Colchicine binding site agent DJ95 overcomes drug resistance and exhibits antitumor efficacy

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1 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 and exhibits antitumor efficacy drug resistance overcomes DJ95 Colchicine binding site agent 4 1 2,3 5 1 Arnst Authors: Kinsie E. -Ning Lei , Dong -Jin Hwang , , Gyanendra Kumar , Yuxi Wang , Zi 5 2 1 6 2 , Tiffany N. Dejian Ma , Qiang Chen e , Deanna N. Park , Stephen W. White , Jinliang Yang 1 1 4 6 , Duane D. Miller* , and Wei Li* , -Sheng Chen Zhe Seagroves 1 Department of Pharmaceutical Sciences, College of Pharmacy, the University of Affiliation: 2 Tennessee Health Science Center, Memphis, TN. State Key Laboratory of Biotherapy and Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from 3 Cancer Center, Collaborative Innovation Center of Biotherapy, Department of Respiratory 4 Department of Me dicine, West China Hospital, Sichuan University, Chengdu, China. molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org molpharm.aspetjournals.org Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, 5 Department of Structural Biology, St. Jude Children’s Research Hospital, Queens, NY. 6 Department of Pathology, the University of Tennessee Health Science Center, Memphis, TN. Memphis, TN at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 at ASPET Journals on May 8, 2019 1

2 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 PAGE RUNNING TITLE Tubulin inhibitor DJ95 overcomes drug resistance in cancer Corresponding author : Wei Li, Ph.D., Address: 881 Madison Avenue, room 561, Memphis, TN 38163. Phone: 901- 448-7532, Email: [email protected] Duane D. Miller, Ph.D., Address: 881 Madison Avenue, room 564, Memphis, TN 38163. Phone: Downloaded from 901-448-6027, Email: [email protected] molpharm.aspetjournals.org Text pages: 30 : 4 Tables Figures : 8 : 89 References at ASPET Journals on May 8, 2019 : 241 Abstract Introduction: 684 : 1152 Discussion List of nonstandard abbreviations ABC, ATP -binding cassette; ANOVA, analysis of variance; AUC, area under the curve; BCRP, breast cancer resistant protein COSMIC, Catalogue of Somatic Mutations in Cancer; DMSO, dimethyl sulfoxide; 2

3 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 FBS, fetal bovine serum; GPCR, G -coupled protein receptor; HUVEC, human umbilical vein endothelial cells; i.p., intraperitoneal; MDR, multidrug resistance; MDR1, multidrug resistant protein 1 Downloaded from associated MRP1; multidrug resistant- protein 1; MTD, maximum tolerable dose; NCI/DTP, National Cancer Institute Developmental Therapeutics Program; molpharm.aspetjournals.org PBS, phosphate buffered saline; PEG, polyethylene glycol; P-gp, P-glycoprotein; RI, resistant index; at ASPET Journals on May 8, 2019 TGI Tumor growth inhibition, total growth inhibition; -TTL; T2R -TTL, Tubulin- RB3_SLD TTL, tubulin tyrosine ligase; resistant TxR, taxane- ; VDA, vascular disrupting agent 3

4 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 ABSTRACT -established strategy for cancer treatment, but Interfering with microtubule dynamics is a well microtubule targeting agents are associated with drug resistance and adverse effects. many binding cassette transporters as critical players in the Substantial evidence points to ATP- development of resistance. Herein, we demonstrate the efficacy of DJ95, a novel tubulin inhibitor, in a variety of cancer cell lines, including malignant melanomas, drug selected resistant Downloaded from expressing cell lines, and in -60 cell line panel . cell lines, specific ABC transporter over- the NCI ell migration cancer c , caused morphological changes to the microtubule DJ95 treatment inhibited . The network foundation, and severely disrupted mitotic spindle - formation of mitotic cells high molpharm.aspetjournals.org resolution crystal structure of DJ95 in complex with tubulin protein and the detailed molecular ed its direct binding to the colchicine site. In vitro pharmacological interactions confirm -target interactions using SafetyScreen44 revealed no significant off screening of DJ95 , and pharmacokinetic analysis showed that DJ95 was maintained at therapeutically relevant plasma at ASPET Journals on May 8, 2019 concentrations for up to 24 hours in ICR mice. In an A375 xenograft model in nude mice, DJ95 inhibited tumor growth and disrupted tumor vasculature in xenograft tumors . These results demonstrate that DJ95 is potent against a variety of cell lines, demonstrated greater potency to ABC transporters over- existing tubulin inhibitors , directly targets the expressing cell lines than , and demonstrates vascular - colchicine binding domain, exhibits significant anti -tumor efficacy disrupting properties. Collectively, these data suggest that DJ95 has great potential as a cancer and warrants further development. therapeutic, particularly for multidrug resistance phenotypes, 4

5 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 INTRODUCTION Microtubules are cytoskeletal structures that are essential for a variety of cellular events including movement, intracellular transport, cell signaling , and mitosis (Jordan, 2002; Kavallaris, 2010; α - and Pasquier and Kavallaris, 2008; Perez, 2009b). Microtubules are composed of -tubulin β proteins, which readily undergo polymerization and depolymerization in a phenomenon known as dynamic instability (Mitchison and Kirschner, 1984). They also form the highly dynamic mitotic Downloaded from spindles that are responsible for the alignment and segregation of chromosomes in the cell during mitosis -Smith and Walczak, 2004) . Interference with microtubule dynamics consequently (Kline disrupts mitotic progression and ultimately leads to apoptosis and cell death (Schmidt and molpharm.aspetjournals.org nterfering with microtubule dynamics by targeting tubulin with small Bastians, 2007) . Therefore, i molecules is a validated anticancer strategy, and many agents are already used clinically or are undergoing development (Dorleans et al., 2009; Gigant et al., 2005; Perez, 2009b; Ravelli et al., 2004). at ASPET Journals on May 8, 2019 categories: microtubule stabilizing agents (e.g. Tubulin inhibitors can be broadly divided into two taxanes) or destabilizing agents (e.g. vinca alkaloids, colchicine binding site agents). Currently, all approved tubulin inhibitors for cancer therapy target the taxane or vinca alkaloid binding site. However, many tubulin inhibitors , including paclitaxel (Taxol®), are associated with multidrug resistance (MDR) mechanisms such as overexpression of drug efflux pumps or the βIII tubulin form (Kamath et al., 2005; Lu et al., 2012; Morris and Fornier, 2008; Orr et al., 2003). Drug iso -binding cassette (ABC) transporters is the most commonly observed efflux mediated by ATP mechanism responsible for inhibiting the intracellular accumulation of therapeutic agents in resistan t cell lines (Szakacs et al., 2006). Of the known human ABC transporters, resistant melanoma cells have been shown to overexpress ABCB1 (MDR1, P-gp), ABCC1 (MRP1), 5

6 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 ABCC2 (MRP2), and ABCB5 amongst others (Chen et al., 2009; Frank et al., 2005; Luo et al., 2012; Schadendorf et al., 1995) well -characterized and it s overexpression . ABCG2 (BCRP) is also resist ance to a variety of anticancer drugs including paclitaxel and docetaxel causes (Vlaming et al., 2009; Wu et al., 2011) . Additionally, studies have demonstrated that alterations in β -tubulin isotypes can lead to resistance to taxanes in melanoma and other cancers (Hari et al., 2003; Kamath et al., 2005; Mhaidat et al., 2008; Ranganathan et al., 1998). The colchicine binding site is located Downloaded from - and β bind to this site may have at the interface between the α -tubulin monomers, and agents that advantages over other tubulin inhibitors targeting the taxane or vina alkaloid binding important domains . Extensive research efforts have address ed the issue of MDR, and numerous studies have molpharm.aspetjournals.org demonstrated that colchicine binding agents can overcome ABCB1 (also known as P -glycoprotein) - mediated drug resistance (Arnst et al., 2017; Devambatla et al., overexpression-, and βIII tubulin 2017; Dong et al., 2016; Gangjee et al., 2013; Gangjee et al., 2010; Li et al., 2017b; Stengel et al., et al., 2016) 2010; Wang et al., 2012; Wu . While colchicine is not employed as an anticancer agent at ASPET Journals on May 8, 2019 due to its toxic side effects, other colchicine binding inhibitors have demonstrated promising are currently being investigated as anticancer candidates (Lu et al., 2012; potential and some Stanton et al., 2011). -disrupting capabilities possessed More recently, there has been increasing interest in the vascular by some microtubule binding agents (Canela et al., 2017; Ji et al., 2015; Schwartz, 2009) . It is well -known that tumor progression is dependent on blood vessels to supply oxygen, essential nutrients, and growth factors. Vascular disrupting agents act on the tumor endothelium and induce initiate hypoxia within the destructive changes that decrease blood flow, induce vascular collapse, (Canela et al., 2017) tumor, and necrosis cause . There is accumulating evidence that microtubule 6

7 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 on tumor endothelial cells supports the pursuit of binding agents can act selectively , and this targeting tumor vasculature (Schwartz, 2009). We previously reported a variety of compounds that inhibit tubulin polymerization through s with the colchicine binding site. (Ahn et al., 2010; Banerjee et al., 2018; Hw interaction ang et al., 2015; Lu et al., 2011; Wang et al., 2012) . This led to the development of a novel class of indolyl- imidazopyridines , and several of these compounds demonstrated superior potency in vitro and Downloaded from depolymerizing effects (Arnst et al., 2017; Hwang et al., 2015) strong tubulin . Herein, we describe one of the most potent of the imidazopyridines, DJ95, and evaluate its potential as a small - molecule chemotherapeutic agent. molpharm.aspetjournals.org against our melanoma panel, as well as many cancer types in the NCI -60 DJ95 is highly a ctive a low resistance index against ABC Additionally, it presents panel. -transporter overexpressing cell lines and outperformed other tubulin targeting agents such as paclitaxel, colchicine, and vincristine . We also evaluated its effect on cancer cell migration, clonogenic potential, and at ASPET Journals on May 8, 2019 demonstrated through endothelial cell tube formation in vitro . Its depolymerization effects were visualization of the microtubule fragmentation a s well as the distortion of mitotic spindles. The of DJ95 to the colchicine site was confidently binding ic -ray crystallograph confirmed through X in a melanoma xenograft model and did not in vivo analyses . Finally, DJ95 inhibited tumor growth . icant off- reveal signif target effects in pharmacological screening 7

8 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 MATERIALS AND METHODS Cell culture and reagents , A375, RPMI -7951, WM -164, WM115 and SK- MEL Human melanoma cell lines -1 (American Type Culture Collection or ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Corning, Manassas , VA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic/antimycotic mixture Downloaded from -Aldrich, St. Louis MO). HUVEC cells were cultured in Endothelial Cell Growth (Sigma 1% Medium containing growth supplement (Promocell, Heidelberg, Germany) and -Aldrich, St. Louis MO). The human epidermoid antibiotic/antimycotic mixture (Sigma molpharm.aspetjournals.org -overexpressing KB -C2 cell line -3-1, its drug- carcinoma cell line KB selected ABCB1 g/mL colchicine) and ABCC1 -overexpressing KB-CV60 cell (maintained in medium with 2 μ nd 60 ng/mL of vincristine) (Wang line (maintained in medium with 1 mg/mL of cepharanthine a at ASPET Journals on May 8, 2019 et al., 2018) were kindly provided by Dr. Shinchi Akiyama at Kagoshima University, Japan. The small cell lung cancer cell line NCI -H460 and its mitoxantrone- selected ABCG2 - human non- -H460/MX20 cells (maintained in medium with 20 nM of mitoxantrone) overexpressing NCI (Robey et al., 2001), and the transfected cell lines HEK293/pcDNA 3.1, HEK293/ABCB1, HEK293/ABCC1, and HEK293/ABCG2-R482 were kindly provided by Drs. Susan E. Bates at Bethesda, MD). These cell lines were Columbia University, NY, and Robert W. Robey (NIH, established by transfecting HEK293 cells with either the empty pcDNA3.1 vector or the vector containing full length ABCB1, ABCC1, and wild type ABCG2, respectively (Patel et al., 2017). (Corning, These cell lines were cultured in DMEM , supplemental with penicillin/streptomycin or Hyclone bovine calf serum (GE Healthcare Life Science, Pittsburgh, and Manassas, VA) FBS PA). All cell lines were auth enticated by ATCC by short tandem repeat profiling. Cultures were 8

9 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 maintained to 80-90% confluency at 37 °C in a humidified atmosphere containing 5% CO . 2 Compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO) to make a stock solution of 20 mM. Compound solutions were freshly prepared by diluting stocks with cell culture medium before use. Cytotoxicity assays The cytotoxic effect against melanoma cell lines was previously described (Arnst e t al., 2017). Downloaded from A375, RPMI-7951, WM-164, WM115, or SK -MEL -well Briefly, -1 cells were seeded in 96 growth rate of the cell line. plates at a density of 1,000–3,500 cells per well, depending on the After overnight incubation, test compounds were added to the wells at 10 concentrations ranging molpharm.aspetjournals.org from 0.03 nM to 1 μM plus a media-only control for 72 h in four replicates. Following treatment, the MTS reagent (Promega, Madison, WI) was added to the cells and incubated in dark at 37 °C for at least 1 hour. Absorbance at 490 nm was measured using a plate reader (BioTek at ASPET Journals on May 8, 2019 Instruments Inc., Winooski, VT). The cytotoxic effects of DJ95 to KB-3-1, KB-C2, KB-CV60, NCI-H460 and NCIH460/MX20 cell lines, and the transfected cell lines HEK293/pcDNA3.1, HEK293/ABCB1, HEK293/ABCC1, and HEK293/ABCG2-R482, were determined using the MTT reagent (Thermo Fisher Scientific Inc., Haverhill, MA) as previously described (Fan et al., 2018). Known tubulin inhibitors with different mechanisms of actions, including paclitaxel (microtubule- stabilizing agent, targeting the taxane -binding site in tubulin), colchicine tine (microtubule- destabilizing agent, targeting the colchicine -binding site in tubulin), and vincris (microtubule- destabilizing agent, targeting the vinca alkaloid -binding site in tubulin) (Li et al., aclitaxel, 2017a; Perez, 2009a), were selected as positive controls for comparison with DJ59. P al., colchicine and vincristine, which are existing substrates of ABCB1 and ABCC1(Chen et 9

10 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 2006; Deeley and Cole, 2006; Hodges et al., 2011), also served as positive substrate controls for the experiments involving ABCB1- or ABCC1 -overexpressing cell lines in this study. Mitoxantrone, a known substrate of ABCG2 (Homolya et al., 2011), was used as positive isplatin was used as negative control since it substrate control in ABCG2-overexpressing cells. C is not a substrate of ABCB1, ABCC1 or ABCG2 (Fan et al., 2018). IC values were calculated by nonlinear regression analysis using GraphPad Prism (GraphPad 50 Downloaded from Software, San Diego, CA). In addition, DJ95 was evaluated in one dose and five dose assays against the NCI -60 cell line panel by the National Cancer Institute Developmental Therapeutics Program (NCI/DTP). molpharm.aspetjournals.org ATPase assay The vanadate- sensitive ATPase activity of ABCB1 and ABCG2 using crude membranes of -95 (0 to 10,000 nM) or positive -five insect cells was determined with the presence of DJ High at ASPET Journals on May 8, 2019 substrate drug by PREDEASY ATPase Kits with modified protocols as previously described (Ambudkar, 1998; Wang et al., 2017b) . Paclitaxel and topotecan were selected as positive substrate drugs for ABCB1 and ABCG2, respectively. Colony forming assay were seeded in 6 well plates (500 cells/well) in replicates of four and incubated at 37 A375 cells °C overnight. Cells were treated with the compound or media only control and incubated for 10 days. Cells were then fixed with methanol and stained with 0.5% crystal violet. Images were taken, and colony area was quantified with ImageJ software (NIH, Bethesda, MD). 10

11 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Scratch migration assay A375 and RPMI7951 cells were seeded in 24 well plates (200,000 cells/well) in replicates of four and incubated overnight. A 200 μL pipette tip was used to scratch a straight line through the cell monolayer to remove an area of cells, then washed several times to remove any debris and uprooted cells. Media was replaced containing equivalent vehicle (DMSO) control or DJ95 at 10 nM or 25 nM concentrations. Images were obtained at the start of the experiment and after 24 hrs Downloaded from with Evos Fl Imaging System (LifeTechnologies, Carlsbad, CA). The analysis was performed with ImageJ software (NIH, Bethesda, MD). Endothelial cell tube formation as say molpharm.aspetjournals.org (Corning, Manassas, VA) was thawed on ice overnight then diluted with serum- free Matrigel n 48 well plates and incubated for a final concentration of 10 mg/mL media . Matrigel was plated i at 37 °C for 1 hour. Low passage number HUVEC cells (<5) in logarithmic growth phase were at ASPET Journals on May 8, 2019 4 trypsinized and suspended in endothelial cell growth media. Cells (7 x10 ) were plated on the Images were quadruplicate containing the desired drug concentrations. in matrigel plates captured after 6 h r incubation with Evos Fl Imaging System (LifeTechnologies, Carlsbad, CA). Analysis was performed with angiogenesis tool plug-in with ImageJ software (NIH, Bethesda, MD). Immunofluorescent staining 5 5 on gl ass coverslip s in 6 well plates and incubated - 5×10 2.5×10 WM -164 cells were seeded and cells were treated with DJ95, paclitaxel, or media only overnight. Media was changed, fixed with 4% paraformaldehyde, permeabilized in 0.1% control for 18 hrs. Cells were then -Aldrich, St. Louis MO) in Phosphate Buffered Saline (PBS) ( Thermo Fisher Triton X (Sigma Scientific Inc., Haverhill, MA) and blocked with 3% Bovine serum albumin ( Cell Signaling 11

12 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Technology, Danvers, MA) prior to staining. Microtubules and mitotic spindles were visualized after incubating with anti α -tubulin antibody (catalog# 62240) (Thermo Scientific, Rockford, IL) - mouse IgG (catalog#A21235) (Molecular Probes, Eugene, OR). and Alexa Fluor 647 goat anti- The coverslips were mounted with Prolong Diamond Antifade mounting media containing DAPI (In vitrogen, Eugene, OR) and images acquired with a Keyence BZ -X700 fluorescence microscope and BZ- X analyzer software (Keyence, Osaka, Japan). Downloaded from X-ray crystallography Protein expression and purification -SLD) was transformed into and over- expressed The stathmin from rat -like domain of RB3 (RB3 molpharm.aspetjournals.org . The protein was purified by anion- exchange chromatography in E. coli (QFF; GE Healthcare, –200 mM NaCl linear gradient in 20 mM Tris-HCl and 1 mM EGTA (pH 8.0)) eluted with a 0 and gel filtration chromatography (Superde x 75; GE-Healthcare, 10 mM HEPES(pH 7.2), 150 at ASPET Journals on May 8, 2019 mM NaCl and 2 mM DTT). The peak fractions from the gel filtration column were concentrated to 10 mg/mL and stored at -80 °C (Charbaut et al., 2001; Dorleans et al., 2009; Wang et al., expression 2016). The TTL protein from chicken was expressed and purified from an E. coli E. coli system as described previous ly (Prota et al., 2013). Briefly, the protein w as expressed in affinity chromatography and gel , purified through Ni- nitrilotriacetic acid using Lysogeny broth , 5 -Tris Propane pH 6.5, 200 mM NaCl, 2.5 mM MgCl filtration chromatography (buffer: Bis 2 mM βMe, 1% glycerol). The peak fractions were concentrated to 20 mg/mL and saved at -80 °C. The sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed to check the atalog # T -238P) (Cytoskeleton Inc., Denver, purity of RB3 and TTL. Porcine brain tubulin (c CO) was supplied at 10 mg/mL in G-PEM (General tubulin buffer:80 mM PIPES pH 6.9, 2 mM MgCl , 0.5 mM EGTA and 1 mM GTP) as a frozen liquid and saved at -80 °C until use. 2 12

13 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Crystallization and crystal soaking The previously published process of obtaining crystals of the tubulin-RB3_SLD- - TTL (T2R TTL) complex was applied (Prota et al., 2013; Wang et al., 2017a). In brief, tubulin (10 mg/mL ), 20 mg/mL ) and RB3 (10 mg/mL ) were mixed TTL ( at the molar ratio of 2:1.3:1.2 (Tubulin: RB3 : TTL ) and incubated on ice with 1 mM AMPPCP acid adenylate ester _SLD , 5 mM tyrosinol and 10 mM DDT , and the mixture was concentrated to 20 mg/mL at 4 °C. The Downloaded from -TTL -drop vapor crystallization of the T2R complex was carried out at 20 °C using the sitting diffusion method by mixing an equal volume of protein complex and crystallization buffer containing 6% PEG 4000, 5% glycerol, 0.1 M MES, 30 mM CaCl , 30 mM MgCl , pH 6.7. 2 2 molpharm.aspetjournals.org was used to Seeding obtain the well diffracting crystals. Initial crystals were observed after two days of incubation and reached a final length of 200-300 μ m within 3 -5 days. Morphologically , cryoprotected with crystallization buffer containing 20% super ior crystals were selected at ASPET Journals on May 8, 2019 and flash frozen in liquid nitrogen. glycerol, X-ray data collection and structure determination Diffraction data were collected at 100K on the beamlines BL19U1 at Shanghai Synchrotron Radiation Facility (SSRF) in Shanghai, China. Data were indexed, integrated and scaled using HKL2000 (Otwinowski and Minor, 1997). The structure of T2R- TTL -DJ95 was determined by TTL structure (PDB ID: 4I55) as a usly published T2R- molecular replacement using the previo search model. The rotation and translation function searches were performed by the program PHASER (McCoy et al., 2007). The model was further built with Coot (Emsley and Cowtan, 2004) and refined using the phenix.refine module Phenix (Adams et al., 2002). The model quality was checked with PROCHECK and shows a good stereochemistry according to the Ramachandran plot. 13

14 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 esonance (SPR) for tubulin binding affinity analyses Surface plasmon r . To evaluate the binding affinities of DJ 95 with tubulin protein, we performed SPR analyses using a Biacore T200 system (GE Healthcare Life Sciences). A Series S Sensor Chip CM5 (GE ife Sciences) was pre-conditioned with three consecutive 1-min injections of 70% Healthcare L normalizing solution. Then 20 μg/mL tubulin (catalog # T -238P) (Cytoskeleton Inc., (w/w) BIA Denver, CO) was immobilized to the sensor chip surface to attain 17,000RU (1,000 R U Downloaded from correspond to an angle change of ~0.1°). One of the four flow cells on the chip was left free as a negative control. DJ95, colchicine or CA-4 (positive controls) was injected over the sensor chip surface for association analysis, followed by dissociation analysis. We adjusted the concentration molpharm.aspetjournals.org gradients for each of the three compounds based on their different affinities to tubulin and different solubility . The experiment data were obtained at 25°C with running a buffer PBS (10 mM phosphate, 2.7 mM KCl, 137 mM NaCl), and 0.01% (v/v) surfactant P20, pH7.4. The flow at ASPET Journals on May 8, 2019 rate was 30 μl/min. The analytes bound on the sensor chips were connected for 120 s and dissociated for 120 s. Regeneration of the sensor chips was performed for 30 s by 10 mM glycine -HCl buffer (pH = 1.5). The equilibrium dissociation constant (Kd) was calculated by a steady state fitting mode with Biacore T200 Evaluation Software, version 2. In vitro pharmacological profiling to assess potential off- target effects t effects to DJ95 binding and enzyme targets was Screening of potentially significant off -targe performed via SafetyScreen44 offered by Eurofins Cerep-Panlabs. DJ95 was tested at 100 nM. Compound binding was calculated as a % inhibition of the binding of a radioactively labeled and specific for each target. Compound enzyme inhibition effect was calculated as a % lig inhibition of control enzyme activity. Results showing an inhibition (or stimulation for assays run in basal conditions) higher than 50% are considered to represent signi ficant effects of the test 14

15 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 compounds. Results showing an inhibition (or stimulation) between 25% and 50% are indicative of weak effects. Results showing an inhibition (or stimulation) lower than 25% are not considered significant and mostly attributable to variability of the signal around the control level. In each experiment , the respective reference compound was tested concurrently with DJ95, and the data were compared with historical values determined at Eurofins. The experiment was accepted in accordance with Eurofins validatio n Standard Operating Procedure. Downloaded from LC -MS/MS plasma concentration analysis LC -MS/MS parameters molpharm.aspetjournals.org The LC -MS/MS system comprised a Sciex (Framingham, MA) 5500 triple quadrupole mass spectrometer, equipped with a Turboionspray™ ionization interface and Analyst software version 1.6.3. Chromatographic separation was carried out using a ZORBAX SB -C18 column of 150 x 4.6 mm i.d., and 3.5 μm particle size (Agilent Technologies, Santa Clara, CA) maintained at 35°C using at ASPET Journals on May 8, 2019 stem and SIL Shimadzu (Columbia, MD) Nexera XR HPLC sy -20ACXR autosampler. The mobile Q water, B: methanol) was eluted at a flow rate of 0.5 mL/min. The gradient started phase (A: Milli- at 50% of mobile phase B and maintained for 0.5 min , then linearly rose to 100 % B over 1 min. Subsequently, the eluent composition was maintained at 100% B from 1.5 to 6 min before it was -equilibration in 0.1 min. The total run decreased to initial condition 50 % mobile phase B for re time was 6.5 min plus a pre-equilibrate of 0.5 min. A switching valve directed the mobile phase to the MS system between 4.5 and 6.1 min. The electrospray ion source was operated in a positive ionization mode for all the experiments. The typical parameters were: capillary 5.5 kV; entrance potential (EP) 10 V; channel electron multiplier (CEM) 1800 V; source temperature 600°C. Other compound specific parameters are listed in Supplement Table 1. 15

16 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 -MS/MS sample preparation LC pitation was used to extract DJ 95 from plasma. Plasma (50 μL) was added in 150 μL Protein preci pre cipitation solution, methanol including 26.5 nM ABI -231 as IS, vortexed for 15 seconds, and ). Supernatant (120 μL) was then then centrifuged for 15 minutes at 4°C (circa 12,000 rpm - well plate and 1 μL sample was injected into the LC -MS/MS system. transferred to a 96 Calibration stand ards were prepared by mixing DJ 95 stock solution in pooled human plasma, Downloaded from resulting in matrix concentrations of 1, 10, 100, 200, 1000, 2000, 10,000 nM. Blank samples were prepared using blank plasma. All samples were stored at -20°C prior to use. The lowest standard of molpharm.aspetjournals.org 1 nM was not detected. The LLOQ was determined to be 10 nM. models and treatments In vivo mouse All protocols and methods, including methods of anesthesia, administration of drugs, blood requiring euthanasia, were approved by the University of Tennessee collection and endpoints at ASPET Journals on May 8, 2019 Health Science Center (UTHSC) Animal Care and Use Committee (ACUC), consistent with the th Guide for the Care and Use of Laboratory Animals , 8 edition as published by the National All animals were maintained in a room with a 12 h light/dark cycle and Academy of Sciences. provided food and water ad libitum. Pharmacokinetic studies Thirty CD Laboratories (Wilmington, MA) of approximately 6 -1 ICR mice from Charles River weeks of age were used for the study. A mix of males and females were used, and at least one animal of each sex was collected at each time point. For each time point cohort, animals (n=3 per time point) were dosed with 15 mg/kg DJ95 via intraperitoneal injection and the drug vehicle was PEG300 -Aldrich, St. Louis MO) and PBS at a 1:1 ratio. (Sigma 16

17 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Blood was collected using heparinized syringes from mice deeply anesthetized with isoflurane during the terminal blood collection via cardiac venipuncture into lithium heparinized tubes. At each time point, blood (~0.6 mL) was collected from a separate cohort of three mice at the following time points: 0, 15, 30, 60, 90, 180, 260, 480, 720 and 1440 minutes. Samples were centrifuged at 3,000 rpm for 10 min. Plasma was collected into 1.5mL centrifuge tubes and frozen at -80 ° C until analysis by LC/MS. Pharmacokinetic parameters were determined by Downloaded from , Princeton, NJ ). These noncompartmental analysis using Phoenix WinNonlin 8.1 (Certara life (t -time profile curve (AUC), half- parameters included area under the concentration ), 1/2 clearance, volume of distribution and maximum concentration (C ). max molpharm.aspetjournals.org MTD and Xenograft study , age 6–8 w eeks old, were purchased from Evigo Laboratories Nude mice, 50:50 male:female (Indianapolis, IN) . A maximum tolerable dose (MTD) study was performed by subjecting nude at ASPET Journals on May 8, 2019 mice up to a dose of 30 mg/kg of DJ95 formulated in the vehicle (equal parts PEG300:PBS) by i.p. injection for 5 consecutive days. Because mice began to show signs of toxicity beyond 30 mg/kg for the 5 day treatment, we scaled the dose back to 15mg/kg for the xenograft study to Logarithmic growth phase A375 cells were prepared in ensure an adequate safety margin. injecting into mice. phenyl red -free, FBS -free media and mixed with thawed matrigel prior to 6 containing 2.5×10 matrigel/cell suspension Tumors were established by injecting 100 μ L of cells subcutaneously in the hind flank of each mouse . After tumors were established , mice were ranked on tumor size and randomized into control or treatment groups, with each group receiving half males and half females . 100 μL of the drug treatment or vehicle control solution was administered via i.p. injection 5 times a week for the duration of the studies. 17

18 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 eek with a caliper and calculated by using the Tumor volume was measured three times a w formula a×b 2×0.5, where a and b represent the larger and smaller diameters, respectively. Tumor growth inhibition (TGI) at the conclusion of the experiments was calculated as 100 – 100 ×((T − T0)/(C − C0)), where T, T0, C and C0 are the mean tumor volume for the specific group on the last day of treatment, mean tumor volume of the same group on the first day of treatment, mean tumor volume for the vehicle control group on the last day of treatment and mean tumor volume Downloaded from for the vehicle control group on the first day of treatment, respectively (Wang et al., 2014). Animal activity was monitored, and body weights were recorded throughout the study to assess and the tumors were potential acute toxicity. At the end of the experiment, mice were sacrificed, molpharm.aspetjournals.org dissected out, weighed , and fixed in in 10% neutral buffered formalin solution prior to pathology staining analysis . Histology and immunohistochemistry at ASPET Journals on May 8, 2019 The fixed tumor xenograft tissues were embedded in paraffin. Serial sections were obtained for immunohistochemistry analysis. Staining was performed with rabbit anti-CD31 (catalog # D8V9E ing ABC -DAB methods. ) (Cell Signaling Technology Inc., Danvers, MA) follow -3300 antigen unmasking solution (Vector Laboratories, Antigen retrieval was performed with H Burlingame, CA). Images were captured with a Keyence BZ -X fluorescent microscope (Keyence at 10x and 20x magnification. Five representative fields of view from Corporation, Itasca, IL) three tumors per group at 20x magnification were analyzed in ImageJ to calculate positive stained area. Statistical analysis In all studies, it was presumed that the null hypothesis would be that the DJ95 treatment had no effect on the biological phenotype measured compared to the control, therefore, significance was 18

19 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 tailed statistics tests. The sample sizes for all in vitro and in vivo studies were pre- based on two- nitiated. In vitro studies included multiple technical determined before the studies were i replicates per assay and were repeated at least three times (biological replicates). values IC 50 were calculated by nonlinear regression and SD was determined from at least 3 independent experiments. For in vivo studies, sample sizes were based on our prior experience from studies testing similar drug compounds in xenograft animal models using the subcutaneous injection Downloaded from method. For in vitro experiments comparing multiple doses of DJ95 multiple doses (Figures 2, 8), the statistical significance ( < 0.05) was calculated by one- way analysis of variance p (ANOVA) followed by Dunnett’s multiple comparison test. For the in vivo experiments (Figure molpharm.aspetjournals.org 7), two cohorts were compared to each other, the control mice (vehicle, n=7) and the DJ95 - test . Data for endpoint tumor treated mice (15 mg/kg, n=6) using the unpaired Student’s t- volume and tumor wet weight were shown as a scatter plot presenting the mean with one SD. All data were analyzed using Prism Software 5.0 (GraphPad Software, Inc., San Diego, CA). at ASPET Journals on May 8, 2019 19

20 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 RESULTS mediated DJ95 is potent against cancer cell lines and overcomes ABC transporter- resistance. We previously reported that DJ95 is active against a variety of melanoma and prostate cancer . To further explore these initial findings, we tested DJ95 against a cell lines (Hwang et al., 2015) panel of malignant melanoma cell lines representing genomic complexity and heterogeneity , Downloaded from -164, WM115, and SK -MEL including A375, RPMI-7951, WM -1 (Supplement Table 2). We and had IC discovered that DJ95 was highly potent in melanoma cell lines values of less than 50 of 24.7 ± 4.9 nM 100 nM (Table 1). It was the most potent against A375, with an average IC 50 molpharm.aspetjournals.org and was similar to colchicine (10.6 ± 1.8 nM ). Paclitaxel was generally the most potent against the parental melanoma cell lines. mediated Because we were particularly interested in the ability of DJ95 to overcome transporter- at ASPET Journals on May 8, 2019 drug resistance, we tested DJ95 against genetically engineered HEK293 cells lines, that stably transfected full length ABCB1, ABCC1, wild type ABCG2, or the pcDNA3.1 blank vector (Supplement Figure 1). Cisplatin, which is not a substrate for any of these ABC transporters, was used as negative control. Greater than 100-fold resistance was observed for the ABCB1 overexpressing HEK293 cells against paclitaxel, and colchicine and vincristine (Table 2). On the other hand, HEK293/ABCB1 resistant cells showed virtually no resistance to DJ95. Slight resistance (RI= 3.7) was revealed for the ABCC1 overexpressing HEK293 transfected cells again only cells, but all other microtubule targeting drugs that st DJ95 compared to the vector- were tested (paclitaxel, colchicine, vincristine) demonstrated significantly increased resistance, s greater than 10,000 nM against HEK293/ABCC1. Similarly, against the with IC 50 -R482 cells, resistance was evident for all tubulin inhibitors, though less HEK293/ABCG2 20

21 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 resistance was developed against the DJ95 compound (about 8.7-fold). This was still lower than mitoxantrone, a known substrate of ABCG2, showing about 11.2-fold decrease in potency in the expressing cell line. ABCG2 over- We also wanted to assess the effects of DJ95 against drug -selected, resistant cell lines. From the -3- parental epidermoid carcinoma cell line KB -overexpressin g 1, the colchicine resistant/ABCB1 (KB -C2) cell line and vincristine resistant/ABCC1 -overexpressing (KB -CV60) cell line were Downloaded from selected ABCG2 -overexpressing cell line NCI developed. The mitoxantrone- -H460/MX20 -H460 was utilized to further -small cell lung cancer cell line NCI generated from the parental non evaluate the effects against ABCG2 upregulation. Overexpression of ABCB1, ABCC1, or molpharm.aspetjournals.org ABCG2 for each of these cell lines is shown in Supplement Figure 1. Compared to existing tubulin inhibitors paclitaxel, colchicine and vincristine, DJ95 exhibited -overexpressing KB -C2 cells and ABCC1 - higher cytotoxic levels in drug- selected ABCB1 at ASPET Journals on May 8, 2019 -CV60 cell lines, with IC overexpressing KB values of 719.54 ± 181.48 nM and 35.90 ± 4.50 50 nM, respectively (Table 3). Although there was still some increase in resi stance against the KB - 1 cells, C2 (colchicine selected/ABCB1 over -expressing) cells compared to the parental KB -3- both KB-C2 and KB-CV60 were much less resistant to DJ95 than they were against the three other tubulin inhibitors tested. Since no resistance to DJ95 was revealed from ABCB1 -selected transfected cell line, the increase in resistance observed for DJ95 against the colchicine -C2 may be related to mechanisms affiliated with the colchicine binding site and not solely KB ABCB1 overexpression. In both NCI -H460 and its mitoxantrone-selected ABCG2 overexpressing cell line, DJ95 -cancer activities than paclitaxel, colchicine and vincristine, with IC demonstrated better anti 50 values of 673.04 ± 111.71 nM and 5361.50 ± 645.45 nM, respectively, whereas IC values of 50 21

22 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 the three other tubulin inhibitors were higher than 10,000 nM in both cell lines. However, low resistance against DJ95 was observed in NCI -H460/MX20 cell line compared to its parental cell line (about 8-fold resistance), though the resistance index was not as high as that of the substrate fold resistance). The resistance index is the ratio of IC control mitoxantrone (about 20- value of 50 the drug resistant cell (or transfected cell overexpressing ABC transporter) over IC value of the 50 parental cell (or vector control for the transfected cell). The results from the drug- selected Downloaded from -overexpressing cell resistant cells are consistent with those obtained from transfected ABCG2 line HEK293/ABCG2 -R482 and its vector control, which indicated that DJ95 might be a weak substrate of ABCG2. molpharm.aspetjournals.org To follow up on these results, the ABCB1 or ABCG2 ATPase activity with the presence of DJ95 were investigated to assess the interaction between DJ95 and these ABC transporters. DJ95 inhibited ABCB1-mediated ATP hydrolysis, while paclitaxel, which is a substrate of ABCB1, at ASPET Journals on May 8, 2019 -sensitive ATPase activity of ABCB1 ( Supplement Figure 2A). This stimulated the vanadate suggests that DJ95 may not be a substrate of ABCB1 and may be able to circumvent ABCB1- medited MDR, which is consistent with the cytotoxicity results from HEK293/ABCB1 and HEK293/pcDNA3.1 cells. On the contrary, DJ95 showed stimulating effects on ABCG2 ATPase activity, similarly to the positive substrate drug, topotecan (Supplement Figure 2B). This is in accordance with the resistance to DJ95 shown by ABCG2-overexpressing cells, which further supports the hypothesis that DJ95 may be a substrate of ABCG2. a diverse selection of cancer types . Finally, DJ95 was tested in the NCI -60 screening, assaying Supplement Figure 3A and S3B - -dose assay ( DJ95 was evaluated in a single ) as well as a 5 ). DJ95 was highly assay to see if it was sensitive in other kinds of cancer (Figure 1 concentration for the majority of melanoma cell lines , and was especially active and demonstrated a low GI 50 22

23 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 effective against leukemia, colon, CNS, and prostate cancers. The average total growth inhibition (TGI) and LC was greatest for colon cancer and melanoma. Additionally , we assessed the dose- 50 response curves of each individual melanoma cell line tested and determined that the cell lines -MEL -5 (Supplement Figure 3C, most sensitive to DJ95 treatment were LOX IMVI and SK Supplement Table 3). T ogether, these results indicate that DJ95 is effective against a multitude of cancer cell lines representing genomic heterogeneity, drug resistan t profiles, and cancer types. Downloaded from DJ95 impedes cancer cell colony formation and migration. molpharm.aspetjournals.org After verifying the potency of DJ95, it was further evaluated for functional inhibition of cell in vitro etermine d its ability to inhibit colony formation in . We first d proliferation and migration a concentration -dependent manner compared to untreated A375 control cells (Figure 2A). A significant decrease was observed for all treated cells, and concentrations as low as 10 nM at ASPET Journals on May 8, 2019 and higher caused a 26.78 ± 1.9% inhibition of colony area compared to control ( P < 0.001) concentrations of 25 nM and 50 nM inhibited colony formation by 44.73 ± 1.9% and 65.01 ± P eliminated ( 3.08%, respectively <0.0001) (Figure 2B). At 100 nM, DJ95 almost completely . DJ95 was colony formation and only 1.25 ± 0.07% of the area compared to the control remained then tested in both A375 and RPMI- 7951 melanoma cell lines to determine its effect on migration ability of the cells in a scratch assay (Figure 2C). After 24 hrs, the untreated cells migrated into 80.73 ± 4.48% of the wound channel, nearly closing the gap. the Treatment to A375 cells with 10 nM and 25 nM of DJ95 decreased the cell migration and led to cells only P < 0.0001) of the scratch area, which occupying 58.23± 2.15% ( P < 0.01) and 35.60 ± 1.35% ( . A similar phenomenon was also observed in was significantly less than the control (Figure 2D) -7951 cell line, where the control cells reclaimed 77.43 ± 3.91% of the scratch area the RPMI 23

24 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 s of 10 nM and 25 nM of DJ95 caused the cells to only migrate into 34.37 (Figure 2E). Treatment P < 0.001) and 23.10 ± 2.92% ( P < 0.0001) of the scratch area, respectively. There was ± 6.09% ( no significant difference in the scratch channel area for each group at the beginning of the experiment (time 0) as determined by two -way ANOVA analysis factoring in both time and treatment group (Supplement Figure 4). To this end, it can be interpreted that DJ95 inhibits cell proliferation and migration at low concentrations for these melanoma cell lines. Downloaded from DJ95 disrupts microtubule networks and mitotic spindle formation. molpharm.aspetjournals.org Previously, we demonstrated that DJ95 is able to potently inhibit polymerization of purified free assay (Hwang et al., 2015). Here, we visually present the effect tubulin protein in a cell- 164 melanoma cells (Figure 3A ). The stabilizing DJ95 has on the microtubule networks of WM- used for comparison. After 18 hrs agent paclitaxel, a potent enhancer of polymerization, was also at ASPET Journals on May 8, 2019 , cells showed dramatically disrupted nM concentration with DJ95 of treatment at a 50 microtubule networks and an increase in soluble, cytoplasmic tubulin. Furthermore, at 200 nM, there appears to be very little polymeric tubulin framework left intact. In contrast, paclitaxel treated cells demonstrated highly condensed, polymeric tubulin. For cells treated at the higher concentration of 200 nM paclitaxel, the fluorescent signal was amplified and the aggregation of of microtubules. the filaments was more pronounced, consistent with increased stabilization Microtubules also make up the mitotic spindle and utilize the d for ynamic properties chromosom e segregation during mitosis (Kline -Smith and Walczak, 2004). Therefore, tubulin also interfere with the formation targeting agents and organization of mitotic spindles (Rai et al., 2012; Rohena et al., 2016; Sakchaisri et al., 2017; Weiderhold et al., 2006) . We investigated the effects of DJ95 in a concentration-dependent manner compared to untreated control cells (Figure 24

25 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 . While control cells exhibited normal, bilateral 3B) spindle formation extruding from centrosomes, DJ95 treatment led to aberrant spindle development as evidenced by multi-polar . These abnormalities were exacerbated with increasing and disorganized spindle morphology drug concentrations . This is further proof that DJ95 inhibits the progression of mitosis by interfering with microtubule dynamics . Downloaded from X-ray crystallography confirms that DJ95 occupies the colchicine binding site. We determined the crystal structure of the T2R -TTL complex bound with compound DJ95 at 2.4 molpharm.aspetjournals.org Å resolution (Figure 4A), and the coordinates and structure factors have been deposited in the overall geometric Protein Data Bank with the PDB ID: 6NNG. The refinement statistics and parameters confirm that the structure quality is excellent; this information together with the data collection parameters are presented in Table 4. DJ95 binds at the interface of the -tubulin α / β at ASPET Journals on May 8, 2019 -tubulin protomer (PDB ID: heterodimer and occupies the same locale as colchicine in the β -tubulin. Unlike colchicine that binds 4O2B) directly opposite the GTP that binds within the α both available interfaces in the T2R -TTL complex, DJ95 only binds to the interface / β α proximal to the TTL subunit. DJ95 is slightly more extended than colchicine and it appears that the single occupancy is not biologically relevant but rather the result of small conformational changes in the assembly that cannot easily be accommodated by the packing within the crystal lattice. The 3 -methoxyphenyl group of DJ95 occupies a deep pocket that is lined with residues Tyr200, Val236, Cys239, Leu240, Leu250, Leu253, Ala314, Ile316, Ala352 and Ile368 (Figure 4B). The central imidazopyridine ring and the distal indole ring are linearly connected and helix β - flanked on one side by residues from the short α -H8, specifically Lys252, Leu253, Asn256 and Met257, and residues Lys350 and Leu246 on the other side. The DJ95 binding 25

26 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 interactions are almost exclusively van der Waals in nature but there are several specific interactions (Figure 4B). The central imidazopyridine ring forms two hydrogen bonds; a nitrogen atom with the main chain amide of Asp249, and an NH group with the main chain of α carbonyl oxygen of Thr179 -methoxyphenyl moiety has -tubulin across the interface. The 3 three interactions; the central methoxy group interacts via a water molecule with the main chain NH of Cys239, and the side chains of Leu253 and Cys239 make flanking pi- H interactions with Downloaded from the phenyl ring. Comparison with the colchicine complex reveals that the binding interactions are very similar, with most of the residues described above in equivalent conformations (Figure 4C). There are molpharm.aspetjournals.org two noticeable differences. First, the equivalent 3-methoxyphenyl moiety of colchicine does not penetrate the pocket as deeply (by some 2.5 Å ) which precludes the water mediated hydrogen bond to the central methoxy group. Second, in the unliganded (PDB ID: 4I55) and colchicine at ASPET Journals on May 8, 2019 liganded T2R-TTL complexes, the side chain of Lys350 is extended and interacts across the interface with Ser178 and Thr179, respectively, in the flexible α -T5 loop of α -tubulin. In α / β the DJ95 complex, however, steric interference with the distal indole ring and favorable van der Waals interactions with the molecule cause Lys350 to swing around, which in turn allows Thr179 to form the main chain hydrogen bond described above. Finally, in a similar fashion to colchicine, the binding of DJ95 elicits substantial conformational changes at the interface, / β α β of tubulin and α - Τ5 -T7 of α -tubulin (Figure 4D). These are including within loops - β incompatible with the ‘straight’ structure of the tubulin filament (PDB ID: 1JFF) and therefore The electron density map consistent with the DJ95 acting as a destabilizer of filament formation. is demonstrated in Supplement Figure 5. 26

27 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 -4. DJ95 shows a lower binding affinity to tubulin than that of colchicine and CA To quantitatively determine the binding affinity of DJ95 with tubulin protein, we performed surface plasmon resonance experiments using Bia core T200 system (GE Healthcare Life Sciences). A CM5 sensor chip with glucan on the surface was used to immobilize tubulin. DJ95, colchicine or CA-4 was injected over the sensor chip surface for binding detection. As 95, determined by a 1:1 kinetics fitting model, the equilibrium dissociation constant Kd of DJ colchicine, and CA-4 was 59.4 , 5.7 μM and 7.7 μM , respectively (Figure 5). These data μM reveal that DJ95 has approximately 10-fold lower binding affinity than colchicine and CA-4, and Downloaded from further modifications will be required to increase the affinity to tubulin and avoid potential binding to additional molecular targets. molpharm.aspetjournals.org DJ95 has negligible interactions with 44 physiologically important targets. studies, we wanted to determine if DJ95 would exhibit a relatively safe profile Prior to in vivo predict if there would be any potential ly detrimental based on interactions with off- targets and at ASPET Journals on May 8, 2019 pharmacological profiling involves screening the compound of interest clinical effects In vitro . against a wide range of targets such as receptors, ion channels, transporters and enzymes other than the intended therapeutic target in order to identify specific interactions that may elicit -related side effects (Bowes et al., 2012). Here, DJ95 was evaluated in binding and adverse drug enzyme uptake assays performed by Eurofins Cerep Panlabs. DJ95 demonstrated minimal ng inhibition specific bindi or stimulation against 37 radioactive labeled ligand targets (Figure 6A). Additionally, DJ95 did not induce significant alterations in enzyme function for COX1, showing an inhibition or esults COX2, PDE3A, PDE4D2, Lck kinase or ACHE (Figure 6B). R stimulation higher than 50% are considered significant for test compound, none of which were observed at any of the targets studied in this screening. Specific binding assay targets and 27

28 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 -based assays are summarized in enzyme , along with the corresponding reference compound, Supplement Table 4. in vivo . DJ95 pharmacokinetics and antitumor activity in vitro data suggesting that DJ95 is potently active against melanoma Based on the preliminary cell lines and minimally interferes with off-t argets, we sought to test the activity of DJ95 in vivo . Downloaded from was well We determined that concentrations up to 30 mg/kg administered by i.p. injection daily - caused a decrease in mouse 5 days in nude mice, but additional treatments tolerated for at least molpharm.aspetjournals.org weight and decline in behavioral activity. Therefore, we scaled back the dose to half of that (15 mg/kg) for the pharmacokinetic and xenograft study. To determine if DJ95 could reach therapeutically relevant biological concentrations at doses of 15 mg/kg, we collected blood samples from 15 minutes to 24 hrs following i.p. injection of the drug and analyzed the plasma at ASPET Journals on May 8, 2019 -MS methods. The C concentration s by LC for DJ95 was 13.65 μ M and the detected max concentrations stayed above 13 μ M for at least 1.5 hrs in mouse plasma (Figure 7A). While the the course of the 24 hrs when samples were concentration of DJ95 gradually decline over 8.1 nM at 24 hrs. This data, along collected, there was still an average of 126.5 nM at 12 hrs and with the AUC (50 ,500 hr*nM) suggests acceptable exposure for DJ95 over the course of a day. We also determined additional pharmacokinetic parameters including half -life (3.28 hrs), ed that these , and clearance (0.744 L/hr/kg). We reason volume of distribution to (3.51 L/kg) supported a dos ing regimen of 5 treatments/wee k, allowing for 2 recovery days to avoid results anticancer efficacy accumulating toxicities. in vivo , x enografts were To test the of DJ95 established by subcutaneous inoculation of A375 cells, and treatment began after viable tumors developed. Groups were dosed by i.p. injection with either 15 mg/kg treatments of DJ95 or 28

29 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 eeks and a total of 10 treatment vehicle solution only. After two w s, tumor growth for the DJ95 treated group was significantl y inhibited compared to the control group, with a tumor growth s T test gave an overall P inhibition (TGI) of 61.4% (Figure 7B). A student’ value of 0.0081 based on final percent change and 0.0382 based on final tumor volume volumes (Figure 7C), compar ed to the control group. Animal behavior and mouse body weights were measured and recorded throughout the course of the experiment to assess for acute toxicities, and major Downloaded from deviations were not observed (Figure 7D). At the end of the study, tumors were resected and weighed, and there was a significant decrease in tumor weight for the DJ95 group ( P = 0.0406) study demonstrates the antitumor efficacy of DJ95 in a melanoma in vivo (Figure 7E). This molpharm.aspetjournals.org . xenograft model and supports its continued investigation as an anticancer agent capabilities DJ95 shows vascular disrupting . at ASPET Journals on May 8, 2019 In recent years, there has been extensive research on the vascular disrupting properties of tubulin targeting agents and their ability to selectively target tumor vasculature (Banerjee et al., 2016; Ji et al., 2015; Martel-Frachet et al., 2015; Su et al., 2016; Wang et al., 2012) . To see whether DJ95 in vitro -like would exhibit these characteristics, we tested the effect on the formation of capillary networks of HUVEC cells plated on m atrigel . The m atrigel basement membrane allows (Benton et endothelial cells to form tubules with tight cell- to-cell and cell -to-membrane contacts al., 2014). After 6 hrs of incubation, it was clearly observed that both DJ95 and colchicine -dependent manner (Figure 8A ). At disrupted the tube cell formation in a concentration concentrations of 100 nM and 200 nM, DJ95 disrupted networks by 35.71 ± 5.62% and 51.81 ± < 0.0001) B). This was similar to the effect observed upon colchicine P (Figure 8 3.25% ( treatment at the same concentrations , inhibiting HUVEC tube cell formation by 60.43 ± 9.04 % 29

30 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 ( < 0.0001). Since this result was observed in a short and 81.59% ± 8.01, respectively P we expect that the drug action on tube cell formation was not a result of timeframe, antiproliferative activity. We also wanted to assess the effect on tumor vasculatu re from the DJ95 treated xenografts. CD31 staining of tumor sections revealed the change in microvessels of the DJ95 treated group compared to the control group (Figure 8C.) It is apparent that the control group has more Downloaded from tact microvessels throughout the tumors, whereas DJ95 treatment induced vessel abundant and in- and decreased overall density and occupied area. Quantification of the positive fragmentation -stained area revealed that there was a 50.47% decrease in total microvessel area, CD31 molpharm.aspetjournals.org P (Figure 8 D) . Detailed i mages used for representing a significant difference ( = 0.0038) results quantification can be found in Supplement Figure 6. This finding corroborates the in vitro contribute to its anticancer and suggests that the anti- vascular capacities portrayed by DJ95 may at ASPET Journals on May 8, 2019 efficacy. 30

31 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 DISCUSSION Importance of overcoming drug resistance. Melanoma is the most aggressive form of skin cancer and is one of the most rapidly increasing cancers worldwide (Linos et al., 2009) . Malignant melanoma is characterized by resistance to chemother apy and is incurable in most affected patients (Kalal et al., 2017; Wu and Singh, 2011) . Despite recent advances in treating melanoma with targeted therapies and Downloaded from immunotherapies, significant obstacles still exist for finding satisfactory treatments. Intrinsic and acquired resistance are the major causes of treatment failure, and it is of utmost importance to discover and develop agents that can overcome drug resistance, improve response rates, and molpharm.aspetjournals.org extend survival for melanoma patients. -molecule synthesized from a series of indolyl- We previously identified DJ95, a small imidazopyridines , that targets the colchicine binding site and showed promising activity in vitro at ASPET Journals on May 8, 2019 (Hwang et al., 2015). Genetic heterogeneity among cancer cell lines of the same cancer type has emphasized the necessity to study multiple cell lines in a panel (Gillet et al., 2011). To expand on its cytotoxic potential, DJ95 was tested against our malignant melanoma cell line panel and values ranging from against a 25-100 nM, confirming its strong activity demonstrated IC 50 , to assess its variety melanoma cell lines. We also tested DJ95 against the NCI -60 cell panel efficacy against other cancers -60 cell panel includes numerous cell lines from nine . The NCI different tumor types which are extensively characterized. DJ95 had an exceptionally in low GI 50 the majority of cell lines for all cancer types. In addition to melanoma, DJ95 was particularly active against colon cancers based on the TGI and LC . 50 One of the most significant factors that limit the efficacy of chemotherapeutics is the development of MDR . One of the major culprits responsible for contributing to MDR is the 31

32 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 rs such as ABCB1 -gp/MDR1), ABCC1 ( MRP 1), and overexpression of ABC transporte (P (BCRP ) (Gottesman et al., 2002). A gents that target the taxane and vinca binding site s ABCG2 of tubulin are particularly susceptible to resistance from the overexpression ABCB1 and are (Callaghan et al., 2014; Fojo and Menefee, 2007; Xia and effectively effluxed from the cell Smith, 2012; Yusuf et al., 2003). T hese MRP agents are also substrates to several ABCC/ members that decrease their intracellular concentration s (Dumontet and Jordan, 2010). We Downloaded from tubulin inhibitors in both discovered that DJ95 had a lower resistance index than the other tested gene- transfected cell lines over expressing selected and the drug- ABCC1. While it did not show -C2 resistance to the transfected ABCB1 overexpressing cells, it did show some resistance to KB molpharm.aspetjournals.org ABCB1. Since DJ95 was considered not as a substrate of drug- selected cell line overexpressing ABCB1 based on the data that it did not stimulate ABCB1 ATPase activity, the resistance showed from KB-C2 might be contributed by the more complex MDR mechanisms in drug- selected MD -C2 cells were established n with gene- R cells in compariso transfected cells. As KB at ASPET Journals on May 8, 2019 they may have incurred additional drug- by continuous selection with colchicine (2 μg/mL), For example, mutations to the resistance mechanisms besides solely ABCB1 upregulation. colchicine binding domain on tubulin could account for the cross -resistance to DJ95 or other . Taken together, we infer 95 may be able to circumvent ABCB1- agents targeting this site that DJ or ABCC1 -mediated MDR but may have reduced efficacy in colchicine- selected drug resistant cases. Targeting mitotic machinery. The main function of microtubule targeting agents is to inhibit mitosis through disruption of such as paclitaxel . While stabilizing agents microtubule dynamics (Mukhtar et al., 2014) 32

33 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 promote polymerization by blocking the disassembly of GDP-bound tubulin and form stable tubulin polymers, destabilizing agents inhibit this assembly of tubulin into microtubules and promote depolymerization (Field et al., 2014). This phenomenon was observed in DJ95 treated , which displayed fragmented microtubules and a dramatic decrease of visible tubulin cells filaments . On the contrary, paclitaxel treated cells demonstrated rigid and condensed polymeric microtubules. Furthermore, microtubules constitute the mitotic spindle in cells undergoing Downloaded from through suppression of treadmilling and dynamic mitosis. Disruption of the mitotic spindle instability are the primary means by which microtubule inhibitors thwart cellular functions and induce apoptosis (Loong and Yeo, 2014). Typical features of mitotic spindle suppression and molpharm.aspetjournals.org aberrant formation such as multiple asters in mitotic cells were evident in the DJ95 treated cells, whereas the control cells demonstrated polar spindle formation and centrally aligned chromosomes. at ASPET Journals on May 8, 2019 Optimizing scaffolds through x-ray crystallography. The crystal structure of the T2R -TTL complex bound with DJ95 provides further evidence to the mechanism of action for the anti-cancer activity of this compound. It binds at the colchicine - and -t ubulin heterodimer. α Colchicine targets the β subunit binding pocket at the interface of β microtubule of tubulin and keeps it from adopting a straight conformation, thus inhibit ing assembly. The binding of DJ95 also blocked the curve- to-straight conformational change of tubulin by the steric clashes between DJ95 and surrounding secondary structure el ements ( Figure The 4C). Therefore, DJ95 likely shares the same inhibition mechanism as that of colchicine. cancer activity of DJ95, it also crystal structure not only provides the structural basis for the anti- presents opportunities for designing better analogues of this compound that may show improved 33

34 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 potency. For example, replacing a carbon atom that faces residue Asn347 by a hydrogen bond donor (NH) could pick up an additional hydrogen bond with the main chain C=O of Asn347. Minimizing failure in drug development. Reducing the attrition rate of potential pharmaceutical drug candidates i s an important step in drug discovery and development. Identification of off- target , adverse drug reactions to important Downloaded from pharmacological targets such as GPCRs, drug transporters, ion channels, nuclear receptors, kinases and non- kinase enzymes can aid in determining if a dr ug may elicit side effects that . Use of predictive safety panels that implement assays advancement would prevent its further to molpharm.aspetjournals.org identify significant off -target interactions of drug candidates is supported by major eca, GlaxoSmithKline, Novartis and Pfizer (Bowes pharmaceutical companies such as AstraZen et al., 2012). DJ95 underwent this pharmacological profiling in Eurofins SafetyScreen44™ at ASPET Journals on May 8, 2019 Panel. DJ95 did not show significant inhibition or activation to any of the 44 physiologically important targets tested in the screening. This suggests that DJ95 minimally interacts with or potentially cause adverse drug reactions. We therefore activates some of the key targets that proceeded with in vivo studies and found that DJ95 effectively inhibited melanoma tumor growth at a 15 mg/kg dose. While colchicine is known to cause significant toxicity, no toxic side effects treated groups. based on animal body weight and behavior were observed in the DJ95 DJ95 disrupts tumor vasculature blood vessels to a reliable system of It is well-known that tumor growth and metastasis require support the tumor microenvironment , and interfering with this process is an attractive strategy for inhibiting tumor growth (Banerjee et al., 2016; Matter, 2001). This has prompted research in 34

35 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 the development of anti-angiogenic and vascular disrupting agents, and many tubulin inhibitors that target the colchicine binding site demonstrate this capability (Arnst et al., 2017; Banerjee et . Microtubule stabilizing al., 2016; Galmarini et al., 2018; Ji et al., 2015; Wang et al., 2012) agents such as paclitaxel, peloruside A, and laulimalide also have reported vascular disrupting properties, but they have dose- limiting other shortcomings, such development of resistance and tox icities (Akiyama et al., 2012; Bocci et al., 2013; Chan et al., 2015; Kanakkanthara et al., Downloaded from . Here, we demonstrate the ability of DJ95 in suppressing capillary-like networks in vitro 2014) in -dependent manner. We also discovered vascular disrupting HUVEC cells in a concentration action evident in mouse xenografts after DJ95 treatment , where there was significant disturbance molpharm.aspetjournals.org in vitro and in vivo of endothelial vasculature and an overall decrease in microvessel area. These exert its anticancer data also support the notion of DJ95 as potential dual inhibitor that may iple mechanisms. effects through mult at ASPET Journals on May 8, 2019 Conclusions In summary , we report the preclinical evaluation of DJ95. DJ95 is effective against a variety of melanoma cell lines and other cancer types, significantly reduces tubulin polymerization, binds without to the colchicine binding site on tubulin, and reduces melanoma tumor growth in vivo causing toxicity ABC - , and has vascular disrupting properties. DJ95 incurred less resistance in transporter overexpressing cell lines and could prove to be an effective alternative treatment . There are currently no when other tubulin targeting agents fail to show efficacy due to MDR colchicine binding site agents approved for chemotherapy, and DJ95 shows great potential for the further development as an anticancer agent. 35

36 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 ACKNOWLEDGMENTS The authors thank Dr. Benoît Gigant (Institute for Integrative Biology of the Cell (I2BC), CEA, Sud, Universite ́ Paris -Saclay, France) and Dr. Michel O. Steinmetz (Paul CNRS, Univ. Paris- -SLD and TTL. We Scherrer Institute, Switzerland) for kindly providing the plasmids of RB3 thank Dr. Shinichi Akiyama (Kagoshima University, Japan) for kindly provided the KB-3-1, KB -C2 and KB-CV60 cell lines. We are thankful to Drs. Susan E. Bates (Columbia University, Downloaded from NY, USA) and Robert W. Robey (NIH, Bethesda, MD, USA) for generously providing the NCI- H460, NCI-H460/MX20, HEK293/pcDNA 3.1 and the transfected cell lines HEK293/ABCB1, HEK293/ABCC1 and HEK293/ABCG2-482R. We also thank Dr. Yi Xue at UTHSC for helping molpharm.aspetjournals.org Dr. Pradeep Lukka for his help wi with the IHC analyses and th the LCMS analysis of the plasma samples . Funding. at ASPET Journals on May 8, 2019 M; NIH grants This work is supported by NIH/NCI grant [R01CA148706] to WL and DD [1S10OD010678-01] and [1S10RR026377-01] to W Other support from grants includes L. [1S10OD016226-01A1]. Its contents are s olely the responsibility of the authors and do not necessarily represent the official views of the NIH. Additional support from the University of partially Tennessee College of Pharmacy Drug Discovery Center. The X-r ay work was supported by National Natural Science Foundation of China (Grant No. [81703553]) to YW, QC, and JY. No NIH funds were used for any experiments contributed by YW, QC and JY to this work. All intellectual properties are owned by the University of Tennessee Health Science acknowledge the support of American Lebanese Syrian Associated GK and SWW Center. Charities . (ALSAC) 36

37 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 AUTHORSHIP CONTRIBUTIONS ch design: Arnst, Wang, Miller, Li Participated in resear Conducted experiments: Arnst, Wang, Lei, Ma, Parke Contributed to reagents or analytical tools: Arnst, Wang, Lei, Hwang, Seagroves, Q. Chen, Yang, ZS Chen, Li Wrote o r contributed to writing of manuscript: Arnst, Lei, Kumar, White, Li Downloaded from Conflict of Interest Disclosure: Dr. Wei Li started in January 2019 to serve as a consultant to u Inc., who licensed the patent portfolios covering DJ95 and its related analogs for Ver molpharm.aspetjournals.org commercial development. Veru Inc. did not provide any financial support or have any influence on research design, experiments, data collection, data analyses, or the writing of this manuscript. at ASPET Journals on May 8, 2019 37

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45 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 -Frachet V, Keramidas M, Nurisso A, DeBonis S, Rome C, Coll JL, Boumendjel A, Martel Skoufias DA and Ronot X (2015) IPP51, a chalcone acting as a microtubule inhibitor Oncotarget 6 (16): 14669- with in vivo antitumor activity against bladder carcinoma. 14686. Matter A (2001) Tumor angiogenesis as a therapeutic target. Drug Discovery Today 6 (19): 1005- 1024. Downloaded from -Kunstleve RW, Adams PD, Winn MD, Storoni LC and Read RJ (2007) McCoy AJ, Grosse 40 (Pt 4): 658-674. Phaser crystallographic software. Journal of applied crystallography Mhaidat NM, Thorne RF, de Bock CE, Zhang XD and Hersey P (2008) Melanoma cell molpharm.aspetjournals.org ivity to Docetaxel -tubulin levels. induced apoptosis is determined by class III β - sensit (2): 267-272. 582 FEBS Letters Mitchison T and Kirschner M (1984) Dynamic instability of microtubule growth. Nature (5991): 237-242. 312 at ASPET Journals on May 8, 2019 Morris PG and Fornier MN (2008) Microtubule active agents: beyond the taxane frontier. Clinical cancer research : an official journal of the American Association for Cancer (22): 7167-7172. Research 14 Mukhtar E, Adhami VM and Mukhtar H (2014) Targeting microtubules by natural agents for (2): 275-284. Molecular cancer therapeutics 13 cancer therapy. Orr GA, Verdier -Pinard P, McDaid H and Horwitz SB (2003) Mechanisms of Taxol resistance 22 (47): 7280-7295. related to microtubules. Oncogene Otwinowski Z and Minor W (1997) [20] Processing of X- ray diffraction data collected in Methods in enzymology 276 : 307-326. oscillation mode. 45

46 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 IUBMB Pasquier E and Kavallaris M (2008) Microtubules: a dynamic target in cancer therapy. 60 (3): 165-170. life el A, Li T -W, Anreddy N, Wang D-S, Sodani K, Gadhia S, Kathawala R, Yang D-H, Cheng Pat C and Chen Z-S (2017) Suppression of ABCG2 mediated MDR in vitro and in vivo by a novel inhibitor of ABCG2 drug transport. Pharmacological research 121 : 184-193. Perez EA ( 2009a) Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on Downloaded from Molecular cancer therapeutics : mechanisms of action, clinical activity, and resistance. 1535-7163. MCT-1509-0366. -inhibiting agents based on Perez EA (2009b) Microtubule inhibitors: Differentiating tubulin molpharm.aspetjournals.org 8 mechanisms of action, clinical activity, and resistance. Mol Cancer Ther (8): 2086- 2095. Prota AE, Bargsten K, Zurwerra D, Field JJ, Diaz JF, Altmann KH and Steinmetz MO (2013) Science Molecular mechanism of action of microtubule-s tabilizing anticancer agents. at ASPET Journals on May 8, 2019 339 (New York, NY) (6119): 587-590. Rai A, Surolia A and Panda D (2012) An antitubulin agent BCFMT inhibits proliferation of 7 (8): cancer cells and induces cell death by inhibiting microtubule dynamics. PloS one e44311. Ranganathan S, Benetatos CA, Colarusso PJ, Dexter DW and Hudes GR (1998) Altered beta- resistant human prostate carcinoma cells. tubulin isotype expression in paclitaxel- British (4): 562-566. journal of cancer 77 Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A and Knossow M (2004) Insight -like domain. into tubulin regulation from a complex with colchicine and a stathmin 428 (6979): 198-202. Nature 46

47 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Robey RW, Honjo Y, van de Laar A, Miyake K, Regis JT, Litman T and Bates SE (2001) A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2). 1512 (2): 171-182. Biochimica et Biophysica Acta (BBA)-Biomembranes Rohena CC, Telang NS, Da C, Risinger AL, Sikorski JA, Kellogg GE, Gupton JT and Mooberry SL (2016) Biological Characterization of an Improved Pyrrole- Based Colchicine Site Agent Identified through Structure -Based Design. Molecular pharmacology 89 (2): 287- Downloaded from 296. Sakchaisri K, Kim SO, Hwang J, Soung NK, Lee KH, Choi TW, Lee Y, Park CM, Thimmegowda NR, Lee PY, Shwetha B, Srinivasrao G, Pham TT, Jang JH, Yum HW, molpharm.aspetjournals.org Surh YJ, Lee KS, Park H, Kim SJ, Kwon YT, Ahn JS and Kim BY (2017) Anticancer PloS one 12 activity of a novel small molecule tubulin inhibitor STK899704. (3): e0173311. Schadendorf D, Herfordt R and Czarnetzki BM (1995) P-glycoprotein expression in primary and at ASPET Journals on May 8, 2019 132 (4): 551-555. metastatic malignant melanoma. The British journal of dermatology Schmidt M and Bastians H (2007) Mitotic drug targets and the development of novel anti- mitotic Drug Resist Updat anticancer drugs. -5): 162-181. (4 10 Schwartz EL (2009) Antivascular actions of microtubule-binding drugs. Clinical cancer (8): research : an official journal of the American Association for Cancer Research 15 2594-2601. gs that target dynamic Stanton RA, Gernert KM, Nettles JH and Aneja R (2011) Dru 31 (3): 443-481. Medicinal research reviews microtubules: a new molecular perspective. 47

48 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Stengel C, Newman SP, Leese MP, Potter BV, Reed MJ and Purohit A (2010) Class III beta- tubulin expression and in vitro resistance to microtubule ta British journal rgeting agents. 102 (2): 316 -324. of cancer Su M, Huang J, Liu S, Xiao Y, Qin X, Liu J, Pi C, Luo T, Li J, Chen X and Luo Z (2016) The anti -angiogenic effect and novel mechanisms of action of Combretastatin A-4. Sci Rep 6 : 28139. Downloaded from Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C and Gottesman MM (2006) Targeting multidrug resistance in cancer. 5 (3): 219-234. Nature reviews Drug discovery Vlaming ML, Lagas JS and Schinkel AH (2009) Physiological and pharmacological roles of molpharm.aspetjournals.org CRP): recent findings in Abcg2 knockout mice. Advanced drug delivery ABCG2 (B 61 (1): 14 -25. reviews -Q, Lei Z -N, Gupta P, Zhao Y- D, Li Z- H, Liu Y, Zhang X -H and Li Wang B, Ma L -Y, Wang J -6-phenylpyrimidin derivatives containing an acy cyano Y-N (2018) Discovery of 5- lurea at ASPET Journals on May 8, 2019 -glycoprotein-mediated mutidrug moiety as orally bioavailable reversal agents against P 61 (14): 5988-6001. resistance. Journal of medicinal chemistry Wang J, Chen J, Miller DD and Li W (2014) Synergistic combination of novel tubulin inhibitor ABI -274 and vemurafenib overcome vemurafenib acquired resistance in BRAFV600E 13 (1): 16 -26. melanoma. Mol Cancer Ther Wang Y, Yu Y, Li GB, Li SA, Wu C, Gigant B, Qin W, Chen H, Wu Y, Chen Q and Yang J Nature (2017a) Mechanism of microtubule stabilization by taccalonolide AJ. communications 8 : 15787. 48

49 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Wang Y, Zhang H, Gigant B, Yu Y, Wu Y, Chen X, Lai Q, Yang Z, Chen Q and Yang J (2016) Structures of a diverse set of colchicine binding site inhibitors in complex with tubulin provide a rationale for drug discovery. The FEBS journal 283 (1): 102-111. Wang YJ, Zhang YK, Zhang GN, Al Rihani SB, Wei MN, Gupta P, Zhang XY, Shukla S, Ambudkar SV, Kaddoumi A, Shi Z and Chen ZS (2017b) Regorafenib overcomes chemotherapeutic multidrug resistance mediated by ABCB1 transporter in colorectal Downloaded from Cancer Lett : 145-154. cancer: In vitro and in vivo study. 396 Wang Z, Chen J, Wang J, Ahn S, Li CM, Lu Y, Loveless VS, Dalton JT, Miller DD and Li W (2012) Novel tubulin polymerization inhibitors overcome multidrug resistance and molpharm.aspetjournals.org reduce melano ma lung metastasis. 29 (11): 3040-3052. Pharmaceutical research Weiderhold KN, Randall-Hlubek DA, Polin LA, Hamel E and Mooberry SL (2006) CB694, a 118 (4): 1032- novel antimitotic with antitumor activities. International journal of cancer 1040. at ASPET Journals on May 8, 2019 mediated multidrug Wu CP, Hsieh CH and Wu YS (2011) The emergence of drug transporter- 8 (6): 1996-2011. Molecular pharmaceutics resistance to cancer chemotherapy. Wu S and Singh RK (2011) Resistance to chemotherapy and molecularly targeted therapies: Current molecular medicine nation therapy in malignant melanoma. rationale for combi 11 (7): 553-563. Wu X, Wang Q and Li W (2016) Recent Advances in Heterocyclic Tubulin Inhibitors Targeting -cancer agents in medicinal chemistry 16 (10): 1325- Anti the Colchicine Binding Site. 1338. 49

50 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Xia CQ and Smith PG (2012) Drug efflux transporters and multidrug resistance in acute leukemia: therapeutic impact and novel approaches to mediation. Molecular 82 (6): 1008-1021. pharmacology Yusuf RZ, Duan Z, Lamendola DE, Penson RT and Seiden MV (2003) Paclitaxel resistance: molecular mechanisms and pharmacologic manipulation. Curr Cancer Drug Targets 3 -19. (1): 1 Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 50

51 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 FIGURES LEGENDS Figure 1. DJ95 is potent against NCI- 60 cell lines. -dose assay mean graphs. Positive values indicate net cell growth, 0 represents no net NCI DTP 5 growth, and negative values indicate cell lethality. Figure 2. DJ95 inhibits cell proliferation and migration of melanoma. A) Colony formation assay of A375 c ells treated with DJ95 in 6 well plates (n=3). Cells were Downloaded from treated with indicated concentrations of DJ95 and media only was used as the control. (B) Quantification of colony area using ImageJ software. Graph is represented as area ± SD. (C) Representative images of A375 (top) and RPMI-7951 cells (bottom) in a wound healing assay molpharm.aspetjournals.org (n=3). A scratch was created through a monolayer of confluent cells, which were then treated t of the with DJ95 or control. The migrating ability was calculated from images taken at the star treatment and after 24 hr incubation with compound. The yellow outline represents the leading at ASPET Journals on May 8, 2019 edge of the area boundary as determined by ImageJ software. (D) Quantification of A375 and (E) -7951 migration represented as a percentage of the ini tial scratch area ± SD. Statistical RPMI significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison < 0.01, test, comparing each treatment group to the control group for the above experiments. (** p *** < 0.0001). < 0.001, **** p p Figure 3. DJ95 interferes with microtubule networks and mitotic spindle organization. -164 cells after (A) Microtubule networks of WM 18 hr treatment with paclitaxel or DJ95 at indicated concentrations compared to untreated cells . The fragmentation and destabilization of the microtubules is evident from DJ95 treatment, whereas paclitaxel increased polymerization and stabilization. (B) The effect on mitotic spindle organization after 18 hr treatment with 100 ession is evidenced by multiple asters and 200 nM concentrations of DJ95. Mitotic spindle suppr 51

52 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 and disorganized spindle morphology. Confocal images were obtained from Keyence BZX microscope. Tubulin (red) is visualized by -tubulin primary antibody and Alexa Fluor 647 α d with DAPI. secondary antibody and DNA (blue) is staine -TTL-DJ95 Complex. Figure 4: The Crystal Structure of the T2R (A) Quaternary structure of the T2R- TTL -DJ95 complex showing the arrangement of the protein components, the bound nucleotides and the bound DJ95. Note that DJ95 only occupies one of the Downloaded from -tubulin interfaces. (B) Close-up view of the bound DJ95 (yellow carbons) and its available α/β interactions with surrounding residues from β -tubulin (cyan). Note the hydrogen bond to the molpharm.aspetjournals.org e α/β interface. (C) Close -up -tubulin Thr179 (green) across th backbone carbonyl oxygen of α view of the superimposed DJ95 complex and the colchicine complex (PDB ID: 4O2B, grey). Note that DJ95 is slightly more extended than colchicine. (D) Close-up view of the ‘curved’ DJ95 complex superimposed on the ‘straight’ Taxol complex (PDB ID: 1JFF, wheat). The red at ASPET Journals on May 8, 2019 arrows indicate the extent of movement of b-tubulin with respect to a-tubulin upon the binding of DJ95. Note that the binding of DJ95 is not compatible with the ‘straight’ complex. core surface plasmon resonance kinetic analyses of DJ95, c -4 Bia Figure 5. olchicine, and CA interacting with tubulin . ( Binding kineti cs of DJ95 (32, 16, 8, 4, 2 μM), Colchicine (20, 10, 5, 2.5, 1.25 μM), and CA -4 20, 10, 5, 2.5, 1.25 μM) to tubulin (17,000 RU) determin ed by SPR technology. The calculated μM, equilibrium dissociation constant Kd values for DJ95, colchicine and CA-4 is 59.4 μM, 5.7 and 7.7 μM, respectively. 52

53 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Figure 6. DJ95 in binding and enzyme and uptake assays. (A) DJ95 control-specific binding as a % inhibition to the binding to a radioactively labeled ligand specific for each target. (B) DJ95 enzyme based functional a % of control enzyme activity. Results showing an inhibition or stimulation higher than 50% are considered to represent significant effects of the test compounds. Graphs are represented as the mean of duplicate assays ± SD. Screening was performed by Eurofins Cerep -Panlabs. Downloaded from in vivo . Figure 7. DJ95 inhibits melanoma tumor growth (A) Plasma concentrations at each timepoint following i. p. injection of DJ95 at 15 mg/kg concentration shown as individual replicates connected by mean (n=3 per time point). (B) A375 molpharm.aspetjournals.org melanoma xenograft model in nude mice. Mice were dosed by i.p. injection 5 times a week for 2 weeks with vehicle solution only or 15 mg/kg DJ95. Mean tumor volume is expressed as % growth compared to tumor volume at the beginning of the experiment ± SEM (n=6 for treated at ASPET Journals on May 8, 2019 group,7 for control). (C) Individual tumor volumes at the end of the experiment shown as mean ± SD. (D) Mouse weight change throughout the study shown as mean ± SD. (E) Resected tumor weight at the end of the experiment expressed as mean ± SD . Statistical significance was determined by student’s t test comparing treatment group to the vehicle control group (* p < 0.05, < 0.01). ** p Figure 8. DJ95 targets tumor vascular and capillary network formation. (A) Representative images of HUVEC capillary formation on matrigel. Images were taken after Quantification of total 6 hr incubation with indicated concentrations of DJ95 or colchicine. (B) tube length using Image J analysis expressed as mean ± SD (n=4). Statistical analysis was performed using one-way ANOVA comparing each group to the control. (C) Representative 53

54 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 immunohistochemistry images of CD31 stained tumor sections taken at 10x. (D) Positive stained area calculated for tumor sections at 20x magnification (5 images per tumor, 3 separate tumors per group) represented as mean pixels ± SD. Statistical significance was determined by a student’s t test without correction for multiple comparisons. (** p < 0.01, *** p < 0.001, **** p < 0.0001). Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 54

55 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 TABLES . Table 1. Cytotoxic effects of DJ95 against malignant melanoma cancer cell lines a ± SD (nM) IC 50 Treatment RPMI -7951 WM A375 WM115 SK -MEL -1 -164 DJ95 24.7 ± 4.9 58.9 ± 13.3 99.8 ± 16.8 78.7 ± 16.5 68.2 ± 12.4 Colchicine 10.6 ± 1.8 11.1 ± 0.7 10.1 ± 0.7 9.3 ± 1.0 11.5 ± 2.5 Paclitaxel 2.5 ± 1.0 3.4 ± 0.8 8.0 ± 1.0 3.3 ± 1.6 1.8 ± 0.8 Downloaded from a IC values represent the mean ± SD of three independent experiments performed in 50 quadruplicate. molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 55

56 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 , ABCC1 - Table 2. Cytotoxic effects of DJ95 against parental and gene transfected ABCB1- -overexpressing cancer cell lines. or ABCG2 b IC ± SD (nM) 50 Treatment HEK293/ABCB1 HEK293/ABCC1 HEK293/ABCG2 - R482 HEK293/pcDNA3.1 DJ95 330.0 ± 44.1 314.7 ± 73.1 1213.9 ± 241.6 2869.2 ± 123.3 34.6 ± 6.3 3496.0 ± 657.8 >10000 >10000 Paclitaxel Colchicine 112.1 ± 41.5 >10000 >10000 >10000 Downloaded from Vincristine 99.4 ± 23.1 >10000 >10000 >10000 — a 108.0 ± 27.5 — 1209.7 ± 111.4 Mitox ntrone 2510.1 ± 362.6 2583.1 ± 474. 5 Cisplatin 1 ± 151.8 2475.3 ± 127.4 2584. b molpharm.aspetjournals.org values represent the mean ± SD of three independent experiments performed in triplicate. IC 50 at ASPET Journals on May 8, 2019 56

57 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 of DJ Table 3. Cytotoxic effects -, ABCC1 - or 95 against parental and drug- selected ABCB1 ABCG2 -overexpressing cancer cell lines. b ± SD (nM) IC 50 - NCI Treatment -C2 KB -CV60 KB NCI -H460 -3-1 KB H460/MX20 (ABCC1) (ABCB1) (ABCG2) 95 23. 5 ± 2.2 719.5 ± 181.5 DJ 35.9 ± 4.5 673.0 ± 111.7 5361.5 ± 645. 5 Paclitaxel 9.2 ± 2.1 1918.5 ± 106.3 120.0 ± 14.6 >10000 >10000 7 Colchicine 7445.3 ± 446.3 91.7 ± 7. 23.9 ± 4.9 >10000 >10000 Downloaded from >10000 748.8 ± 99.8 218.6 ± 1.9 >10000 Vincristine 1.3 ± 0.2 a ntrone — — — 118.7 ± 40.2 2456.5 ± 420.4 Mitox 7 2871.2 ± 115. 3 1796.5 ± 198.8 2226.6 ± 179.5 2870.6 ± 281.7 2024.5 ± 463. Cisplatin b values represent the mean ± SD of three independent experiments performed in triplicate. IC 50 molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 57

58 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 ray data collection and refinement statistics. Tubulin-RB3_SLD- Table 4. X- TTL complex resolution shell. bound with DJ95 (PDB ID: 6NNG). Values in parentheses are for highest- Data collection P2 2 2 Space group 1 1 1 Cell dimensions a , , c (Å) 105.42 157.98 182.84 b Downloaded from 90 90 90 α, β, γ ( ) ° 50 - 2.4 (2.44 - 2.4) Resolution (Å) R 0.092 (0.922) meas, 20.89 (2.14) I ) I/ σ ( molpharm.aspetjournals.org 99.2 (99.5) Completeness (%) 6.6 (6.4) Redundancy Refinement Resolution (Å) 48.25 - 2.4 (2.48 - 2.4) 117909 (10915) No. reflections at ASPET Journals on May 8, 2019 / 0.1866 / 0.2253 R R free work 17724 No. atoms Protein 16956 215 Ligand/ion Water 553 B factors 49.92 Protein 52.57 Ligand/ion Water 43.07 R.m.s. deviations Bond lengths (Å) 0.008 1.05 ) Bond angles ( ° Ramachandran Plot 98.04 Favored (%) 1.91 Allowed (%) 0.05 Outliers (%) 58

59 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 FIGURES Figure 1. Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 59

60 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Figure 2. Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 60

61 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Figure 3. Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 61

62 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Figure 4. Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 62

63 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Figure 5. Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 63

64 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Figure 6 . Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 64

65 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Figure 7 . Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 65

66 Molecular Pharmacology Fast Forward. Published on May 1, 2019 as DOI: 10.1124/mol.118.114801 This article has not been copyedited and formatted. The final version may differ from this version. MOL#114801 Figu re 8 . Downloaded from molpharm.aspetjournals.org at ASPET Journals on May 8, 2019 66

67 Supporting information C olchicine binding site agent DJ95 overcomes drug resistance and exhibits antitumor efficacy 2 ,3 1 4 1 5 , , Yuxi Wang Authors: Kinsie Zi - Ning Lei E. , Dong - Jin Hwang , Gyanendra Kumar Arnst , 5 1 6 2 2 , , Qiang Chen Dejian Ma , Jinliang Yang Tiffany N. , , Deanna N. Parke Stephen W. White 4 1 6 1 Duane D. Miller* , , Zhe - Sheng Chen , and Wei Li* Seagroves 1 Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, the University of 2 Tennessee Health Science Center, Memphis, TN. State Key Laboratory of Biotherapy and 3 Cancer Center, Collaborative Innovation Center of Biotherapy, Department of Respiratory 4 Medicine, West China Hospital, Sichuan University Department of , Chengdu, China. Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s U niversity, 5 Queens, NY. Department of Structural Biology, St. Jude Children’s Research Hospital, 6 Department of , the University of Tennessee Health Science Center, Memphis, T N. Pathology . Memphis, TN - 1 S

68 Supplement Table . Multiple reaction - monitoring (MRM) parameters based compound 1 . optimization Transition (m/z) T Analyte DP (V) CE (V) CXP (V) (min) R DJ95 400.7/340.3 5.38 196 39 22 37 ABI 231 (IS) 378.3/210.2 5.10 186 - 12 T Retention time; DP, declustering potential; CE, collision energy; CXP, collision cell exit R potential; IS internal standard Supplement . Melanoma cell lines contain genomic mutations in the following genes 2 Table according to the Sanger COSMIC database: BRAF , CKD4, CTNNB1, CDKN2A, NRAS, PTEN, TP53, and PPP6C. Cell line site Reported gene mutations A375 primary BRAF, CDKN2A, PPP6C 7951 RPMI - metastatic BRAF, CDKN2A, PTEN, TP53 WM 164 metastatic BRAF, CDKN2A, TP53, - - 115 primary BRAF, NRAS, CDKN2A, PTEN, WM BRAF, CDK4, CTNNB1, PPP6C SK - MEL - 1 primary k http://cancer.sanger.ac.u - 2 S

69 Supplement Table Cytotoxic activities of DJ95 against NCI60 human cancer cell lines. GI 3 , . 50 TGI, and LC - DTP five - dose assay and the mean growth % values were obtained from the NCI 50 - was obtained from the one dose assay. (NM) TGI (μM) GI (μM) MEAN PANEL/CELL LINE LC 50 50 GROWTH % Leukemia <10.0 12.3 5.3 >100.0 - CEM CCRF 48.7 - <10.0 <0.01 >100.0 60(TB) HL - 2.3 - 14.4 <10.0 >100.0 K - 562 >100.0 <10.0 7.2 18.5 - MOLT 4 - 43.0 44.7 5.1 >100.0 - RPMI 8226 12.0 >100.0 <10.0 - 4.9 SR NSCLC 28.5 - 11.2 1.8 7.1 A549/ATCC 5.9 40.7 30.9 EKVX <10.0 2.7 11.2 - 19.6 - HOP 62 8.6 - 309.0 5.0 70.8 92 HOP - 1174.9 19.3 3.0 >100.0 H226 NCI - 26.1 81.3 <10.0 4.2 - NCI H23 44.7 <10.0 11.7 45.0 NCI - H322M 2.6 <10.0 1.0 6.3 - NCI 460 <10.0 23.4 - 59.5 NCI - H522 Colon cancer 3.2 <10.0 <0.01 71.5 - COLO 205 <10.0 1.4 5.1 - 19.2 2998 HCC - <10.0 1.1 5.5 4.4 - HCT 116 <10.0 1.3 6.2 - 7.0 - 15 HCT <10.0 1.1 10.7 - 37.8 HT29 1.5 <10.0 9.8 3.6 KM12 5.0 <10.0 1.6 - 15.6 620 SW - CNS cancer 60.3 <10.0 3.2 19.3 - SF 268 59.4 - <10.0 3.6 <0.01 - SF 295 <10.0 12.9 - 19.5 <0.01 SF - 539 34.8 <10.0 15.8 93.3 19 - SNB <10.0 6.6 61.7 18.9 75 SNB - 1.9 8.3 - 16.3 <10.0 U251 Melanoma 67.5 <10.0 1.5 4.5 - LOX IMVI 3.2 1288.2 7.9 21.7 MALME - 3M <10.0 5.9 4.3 M14 <10.0 <0.01 >100 5.2 MDA - MB - 435 <10.0 3.1 20.4 15.2 MEL - 2 - SK 2.9 7.9 42.1 MEL SK - 28 - 38.4 - 3.1 0.6 <10.0 5 - MEL - SK 8.1 27.4 2.6 <10.0 62 - UACC - 3 S

70 Supplement continued 3 Table mean growth % (n M) TGI (μM) GI (μM) Panel/Cell line LC 50 50 Ovarian cancer 29.5 15.1 61.7 3.8 IGROV1 <10.0 4.1 2.2 3 - OVCAR 30.9 17.0 56.2 39.3 4 OVCAR - 1288.2 12.0 44.7 41.5 5 OVCAR - <10.0 11.2 >100.0 12.4 8 OVCAR - 12.7 >100.0 <10.0 10.7 - NCI/ADR RES <10.0 5.6 30.9 1.3 SK - OV - 3 Renal cancer 56.0 - 9.1 <10.0 2.0 0 – 786 2.8 21.4 <10.0 3.0 ACHN 29.7 38.9 11.2 <10.0 1 CAKI - <10.0 10.0 45.7 30.9 SN12C 7.9 1122.0 49.0 10.7 TK 10 - 13.6 35.5 <10.0 3.0 31 UO - Prostate Cancer 0.9 - 34.7 <10.0 4.8 - PC 3 18.2 38.0 <10.0 11.5 145 - DU Breast cancer 2.9 56.2 2.1 49.0 MCF7 20.3 - 30.2 1.9 <10.0 231 - MB - MDA 31.1 >100.0 10.2 <10.0 HS 578T 18.4 10.5 2.7 <10.0 - BT 549 53.7 1258.9 4.4 30.5 T - 47D - 36.7 17.8 1.5 6.9 MDA - MB - 468 - 4 S

71 Table Supplement Assay and reference compound for safety screen data . 4 REFERENCE COMPOUND ASSAY NECA A2A (h) (agonist radioligand) alpha 1A (h) (antagonist radioligand) WB 4101 alpha 2A (h) (antagonist radioligand) yohimbine beta 1 (h) (agonist radioligand) atenolol beta 2 (h) (agonist radioligand) ICI 118551 BZD (central) (agonist radioligand) diazepam CB1 (h) (agonist radioligand) CP 55940 CB2 (h) (agonist radioligand) WIN 55212 - 2 CCK 8s - CCK1 (CCKA) (h) (agonist radioligand) SCH 23390 D1 (h) (antagonist radioligand) - 7 - OH DPAT D2S (h) (agonist radioligand) ETA (h) (agonist radioligand) endothelin - 1 NMDA (antagonist radioligand) CGS 19755 H1 (h) (antagonist radioligand) pyrilamine cimetidine H2 (h) (antagonist radioligand) clorgyline MAO - A (antagonist radioligand) M1 (h) (antagonist radioligand) pirenzepine M2 (h) (antagonist radioligand) methoctramine specific M3 (h) (antagonist radioligand) 4 - DAMP binding assay alpha 4beta 2 (h) (agonist radioligand) nicotine delta 2 (DOP) (h) (agonist radioligand) DPDPE kappa (KOP) (agonist radioligand) U 50488 DAMGO mu (MOP) (h) (agonist radioligand) HT1A (h) (agonist radioligand) 8 - OH 5 DPAT - - - HT1B (antagonist radioligand) serotonin 5 - HT2A (h) (agonist radioligand) (±)DOI 5 - (±)DOI HT2B (h) (agonist radioligand) 5 - HT3 (h) (antagonist radioligand) MDL 72222 5 dexamethasone GR (h) (agonist radioligand) AR (h) (agonist radioligand) mibolerone [d(CH2)51,Tyr(Me)2] - V1a (h) (agonist radioligand) AVP Ca2+ channel (L, dihydropyridine site) (antagonist radioligand) nitrendipine - dendrotoxin KV channel (antagonist radioligand) alpha (site 2) (antagonist radioligand) veratridine Na+ channel NE transporter (h) (antagonist radioligand) protriptyline DA transporter (h) (antagonist radioligand) BTCP 5 - HT transporter (h) (antagonist radioligand) imipramine COX1 (h) diclofenac NS 398 COX2 (h) - enzyme milrinone PDE3A (h) based rolipram PDE4D2 (h) assay Lck kinase (h) staurosporine acetylcholinesterase (h) neostigmine - 5 S

72 Supplement Figure 1 Western blot showing the elevated protein concentrations for each of the . - selected and expressing ABCB1, ABCC1, and ABCG2. - transfected cell lines over - drug gene Primary antibodies against ABCB1 (#P7965, Sigma - Aldrich, St. Louis, MO ) , ABCC1 (#72202, Cell Signaling Technology, Danvers, MA ) , ABCG2 (#MAB4146, Sigma - Aldrich, St. Louis, Rockford, IL - MO ) and GAPDH ( # MA5 - 15738 , Thermo Fisher Scientific , HRP ) were used. linked s - mouse IgG (#7076) and anti - rabbit IgG (#7074) from econdary antibodies used were anti Cell Signaling Technology, Danvers, MA . 95 on ABCB1 or ABCG2 Supplement Figure 2 . Effect of DJ vanadate - sensitive ATPase activity. (A) ABCB1 mediated ATP h ydrolysis in the presence of DJ 95 or positive substrate - paclitaxel (0 - 10,000 nM). (B) ABCG2 - mediated ATP h ydrolysis in the presence of DJ 95 or positive substrate topotecan (0 - 10,000 nM). Data are plotted with mean and error bars depict SD obtained from three independent experiments. - 6 S

73 Supplement Figure 3 . NCI - 60 data for DJ95 . (A) Sensitivity of NCI cancer cell lines to DJ95. each cell line 10 μM of DJ95 was used against cell lines to compare the growth percentage of - drug control, and relative to the time zero number of cells. The percentage relative to the no growth is represented by the horizontal bar, showing both growth inhibition (values between 0 < and 100) and lethality ( 0). (B) Average growth per centage for each cancer type represented as - response curves for DJ95 against melanoma cancer cell lines. Dose mean ± SEM. (C) - 7 S

74 - Supplement Figure 4 . Scratch assay quantification . A375 and RPMI 7951 migration - ical significance was determined by two represented as total area in scratch channel ± SD. Statist way ANOVA followed by Dunnett’s multiple comparison test, comparing each treatment group to the control group at that specific time point. There was no significant difference for the area at < 0.0001). time zero for any group. (* p < 0.05, **** p - 8 S

75 . Electron density maps for DJ95. (A) 2Fo Fc map contoured at 1 sigma. Supplement Figure 5 - (B) Simulated annealing omit map contoured at 3 sigma. - 9 S

76 . Supplement Figure 6 Images obtained from immunohistochemistry CD31 stained tumor 20x magnification used for quantification of positive stained area for (A) control sections at group and (B) DJ95 treated group. Five images were obtained from 3 separate tumors per group. Each row represents a separate tumor. Necrotic tissue area was avoided. - 10 S

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