole for the treatment of cancer. Oncol Lett 2017;14:2587-97.
Coelho ED, Arrais JP, Oliveira JL Computational discovery of putative leads for drug repositioning through drug-target interaction prediction. PLoS Comput Biol 2016;12:e1005219.
Oprea TI, Overington JP Computational and practical aspects of drug repositioning. Assay Drug Dev Technol 2015;13:299-306.
Aarthy M, Panwar U, Selvaraj C, Singh SK Advantages of structure-based drug design approaches in neurological disorders. Curr Neuropharmacol 2017;15:1136-55.
Tsubamoto H, Sonoda T, Inoue K Impact of itraconazole on the survival of heavily pre-treated patients with triple-negative breast cancer. Anticancer Res 2014;34:3839-44.
Akare UR, Bandaru S, Shaheen U, Singh PK, Tiwari G, Singare P, et al
. Molecular docking approaches in identification of high affinity inhibitors of human SMO receptor. Bioinformation 2014;10: 737-42.
Sliwoski G, Kothiwale S, Meiler J, Lowe EW Jr. Computational methods in drug discovery. Pharmacol Rev 2014;66:334-95.
Berridge MV, Tan AS Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): Subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Archiv Biochem Biophys 1993;303:474-82.
Penault-Llorca F, Viale G Pathological and molecular diagnosis of triple-negative breast cancer: A clinical perspective. Ann Oncol 2012;23(Suppl 6):vi19-22.
Sims-Mourtada J, Opdenaker LM, Davis J, Arnold KM, Flynn D Taxane-induced hedgehog signaling is linked to expansion of breast cancer stem-like populations after chemotherapy. Mol Carcinog 2015;54:1480-93.
Tsubamoto H, Ueda T, Inoue K, Sakata K, Shibahara H, Sonoda T Repurposing itraconazole as an anticancer agent. Oncol Lett 2017;14:1240-6.
March-Vila E, Pinzi L, Sturm N, Tinivella A, Engkvist O, Chen H, et al
. On the integration of in silico
drug design methods for drug repurposing. Front Pharmacol 2017;8:298.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]