Translational Science

A Potent, Metabolically Stable Tubulin InhibitorTargets the Colchicine Binding Site andOvercomes Taxane ResistanceKinsie E. Arnst1, Yuxi Wang2,3, Dong-Jin Hwang1, Yi Xue1, Terry Costello4,†,David Hamilton4, Qiang Chen2, Jinliang Yang2, Frank Park1, James T. Dalton5,Duane D. Miller1, and Wei Li1

Abstract

Antimitotics that target tubulin are among the most usefulchemotherapeutic drugs, but their clinical activity is oftenlimited by the development of multidrug resistance. We recent-ly discovered the novel small-molecule DJ101 as a potent andmetabolically stable tubulin inhibitor that can circumvent thedrug efflux pumps responsible for multidrug resistance ofexisting tubulin inhibitors. In this study, we determined themechanism of action of this drug. The basis for its activity wasilluminated by solving the crystal structure of DJ101 in com-plex with tubulin at a resolution of 2.8Å. Investigations of thepotency of DJ101 in a panel of human metastatic melanomacell lines harboring major clinically relevant mutations definedIC50 values of 7–10 nmol/L. In cells, DJ101 disrupted micro-

tubule networks, suppressed anchorage-dependent melanomacolony formation, and impaired cancer cell migration. Inmelanoma-bearing mice, DJ101 administration inhibitedtumor growth and reduced lung metastasis in the absence ofobservable toxicity. DJ101 also completely inhibited tumorgrowth in a paclitaxel-resistant xenograft mouse model ofhuman prostate cancer (PC-3/TxR), where paclitaxel was min-imally effective. Our findings offer preclinical proof of conceptfor the continued development of DJ101 as a next-generationtubulin inhibitor for cancer therapy.

Significance: These findings offer preclinical proof of concept forthe continued development of DJ101 as a next-generation antitu-bulin drug for cancer therapy. Cancer Res; 78(1); 265–77. �2017 AACR.

IntroductionMicrotubules are components of the cytoskeleton that are

involved in a multitude of essential cellular functions includingmitosis, maintenance of cell shape, intracellular transport,motility, and cell signaling (1). They are composed of a- andb-tubulin heterodimers that readily polymerize and depolymer-ize in cells. Tubulin polymerization dynamics is an attractivecancer drug target (2). Tubulin inhibitors are generally classifiedas microtubule-stabilizing or -destabilizing agents based onwhether they promote tubulin polymerization or depolymeri-zation. FDA-approved stabilizing agents targeting the taxane-binding site (e.g., paclitaxel, docetaxel, and epothilones) and

destabilizing agents targeting the vinca alkaloid binding site(e.g., vinblastine, vincristine, and vinorelbine) are already avail-able for clinical use, while compounds targeting the colchicinebinding site (e.g., CA-4P) are being evaluated in preclinicalstudies (3–6). However, the clinical efficacy of the taxanes andvinca alkaloids is often limited by ATP-binding cassette (ABC)transporter mediated drug efflux pumps, including P-glycopro-tein (P-gp), breast cancer–resistant proteins (BCRP), and mul-tidrug-resistant proteins (MRP1 or MRP2; refs. 3, 7–9). Tumorcells exhibiting overexpression of the class III b-tubulin isoformalso demonstrate resistance to these agents (10–12). Extensiveliterature reports indicate that compounds interacting withthe colchicine binding site are much less sensitive to theseclinically observed mechanisms of resistance (13), suggestingthat the development of anti-tubulin agents targeting thecolchicine-binding site can overcome limitations associatedwith existing tubulin inhibitors and improve clinical outcome.

While colchicine itself is an approved drug for gout, it is notapproved for cancer therapy due to its toxic side effectswhich include neutropenia, gastrointestinal upset, bone mar-row damage, and anemia (14). Other compounds targeting thecolchicine site have been developed and many of them havebeen or are currently being evaluated in clinical trials forcancer. We previously reported the discovery of diaryl-ketonechemotypes, including a phenyl ring as linker (I-387; ref. 15),4-substituted methoxybenozyl aryl thiazoles (SMART; ref. 16),phenylaminothiazoles (PAT; ref. 17), arylbenzoylimidazoles(ABI; ref. 9), and reverse arylbenzoylimidazoles (RABI; ref. 18)that interfere with tubulin polymerization by binding to thecolchicine domain. While these compounds displayed potent

1Department of Pharmaceutical Sciences, College of Pharmacy, University ofTennessee Health Science Center, Memphis, Tennessee. 2State Key LaboratoryofBiotherapyandCancerCenter, Collaborative InnovationCenter of Biotherapy,West China Hospital, Sichuan University, Chengdu, China. 3Department ofRespiratory Medicine, West China Hospital, Sichuan University, Chengdu, China.4Department of Comparative Medicine, College of Medicine, the University ofTennessee Health Science Center, Memphis, Tennessee. 5College of Pharmacy,The University of Michigan, Ann Arbor, Michigan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

†Deceased.

Corresponding Author:Wei Li, University of Tennessee Health Science Center,881 Madison Avenue, Room 561, Memphis, TN 38163. Phone: 901-448-7532; Fax:901-448-6828; E-mail: wli@uthsc.edu; and Duane D. Miller, dmiller@uthsc.edu

doi: 10.1158/0008-5472.CAN-17-0577

�2017 American Association for Cancer Research.

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anticancer activity in a number of human melanoma andprostate cancer xenograft models, the ketone moiety in theirstructure presented a metabolically labile site (19). Furtherstructural optimization to incorporate this metabolic softspot into a stable ring produced a novel class of indolyl-imidazopyridines (IIP), with DJ101 (structure shown inFig. 1A) identified as the lead compound (20, 21). DJ101 notonly possessed excellent metabolic stability as we designed,it also showed improved anticancer potency and effectivenessin overcoming P-gp–mediated multidrug resistance (MDR) anda number of additional mechanisms responsible for paclitaxelresistance (21–23).

To further preclinically evaluate DJ101 and develop it as thefirst member of this new generation of tubulin inhibitors, wedescribe our efforts in this report to determine its molecularinteractions by obtaining the high-resolution crystal structureof DJ101 in complex with tubulin proteins; demonstrate thebroad in vitro potency of DJ101 against a diverse panel of cancercell lines; confirm its mechanism of action by examining itseffects on microtubule morphology, cell migration, and clono-genic potential; evaluate its preclinical efficacy in suppressingmelanoma tumor growth and metastasis using melanomamouse models; show that DJ101 possesses a good safety profilewith off-target primary screening, which suggested it may havenegligible off-target effects for major physiologically importanttargets; and finally, demonstrate its efficacy in vivo for over-coming resistance using parental PC-3 and paclitaxel-resistantPC-3/TxR xenograft models.

Materials and MethodsCell culture and reagents

Humanmelanoma cell lines A375, SK-MEL-1, RPMI 7951, andWM-115 (ATCC) were cultured in DMEM supplemented with10% (v/v) FBS (Atlanta Biologicals) and 1% antibiotic/antimy-cotic mixture (Sigma-Aldrich). Murine melanoma B16F10 cells(ATCC) were cultured in minimum essential medium (Invitro-gen), supplemented with 5% heat-inactivated Hyclone FBS(Thermo Scientific), 1% antibiotic/antimycotic mixture (Sigma-Aldrich), 1% MEM-sodium pyruvate (Invitrogen), 1% MEM-vitamin (Invitrogen), L-glutamine (2mmol/L final concentration;Invitrogen), and 1% MEM NEAA (Invitrogen). Parental prostatecancer PC-3, its paclitaxel-resistant daughter linePC-3/TxR, paren-tal prostate cancer DU-145, and its docetaxel-resistant daughterline DU-145/TxR were gifts from Dr. Evan Keller at the Universityof Michigan Medical School (Ann Arbor, MI). PC-3 and DU-145cell lines were cultured in RPMI1640 media (Gibco by LifeTechnologies) supplemented with 10% (v/v) FBS (Atlanta Bio-logicals) and 1%antibiotic/antimycoticmixture (Sigma-Aldrich).Taxane-resistant PC-3/TxR and DU-145/TxR cell lines were cul-tured in the same media and additionally supplemented with10 nmol/L paclitaxel or docetaxel, respectively. Paclitaxel ordocetaxel was not included in the media for PC-3/TxR or DU-145/TxR for at least oneweek prior to in vitro and in vivo testing. Allcell lines were authenticated by ATCC by short tandem repeatprofiling. Cultures were maintained to 80%–90% confluency at37�C in a humidified atmosphere containing 5% CO2. Com-pounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) to make a stock solution of 20 mmol/L. Compoundsolutionswere freshly prepared by diluting stockswith cell culturemedium before use.

Protein purification and crystallizationThe clones of the stathmin-like domain of RB3 and TTL were

generous gifts provided by Dr. Beno�t Gigant (CNRS, France)and Dr. Michel O. Steinmetz (PSI, Switzerland). The expres-sion and purification of proteins were described previously(5, 24, 25). The method of vapor diffusion was applied togenerate crystals of T2R-TTL, following the detailed procedurewhich was reported previously (24, 26, 27). The compoundDJ101 was dissolved in DMSO to 10 mmol/L concentration.A small amount of DJ101 solution (0.1 mL) was soaked intocrystals under microscope, and the soaked crystals were trans-ferred into an incubator. After overnight incubation at 20�C,the soaked crystals were flash cooled in liquid nitrogen fordata collection.

X-ray data collection, structure solution, refinement, andanalysis

X-ray diffraction data were collected at beamline BL19U1 atSSRF (The Shanghai Synchrotron Radiation Facility, NationalCenter for Protein Science Shanghai, Institute of Biochemistryand Cell Biology, Chinese Academy of Sciences, China). Thenative dataset was collected at a wavelength of 0.97853 Å usingMX225 CCD detector. Data were indexed, integrated, and scaledusing the programHKL2000. The initial phase was determined bymolecular replacement using the apo structure T2R-TTL (PDBcode: 4I55) as the searching model. The model was further builtmanually with COOT and refined with Refmac5. The quality ofthe final model was checked by PROCHECK and showed goodstereochemistry according to the Ramachandran plot (28, 29).The data collection and refinement statistics are summarizedin Table 1. PyMol was used to generate all the figures.

Cytotoxicity assayA375, RPMI7951, WM115, SK-MEL-1, PC-3, DU-145, PC-3/

TxR, and DU-145/TxR cells were seeded in 96-well plates at aconcentrationof 1,000–5,000 cells perwell, dependingon growthrate of the cell line. After overnight incubation, the media wasreplaced and cells were treated with the test compounds at tenconcentrations ranging from 0.03 nmol/L to 1 mmol/L plus avehicle (DMSO) control for 72 hours in four replicates. Followingtreatment, the MTS reagent (Promega) was added to the cells andincubated in dark at 37�C for at least 1 hour. Absorbance at 490nm was measured using a plate reader (BioTek Instraments Inc.).IC50 values were calculated by nonlinear regression analysis usingGraphPad Prism (GraphPad Software). In addition, DJ101 wasevaluated at five concentrations on the NCI-60 cell line panel bythe National Cancer Institute Developmental Therapeutics Pro-gram (NCI/DTP).

Microtubule imaging using immunofluorescencemicroscopy

A375 and RPMI7951 cells were seeded on glass coverslips in 6-well plates (5 � 105 cells/well) and incubated overnight. Cellswere treated with specified concentrations of DJ101, docetaxel, orvehicle (DMSO) control for 18 hours. Microtubules were visual-ized after incubating with anti-a-tubulin antibody (Thermo Sci-entific) and Alexa Fluor 647 goat anti-mouse IgG (MolecularProbes). The coverslips were mounted with Prolong DiamondAntifade mounting media with DAPI (Invitrogen) and imagesacquired with a Zeiss 710 Confocal microscope and Zen imagingsoftware (Zeiss).

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Colony-forming assayA375 and RPMI7951 cells were seeded in 6-well plates (500

cells/well) in replicates of four and incubated at 37�C overnight.Cells were treated with the compound or equivalent vehicle(DMSO) control and incubated for another week. Cells were thenfixed with methanol and stained with 0.5% crystal violet. Imageswere taken and colony area was quantified with ImageJ software(NIH, Bethesda, MD).

Scratch migration assayA375 and RPMI7951 cells were seeded in 24-well plates (2 �

105 cells/well) in replicates of four and incubated overnight.Using a 200-mL pipette tip, a straight line was scratched throughthe cell monolayer to remove an area of cells, and cells werewashed several times to remove any debris and uprooted cells.Media was replaced with media containing vehicle (DMSO),25 nmol/L of DJ101, or 25 nmol/L colchicine. Images wereobtained after 0, 12, and 24 hours with Evos Fl Imaging System(Life Technologies). Analysis was performedwith ImageJ software(NIH, Bethesda, MD).

In vivo xenograft models and treatmentsAll animal experiments were performed in accordance

with the NIH animal use guidelines and protocols approv-ed by the Institutional Animal Care and Use Committee(IACUC) at the University of Tennessee Health ScienceCenter (UTHSC, Memphis, TN). We first estimated the acutemaximum tolerable dose (MTD) for DJ101 formulated inPEG300. By progressively increasing injection doses via intra-peritoneal route to ICR mice (two mice in a group; EvigoCorporation), the MTD was estimated to be at least 65 mg/kg.To ensure a safety margin during the repeated treatment, themaximum dose was scaled down to 30 mg/kg in the animalexperiments.

Nude mice, 6–8 weeks old, were purchased from Evigo. Log-arithmic growth phase A375, PC-3, or PC-3/TxR (5� 107 cells permL) cells were prepared in phenyl red-free, FBS-free media andmixed with Matrigel immediately before injection into mice.Tumors were established by injecting 100 mL of this mixturesubcutaneously in the dorsal flank of each mouse (2.5 � 106

cells). After 2 weeks, mice were randomly divided into control ortreatment groups. DJ101 or paclitaxel was dissolved in a 1:1 ratioof PEG300:PBS solution to produce desired concentrations. Thevehicle control solution was formulated with equal parts PEG300and PBS only. Doses (100 mL) of the drug treatment or vehiclecontrol were administered via intraperitoneal injection everyother day for the duration of the studies.

Tumor volume was measured three times a week with acaliper and calculated using the formula a � b2 � 0.5, wherea and b represented the larger and smaller diameters, respec-tively. Tumor growth inhibition (TGI) at the conclusion of theexperiments was calculated as 100 – 100� ((T� T0)/(C� C0)),where T, T0, C, and C0 are the mean tumor volume for thespecific group on the last day of treatment, mean tumor volumeof the same group on the first day of treatment, mean tumorvolume for the vehicle control group on the last day of treat-ment, and mean tumor volume for the vehicle control group onthe first day of treatment, respectively (30). Animal activity andbody weights were monitored during the entire experimentperiod to assess potential acute toxicity. At the end of theexperiment, mice were sacrificed and the tumors and tissues

were dissected out and fixed in 10% buffered formalin phos-phate solution prior to pathology staining analysis.

In vivo B16F10 melanoma lung metastasis model andtreatment

C57BL/6 mice from Charles River Laboratories Interna-tional, Inc., age 7–8 weeks old, were used to study theinhibition effect of DJ101 on lung metastasis of melanomacells.

Murine B16F10 melanoma cells growing in a logarithmicgrowth phase were suspended in the conditioned media at adensity of 1 � 106 per mL. Each mouse was inoculated withthe tumor cells (100 mL containing 1 � 105 cells) via thelateral tail vein. The treatment started on the third day afterthe inoculation to ensure the initiation of metastasis beforebeginning treatment. DJ101 (30 mg/kg) and vehicle wereformulated as described above. Doses (100 mL) of DJ101 orvehicle solution were administered via intraperitoneal injec-tion every other day for 2 weeks. Animal activity and bodyweights were monitored during the entire experiment periodto assess acute toxicity. Mice were sacrificed 15 days after theinitiation of the experiment, and the lungs were separated,expanded, and preserved in 10% neutral buffered formalin.The number of lung metastasis nodules was recorded. Majororgans were also preserved in the same manner for subsequenttoxicologic examination.

Histology and IHCFixed tumor xenograft tissues were embedded in paraffin.

Serial sections were obtained and stained with hematoxylinand eosin (H&E) and IHC. Staining was performed withrabbit anti-cleaved caspase-3 antibody (Cell Signaling Tech-nology Inc.) and rabbit anti-CD31 (Cell Signaling TechnologyInc.) following ABC-DAB methods. Antigen retrieval was per-formed with H-3300 antigen unmasking solution (VectorLaboratories). Microscopic images were captured with a digitalcamera at �20 magnification. Twenty to thirty images fromeach section were analyzed. Pathologic tissue sections frommajor organs (heart, liver, kidney, lung and spleen) wereexamined similarly to identify any potential drug-relatedeffects. Images were obtained with EVOS XL Core microscope(Life Technologies).

In vitro pharmacologic profiling to assess potential off-targeteffects

Assessment of potentially significant off-target effects ofDJ101 to 47 major physiologically important targets was per-formed by DiscoverX (DiscoverX Corporation) in 78 assaysusing its Safety47 Panel and standard protocols. DJ101 wasassayed at 1 mmol/L concentrations, representing at least 100�the IC50 value as determined in melanoma cell lines by thecurrent and our earlier studies for DJ101 (21).

Statistical analysisData was analyzed using Prism Software 5.0 (GraphPad

Software, Inc.). Data were provided as mean � SEM unlessotherwise indicated. The statistical significance (P < 0.05) wascalculated by one-way ANOVA followed by Dunnett multiplecomparison test, comparing each treated group to the corre-sponding control group for the in vitro colony and migration

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assays and the in vivo xenograft studies. An unpaired Studentt test was used to calculate significance for the lung metastasisstudy.

ResultsDJ101 interacts at the colchicine-binding site on tubulin

We previously reported the design and synthesis of DJ101(structure shown in Fig. 1A; ref. 21) and a number of potenttubulin inhibitors related to this scaffold (9, 16, 18). Whilemechanistic studies suggested that these molecules producetheir antitumor activities by interacting with the colchicine-binding site in tubulin, the detailed molecular interactionshave not previously been fully elucidated. Recently, high-res-olution (<3.0 Å) crystal structures of tubulin in complex withseveral known colchicine site inhibitors were reported (26, 27).Using these newly established procedures, we obtained thehigh-resolution X-ray crystal structure of DJ101 in complexwith ab-tubulin (deposited to the Protein Databank with PDBcode: 5H7O, resolution 2.8 Å). The X-ray parameters and thecrystal structure information are summarized in Table 1. Thishigh-resolution crystal structure confirms the direct binding ofDJ101 in the colchicine-binding site (Fig. 1B). DJ101 formsthree hydrogen bonds with the ab-tubulin dimer: one hydro-gen bond from T179 in loop five in the a-monomer to theimidazole nitrogen, one hydrogen bond from N349 in loopnine in the b-monomer to the indole nitrogen, and one hydro-gen bond between the oxygen of the 4-methoxyl moiety andthe thiol moiety in C241 in helix seven of the b-monomer(Fig. 1C). A tight hydrophobic "sandwich" formed by side-chains from C241, L255, L248, and N258 wraps the trimethox-yphenyl and the imidazopyridine moieties firmly in the col-chicine-binding domain. In addition, the sidechain M259 inhelix eight of the b-monomer serves as a "wedge" betweensheet 9 and DJ101 to lock its curved conformation (Fig. 1C). Itis also evident that DJ101 overlaps with colchicine at its

binding site (Fig. 1D). These results provide the first directevidence of DJ1010s direct interaction with the colchicine-binding site in tubulin to inhibit tubulin polymerization.

Figure 1.

X-ray crystal structure of DJ101 in complex with tubulinand molecular interactions between DJ101 and tubulinproteins. A, Chemical structure of DJ101. B, Surfacerepresentation of the overall structure of a-tubulin andb-tubulin. a-Tubulin is shown in black and b-tubulin isin gray. DJ101 (D01) is marked with the red circle and isshown in sphere representation (cyan). C, Detailedmolecular interactions between the DJ101 molecule(cyan sticks) and the tubulin dimer (gray cartoon).Hydrogen bonds are indicated with black dashed lines.D, Superimposition of the binding sites for DJ101–tubulincomplex (5H7O) with colchicine–tubulin complex(4O2B). DJ101 molecule is shown in cyan and colchicineis shown in yellow.

Table 1. Crystallographic data and structure refinement statistics

Ligand D01(PDB Code: 5H7O)

X-ray source SSRF-BL19U1Data collectionWavelength (Å) 0.97853Resolution range (Å) 50–2.80 (2.85–2.80)a

Space group P 212121Unit cell (Å, �) 105.2, 157.0, 182.6Total reflections 503678Unique reflections 75002Multiplicity 6.7 (6.3)Completeness (%) 100 (100)Mean I/sigma (I) 19.3 (3.0)Rmerge 0.109 (0.576)

Structure refinementR-factor/R-freeb 0.2174/0.2567RMS (bonds) 0.007RMS (angles) 1.205No. of atoms 17520Macromolecules 17435Ligand 60Waters 25Average B-factor 55.92Macromolecules 55.91Ligands (TAJ) 62.6Waters 44.0

Ramachandran plot statisticsMost favored regions (%) 92.3Allowed regions (%) 7.5Generously allowed regions (%) 0.2Disallowed regions (%) 0.0

aThe values for the data in the highest resolution shell are shown in parentheses.bRfree ¼ P

Test||Fobs| � |Fcalc||/P

Test |Fobs|, where "Test" is a test set ofabout 5% of the total reflections randomly chosen and set aside prior torefinement for the structure

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DJ101 potently inhibits cell proliferation in cell linespresenting clinically relevant gene mutations and in theNCI-60 panel

Our preliminary studies showed that DJ101 has potentcytotoxic activity against a variety of melanoma and prostatecancer cell lines (21). To expand upon this observation anddetermine its in vitro efficacy against additional metastaticmelanoma cell lines that harbor clinically relevant mutations,we first performed cytotoxicity assays for DJ101 against apanel of human metastatic melanoma cell lines includingA375, RPMI7951, WM-115, and SK-MEL-1 (Table 2). Thesecell lines possess diverse genetic complexity and containgenomic mutations in one or more of the following genes:BRAF, CTNNB1, CDKN2A, PTEN, and TP53. DJ101 demon-strated comparable potency against these lines to colchicine,with IC50 values ranging from 7 to 10 nmol/L in each of thetested cell lines. In addition, DJ101 was tested against NCI-60cell lines representing diverse types of cancer cells, where itsGI50 values were generally less than 10 nmol/L (Supplemen-tary Fig. S1).

DJ101 disrupts microtubule networksWe revealed in our previous studies that DJ101 induces a

strong depolymerizing effect on purified tubulin protein in acell-free assay (21). To characterize its effects on microtubulenetworks and cell morphology, we utilized confocal micros-copy to visualize A375 and RPMI7951 cells treated for 18hours with either DJ101 or docetaxel, a potent microtubule-stabilizing agent that promotes microtubule polymerization.The alteration in microtubule rearrangement in the treatedcells can clearly be observed (Fig. 2A). Control cells appearedto exhibit well-organized microtubule networks extendingthroughout the cell to the cell periphery. Treatment withDJ101 led to reduced and fragmented microtubule networksincorporating polymeric tubulin proteins and emitted aweaker fluorescent signal. This effect in DJ101-treated cellswas accompanied by an increase in dispersed cytoplasmicfluorescence, representing a shift to the depolymerized, solu-ble tubulin form. Conversely, treatment with docetaxel led tohyperpolymerized tubulin and resulted in the formation ofextensive microtubule bundles concentrated toward the nucle-us. The microtubule fragmentation and disorder observed byDJ101 is consistent with its mechanism of action (i.e., inhibit-ing tubulin polymerization).

DJ101 inhibits colony growth and cell migrationDJ101 was tested in a colony-forming assay to evaluate its

long-term growth-inhibitory effects on two different metastaticmelanoma cell lines (Fig. 2B). In A375 cells, treatment with a

low concentration (4 nmol/L) of DJ101 resulted in coloniescovering only 30.6% � 0.8% of the total surface area, whichwas significantly lower than the cells exposed to only thevehicle (DMSO; 75.6% � 3.7%; Fig. 2C). Colchicine wasused as a reference control because its ability to potentlyand persistently inhibit colony formation is well-documented(31, 32). The effect of DJ101 on A375 cell colony formationwas similar to colchicine at the same concentration, whichhad colonies covering a total area of 34.7% � 1.5% (Fig. 2C).Comparable reductions in colony formation were measuredin the RPMI7951 cell line treated with either DJ101 (47.1% �5.7%) or colchicine (50.9% � 3.4%), whereas controlRPMI7951 cells formed colonies occupying a surface areaof 83.2% � 2.1%. Higher concentrations (20 nmol/L) ofDJ101 or colchicine resulted in complete colony obliteration.Dunnett multiple comparison tests after one-way ANOVAanalysis gave an overall P value of less than 0.0001 for eachtreated group compared with the control, indicating that theyare statistically different.

To demonstrate that DJ101 interferes with cell migrationthrough microtubule destabilization, we performed a scratchmigration assay (Fig. 2D). After 24 hours, untreated A375 andRPMI7951 cells had nearly achieved complete closure of thewound by migrating into 86.2% � 2.2% and 93.0% � 3.6% ofthe scratch area, respectively (Fig. 2E). A375 and RPMI7951cells treated with DJ101 demonstrated impaired cell migra-tion, occupying only 40.8% � 6.0% and 24.6% � 2.4% of thescratch area, respectively. Similar but less inhibition of thescratch area was observed colchicine-treated cells, achieving52.0% � 2.7% closure for the A375 cell line and 52.3% �3.4% for the RPMI7951 cell line. The one-way ANOVAP values in both cell lines were less than 0.0001, and Dunnettmultiple comparison test against the control indicatedP values of no more than 0.001 (Fig. 2E). Taken together,our in vitro studies demonstrate that DJ101 strongly reducesaberrant cancer cell proliferation at low concentrations andhinders cell migration at least as efficiently as colchicine inmetastatic melanoma.

DJ101 inhibits melanoma tumor growth and lung metastasisin vivo

The antitumor efficacy of DJ101 was first tested in anA375 xenograft model in nude mice. After 2 weeks of treat-ment, the group of mice receiving 15 mg/kg doses of DJ101had an average tumor growth inhibition (TGI) of 66.4%,while the group receiving 30 mg/kg doses of DJ101 averaged92.8% inhibition compared with vehicle control (Fig. 3A).Dunnett multiple comparison test indicated that treatmentwith 15 mg/kg and 30 mg/kg doses of DJ101 was significantlybetter than the vehicle (P < 0.001 and 0.0001, respectively)based on the percent increase in final tumor volume. One-wayANOVA analysis gave a P value of < 0.0001 suggesting a signi-ficant difference among all groups (Fig. 3A). Mice exhibitednormal physical activity during the study and body weightsincreased slightly (Fig. 3B), indicating negligible acute toxi-cities. H&E stains of representative tumor sections in thevehicle control group showed aggressive growth with numer-ous mitotic cells in different stages (Fig. 3C). The increase ofmetaphase cells and decrease of anaphase cells in the DJ101treatment groups indicate mitotic blocking in the G2–M phase,consistent with our previous cell-cycle analysis (21). Tumor

Table 2. DJ101 showed excellent potency against a panel of metastaticmelanoma cell lines

Cell Lines IC50 � SEM (nmol/L)DJ101 Colchicine

A375 7.6 � 0.5 9.1 � 2.1RPMI7951 10.1 � 0.9 8.3 � 0.5WM115 10.3 � 1.8 8.2 � 0.7SK-MEL-1 9.6 � 0.4 9.0 � 2.5

NOTE: The cell viability after 72-hour treatment was determined using theMTS assay (n ¼ 4). Results are given as IC50 values � SEM. IC50 values werecalculated in GraphPad Prism using nonlinear regression.

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sections from both low- and high-dose treatment groupsshowed significantly less proliferation, and there were abun-dant apoptotic cells showing dense nuclear pyknosis

and cytoplasmic karyorrhexis. There was also extensive centraltumor necrosis in the treatment groups. In addition, CD31-labeled endothelial cells in the vehicle control tumor

Figure 2.

DJ101 disrupts microtubule structure and inhibits melanoma proliferation and migration. A, Confocal images of A375 (top) and RPMI7951 (bottom) melanomacells exposed to different concentrations DJ101, docetaxel, or media containing only the vehicle (DMSO) for 18 hours. Tubulin (red) is visualized by a-tubulinprimary antibody and Alexa Fluor 647 secondary antibody. DNA (blue) was stained with DAPI. Images were obtained by confocal microscopy at �63magnification. B, Representative pictures of control and compounds tested at different concentrations on A375 (top) or RPMI7951 (bottom) and cell lines.The diameter of each well was 35 mm. C, Quantification of colony area � SEM (n ¼ 4). Total colony area was determined using ImageJ software. D, Scratchassay was carried out in A375 (top) and RPMI7951 (bottom) cell lines. Wound closure was assessed at 0, 12, and 24 hours after treatment. E, Cell migrationpresented as percent wound closure � SEM (n ¼ 4). ImageJ software was used to calculate the total wound area at each of the time points. Scale bar,1,000 mm. Statistical analysis was performed by Dunnett multiple comparison test, comparing each treatment group with the corresponding control group.

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sections exhibited well-developed networks of capillaries orsmall blood vessels around tumors, while the DJ101 treatmentgroups displayed severely distorted blood vessels or absence ofCD31-positive stains, suggesting potential vascular disruptingproperties of DJ101. Finally, caspase-3 staining of tumorsections in the control group showed very few positive regions,but tumor sections from the DJ101 treatment groups showedan increase in caspase-3–positive labeled cells, confirmingenhanced apoptosis due to DJ101 treatment.

As a major obstacle in treating melanoma is tumor metas-tasis, we next assessed the efficacy of DJ101 in suppressingmelanoma metastasis using a B16F10 experimental lung metas-

tasis model in mice. After two weeks of treatment with 30mg/kgdoses of DJ101, the average number of tumor nodules thatdeveloped on the lungs was 1.7 � 0.3, whereas the vehicle-treated group averaged 10.5 � 2.0 nodules, demonstratinga 6.2 times decrease in lung metastasis for those receivingDJ101 treatment (Fig. 3D). An unpaired Student t test gave aP value of <0.001, representing a significant decrease in lungmetastasis for the treatment group (Fig. 3D). Body weights(Fig. 3E) and physical activities of mice were normal in bothgroups. These results show that DJ101 is well-tolerated fordoses up to 30 mg/kg in mice and can efficiently reduce thepotential for lung metastasis of murine melanoma.

Figure 3.

DJ101 inhibitsmelanoma tumor growthand lung metastasis in vivo. A, A375xenograft model in nude mice. Graphrepresents mean tumor volumepercent increase � SEM (n ¼ 6).Statistical significance for final tumorvolumes was determined by one-wayANOVA (P<0.0001) analysis, followedby Dunnett multiple comparison test.B, Mouse weight change in thexenograft model � SEM. C, IHC stainsof A375 tumors.A1–A3,H&E of tumors.White arrow, metaphase of mitosis;black arrow, anaphase of mitosis. Inset(2-fold) shows high power view ofanaphase and metaphase mitosis.B1–B3, CD31 expression showingblood vessel disruption in control andtreated groups. C1–C3, Expression ofcleaved caspase-3 indicative ofapoptosis in tumor tissues.Scale bar, 50 mm inA1–B3 and 25 mm inC1–C3.P¼0.007.D,B16F10melanomalung metastasis model in C57BL/6mice. Graph represents mean numberof nodules, with individual number foreachmouse plotted� 95% CIs (n¼ 11).Representative photos of lungs withmelanoma nodules (black dots) areshown below. Statistical significance(P ¼ 0.0001) was determined with anunpaired Student t test. E, Mouseweight change � SEM in themetastasis model.

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DJ101 shows no drug-related toxicity to major organs andnegligible inhibitions to major physiologically importanttargets

To assess whether DJ101 treatment produces potential organtoxicities, pathologic analysis was performed on major organscollected from both melanoma in vivo studies including theheart, kidney, liver, lung and spleen. Sections stained withhematoxylin and eosin revealed no apparent drug-related inju-ry or pathologic changes in the cellular structure of the various

tissues in both xenograft mouse models (Fig. 4A) and theexperimental lung metastasis mouse model (Fig. 4B).

In vitro pharmacologic profiling is increasingly being used toidentify undesirable off-target activity profiles early in the drugdiscovery process. To further test for potentially significant off-target effects and determine the safety profile, DJ101 wasevaluated at 1 mmol/L in the Safety47 Panel using functionalassays with all human targets for safety screening (Fig. 4C;Supplementary Table S1). This in vitro pharmacologic profiling

Figure 4.

Toxicity profile and off-target effects of DJ101. A and B, Pathologic sections of major tissues (heart, kidney, liver, lung, and spleen) obtained from in vivoA375 xenograft (A) and B16F10 lung metastasis (B) studies. Organs were stained with H&E and representative images were captured. C, In vitro pharmacologicprofiling of DJ101 to assess potential off-target effects to major targets at 1 mmol/L concentrations of DJ101 (n¼ 2). Graph represents mean percent response�range. Values in between �70% and þ70% (indicated with dashed lines) are considered insignificant.

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service includes the assessment of the functional response of 47major physiologically important targets across 78 assays to atest compound and normalized to respective controls. Only avalue higher than 70% response is considered significant.DJ101 only showed significant responses in 2 of the 78 assays,namely norepinephrine transporter (NET) blocking and gluco-corticoid receptor (GR) antagonism. As the concentration test-ed was more than 100-fold of the IC50 value for DJ101, which isexpected to be well above its practical physiologic concentra-tion, results from this Safety47 Panel screening strongly suggestthat DJ101 has minimal potential off-target effects. Collective-ly, these in vivo and in vitro findings support a good safety profilefor DJ101.

DJ101 overcomes paclitaxel resistance in vivoWe first confirmed our previous results that DJ101 maintains

efficacy in paclitaxel resistant cell lines in vitro (21), by deter-mining the potency of DJ101, paclitaxel, docetaxel, and col-chicine in the parental PC-3, DU-145, paclitaxel-resistant PC-3/TxR, and docetaxel-resistant DU-145/TxR cell lines (Supple-mentary Table S2). Paclitaxel and docetaxel outperformedDJ101 and colchicine in the drug-sensitive PC-3 and DU-145cell line. However, in the PC-3/TxR- and DU-145/TxR–treatedcells, DJ101 was the most efficacious among the four and wasroughly equipotent in its cytotoxicity to the parental cell lines,while the other three tubulin inhibitors have a significantlylarge drug-resistant index. We next compared the efficacy ofDJ101 and paclitaxel in vivo using both PC-3 and PC-3/TxRxenograft models. Both paclitaxel (15 mg/kg) and DJ101 (30mg/kg) inhibited tumor growth as determined by tumor vol-ume growth (Fig. 5A). The TGI for the group receiving paclitaxeltreatment was 101.1% and 78.8% for the DJ101-treated groupin PC-3 xenografts. ANOVA analysis followed by Dunnettmultiple comparison test of final volumes resulted in P valuesof <0.0001, suggesting significant differences. Their efficacyagainst tumors was also evident based on final tumor weight(Fig. 5B) and representative tumor images (Fig. 5C), where theDJ101- and paclitaxel-treated groups showed a 47.1% (P <0.001) and 79.3% (P < 0.0001) reduction in tumor weightcompared with the control group, respectively. No acute toxi-cities were observed on the basis of physical activity and bodyweights (Fig. 5D). In contrast, using the same dosing scheduleand frequency as we did in the parental PC-3 model, DJ101caused a TGI of 104.0%, remarkably demonstrating an overallreduction in tumor volume in the PC-3/TxR xenograft model,whereas paclitaxel only modestly inhibited tumor growth by37.8% (Fig. 5E). These results were corroborated by final tumorweights, where DJ101 caused a 77.3% reduction and paclitaxelshowed only a 35.6% reduction compared with the controlgroup (Fig. 5F). Representative tumor images are shown in Fig.5G. For both the tumor volumes and tumor weights, one-wayANOVA analysis demonstrated a difference amongst groups ofP < 0.0001, and Dunnett multiple comparison test showed amuch greater significance between control versus DJ101 (P <0.0001) and vehicle control versus paclitaxel (P < 0.05). Sim-ilarly, no acute toxicities were observed in this model (Fig. 5H).

DiscussionInterfering with tubulin dynamics is a validated approach

for anticancer treatment and many antimitotic microtubule

stabilizers and destabilizers are widely used clinically or areundergoing clinical development (33). However, many ofthese agents cause neurotoxicity, exhibit chemical instability,or have elevated metabolic clearance (19). In addition, theclinical efficacy of many FDA-approved tubulin inhibitors isoften limited by drug efflux pumps or overexpression of certaintubulin subtypes, most notably the bIII tubulin isotype (34).Design and development of new tubulin inhibitors targetingthe colchicine binding site represent an attractive approach forimproving and advancing tubulin inhibitors. We previouslyreported several indole derivatives interacting with the colchi-cine-binding site that are highly potent in vitro and metabol-ically stable (21). Herein, we focused on the most promisingcompound in this class, DJ101, and further demonstrated thehigh potency of DJ101 against a broader panel of metastaticmelanomas with varying degrees of genetic complexity basedon genomic mutations (BRAF, CTNNB1, CDKN2A, PTEN, orTP53) and the NCI-60 panel, providing additional evidence tosupport its anticancer activities.

Tubulin inhibitors interacting with the colchicine-bindingsite have diverse structures. Thus it has been very challenging toreliably decipher the molecular interactions between an inhib-itor and tubulin using molecular modeling with crystal struc-tures containing a different class of tubulin inhibitors. Anotherchallenge is that available X-ray crystal structures often havelow resolution which further impedes reliable molecularmodeling studies. In this report, we obtained the high resolu-tion X-ray crystal structure of DJ101 in complex withab-tubulin. The crystal structure confirmed the binding ofDJ101 at the colchicine-binding site in tubulin, overlappingwell with that of colchicine. The three hydrogen bonds formedbetween DJ101 and the tubulin dimer and additional stronghydrophobic interactions from surrounding residues firmlyanchored DJ101 in this colchicine binding site. Interestingly,our previous molecular modeling studies using the crystalstructure of 1SA0 showed a different binding pose for DJ101,in which the top portion of the DJ101 has a 180 degree flip(21). Examination of the crystal structures of 1SA0 and thecurrent DJ101 complex clearly revealed significant conforma-tional changes for the T4 loop in the tubulin a-subunit and T7loop in the tubulin b-subunit. It is apparent that the "closingup" of the T4 to the colchicine-binding pocket in the 1SA0structure prevented the top moiety of DJ101 in adopting its truebinding pose, and thus forced its top moiety to rotate 180degrees. It is well known that while the colchicine site canaccommodate a wide range of structurally distinct molecules(34, 35), seemingly insignificant changes to many of thesemolecules can result in a total loss of activity. Results fromthis study underline the variation of conformations accommo-dated for by a-T4 and b-T7 and their critical contributions tothe optimal bindings of different scaffolds at the colchicine sitein tubulin, as well as the importance of high-resolution crystalstructures over molecular modeling in guiding structure opti-mizations for tubulin inhibitors interacting at this site.

Taxanes, such as docetaxel, are microtubule-stabilizing agentsthat target the taxane site within the lumen of polymerizedmicrotubules and alter their conformation to the more stableGTP-bound b-tubulin structure, thereby locking them in thepolymerized state (36). Microtubule-destabilizing agents, on theother hand, exert their antimitotic effects by interfering withtubulin dynamics as opposed to simply reducing polymerized

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tubulin, leading to mitotic arrest and eventual apoptosis (36). Tofurther confirm the mechanism of action of DJ101 and evaluatethe physical change in microtubule networks, we performedimmunofluorescent imaging studies of tubulin structure using

melanoma cells that had been treated with our microtubuledestabilizing compound DJ101 or docetaxel which potentlystabilizes microtubules. In the control cells, the microtubulenetworks displayed an arrangement of organized, fibrous

Figure 5.

DJ101 inhibits PC-3 and PC-3/TxR tumor growth in vivo. A and E, Tumor volumes for PC-3 (A) and PC-3/TxR (E) xenograft model in nude mice. Graphsrepresent mean tumor volume percent increase � SEM (n ¼ 7). B and F, Final tumor weights � SEM for PC-3 (B) and PC-3/TxR (F) mice. C and G,Representative images for PC-3 (C) and PC-3/TxR (G) tumors. Mouse weight change � SEM for PC-3 (D) and PC-3/TxR (H) mice. One-way ANOVA analysiswas performed for final tumor volumes (P < 0.0001 in all cases) and weight, followed by Dunnett multiple comparison test of each treated group with thecorresponded results of vehicle group.

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microtubules extending throughout the elongated cells. Cellstreated with DJ101 displayed dispersed and disordered tubulinfragments and greater cytoplasmic fluorescence consistent withdepolymerization and elevated free tubulin. Docetaxel-treatedcells displayed dense and abundant arrays of tubulin accompa-nied by a strong fluorescence signal. The immunofluorescentimages clearly differentiated the stabilizing and destabilizingeffects on microtubule structure and cellular framework by thesetwo opposing classes of compounds.

In addition to their well-defined roles in supporting cellularstructure and their involvement in mitosis, microtubules arealso implicated in cell migration and motility. It has beensuggested that dynamic instability imposed on the microtu-bules by suppressing and interfering with tubulin behaviorcauses cell immobility, as the cell is less able to remodel andrespond to change the cell shape demanded for cell migration(32). The antimitotic and antimigratory effects of DJ101 weredemonstrated by its ability to inhibit anchorage-dependentcolony propagation and suppress cell motility in a woundhealing assay. The cessation of colony formation and cellmovement was attributed to disruption of microtubule dynam-ics, although additional studies are needed to further elucidatethe mechanism of microtubule-binding drugs on differentfacets of cellular function.

As a further validation of its anticancer potential, DJ101effectively inhibited melanoma tumor growth and lung metas-tasis in vivo in mouse models. Because antimitotic drugs exploitdifferent sites on tubulin and microtubules, synergistic combi-nations could be investigated to optimize their clinical useful-ness. This assertion is supported by a number of reportedstudies that combined paclitaxel with other tubulin targetingdrugs (37–40). It will also be important to combine a noveltubulin inhibitor such as DJ101 with a different targeted agent.Toward this end, we previously demonstrated that strongsynergy can be achieved by combining ABI-274 (a less meta-bolically stable analogue of DJ101) and vemurafenib in a BRAFinhibitor–resistant melanoma xenograft model (30). Ourongoing efforts will continue to investigate combination ther-apies to maximize the clinical potential of DJ101.

Off-target adverse drug reactions (ADR) often contribute tothe high attrition rate in the drug discovery and developmentprocess. Reducing potential off-target ADRs early on will helpto mitigate the risk of expensive failure in late stages. To assessthe safety of DJ101 and its potential off-target effects, theinhibition of DJ101 was evaluated against 47 major physio-logically important GPCRs, nuclear hormone receptors, trans-porters, ion channels, kinases, and nonkinase enzymes withSafety47� Panel. This pharmacologic profile screeningincludes the assays recommended by major pharmaceuticalcompanies including AstraZeneca, GlaxoSmithKline, Novartisand Pfizer (41). Of the 78 assays tested, DJ101 at a very highconcentration only elicited a functional response to NET andGR antagonism, suggesting a good safety profile for thisscaffold.

Finally, while taxanes are some of the most clinically effectivechemotherapy drugs available, their clinical success is oftenlimited by the emergence of intrinsic and acquired drug resis-tance (42). The most common mechanisms of resistance totaxanes are derived from the overexpression of ATP-bindingcassette proteins such as P-gp and alterations in tubulin–iso-form expression, particularly of bIII-tubulin (13). There is

evidence that suggests that tubulin-binding agents that specif-ically target the colchicine-binding site may circumvent theseresistance mechanisms (43). In the paclitaxel-resistant PC-3/TxR prostate cancer cell line, more than 200 genes are upre-gulated in addition to P-gp overexpression, which represent alarge number of paclitaxel resistance mechanisms (22, 23). Wedemonstrated that DJ101 maintains its potency in both pacli-taxel sensitive PC-3 and paclitaxel-resistant PC-3/TxR prostatecancer xenograft models in mice. Other groups have alsoreported elevated sensitivity of paclitaxel-resistant cancer cellsto colchicine-binding site drugs. One such study investigated avariety of microtubule targeting agents that bind to the taxanesite, vinca alkaloid binding site, and colchicine binding site fordocetaxel resistant MCF-7 breast cancer cells. They reported thatwhile MCF-7TXT cells demonstrated cross-resistance to vincaalkaloids, they were more sensitive to colchicine-binding siteagents including 2MeOE2, ABT-751, CA-4P, and colchicinethan the nonresistant counterpart MCF-7 cells (44). This alsosupports the notion that colchicine-binding agents such asDJ101 may be an alternative treatment when tumors acquireresistance to treatment by taxanes.

In summary, we have obtained the high-resolution X-raycrystal structure of DJ101 which represents a novel class oftubulin inhibitors targeting the colchicine-binding site. DJ101depolymerizes microtubules in vitro and disrupts micro-tubule morphology distinctly from agents that stabilize tubu-lin polymerization. It is effective against a broad panel ofmetastatic melanomas representing different gene mutationsas well as other types of cancers as revealed by the NCI-60screening. DJ101 demonstrates strong antitumor efficacy anddecreases metastasis in two melanoma mouse models withoutcausing apparent toxicity to major organs and possesses agood safety profile. Furthermore, DJ101 maintains potencyand efficacy in the paclitaxel-resistant PC-3/TxR xenograftmodel. Collectively, this preclinical evaluation and our pre-vious studies of DJ101 strongly support its further develop-ment as a new generation of tubulin inhibitor targeting thecolchicine-binding site.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerThe contents of this article are solely the responsibility of the authors

and do not necessarily represent the official views of the NIH/NCI.

Authors' ContributionsConception and design: K.E. Arnst, D.-J. Hwang, J.T. Dalton, D.D. Miller,W. LiDevelopment of methodology: K.E. Arnst, D.-J. Hwang, Y. Xue, J. Yang,J.T. Dalton, W. LiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.E. Arnst, Y. Xue, T. Costello, D. Hamilton, Q. Chen,J. Yang, W. LiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.E. Arnst, Y. Wang, D.-J. Hwang, Y. Xue, J. Yang,F. Park, J.T. Dalton, D.D. Miller, W. LiWriting, review, and/or revision of the manuscript: K.E. Arnst, Y. Wang,D.-J. Hwang, Y. Xue, F. Park, J.T. Dalton, D.D. Miller, W. LiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.E. Arnst, Y. Xue, J. Yang, W. LiStudy supervision: W. Li

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AcknowledgmentsThis work is supported by NIH/NCI grants R01CA148706 (to W. Li and

D. D. Miller) and National Natural Science Foundation of China (81572995,81703553 to J. Yang and Y. Wang).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 9, 2017; revised September 1, 2017; accepted November 1,2017; published OnlineFirst November 27, 2017.

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2018;78:265-277. Published OnlineFirst November 27, 2017.Cancer Res   Kinsie E. Arnst, Yuxi Wang, Dong-Jin Hwang, et al.   Colchicine Binding Site and Overcomes Taxane ResistanceA Potent, Metabolically Stable Tubulin Inhibitor Targets the

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