The synthetic diazonamide DZ-2384 has distinct effects on ...DZ-2384 has a wide safety margin...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

CANCER

1Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada.2Laboratory for Therapeutic Development, Rosalind and Morris Goodman CancerResearch Centre and Department of Biochemistry, McGill University, Montreal,Quebec H3G 1Y6, Canada. 3Laboratory of Biomolecular Research, Departmentof Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland.4McGill SPR-MS Facility,McGill University,Montreal, QuebecH3G1Y6, Canada. 5Depart-ment of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10561, USA. 6De-partment of Pharmacology, UT Southwestern Medical Center, Dallas, TX 75390, USA.7Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal,Quebec H3A 1A3, Canada. 8Research Institute of the McGill University Health Centre,Montreal, Quebec H4A 3J1, Canada. 9Department of Chemistry and Biochemistry, Uni-versity of California at Los Angeles, Los Angeles, CA 90095, USA.*Corresponding author. Email: [email protected] (G.J.B.); [email protected] (A.R.)

Wieczorek et al., Sci. Transl. Med. 8, 365ra159 (2016) 16 November 2016

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The synthetic diazonamide DZ-2384 has distinct effects onmicrotubule curvature and dynamics without neurotoxicityMichal Wieczorek,1 Joseph Tcherkezian,2 Cynthia Bernier,2 Andrea E. Prota,3 Sami Chaaban,1

Yannève Rolland,2 Claude Godbout,2 Mark A. Hancock,4 Joseph C. Arezzo,5 Ozhan Ocal,6

Cecilia Rocha,1 Natacha Olieric,3 Anita Hall,7,8 Hui Ding,9 Alexandre Bramoullé,2 Matthew G. Annis,7

George Zogopoulos,7,8 Patrick G. Harran,9 Thomas M. Wilkie,6 Rolf A. Brekken,6 Peter M. Siegel,7

Michel O. Steinmetz,3 Gordon C. Shore,2 Gary J. Brouhard,1* Anne Roulston2*

Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit theirclinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384,a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an un-usually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds thevinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubuledynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studiesdemonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymeriza-tion of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons.This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.

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INTRODUCTIONMicrotubules are essential in rapidly dividing cells where they form themitotic spindle, an apparatus that ensures proper chromosome segre-gation during mitosis (1, 2). As such, microtubules are the direct targetof a large group of chemotherapeutics, known as microtubule-targetingagents (MTAs), that effectively kill cancer cells as they divide [reviewedin (3)]. MTAs can bind to five distinct sites on tubulin, with the taxanesite and the vinca alkaloid site being the most prominent targets. Al-though different MTAs bind to distinct sites, cancer cell death occursby a similar mechanism with all of them: an arrest in mitosis causesprolonged activation of the spindle assembly checkpoint, which ulti-mately triggers apoptosis (4, 5).

Despite the success of MTAs in the clinic, toxic side effects oftenforce discontinuation of treatment or reduction in dose. Themost com-mon problems are neutropenia (6) and peripheral neuropathy (7). Thelatter can be especially severe when MTAs are combined with otherchemotherapeutics such as cisplatin or bortezomib, a common practice(8). Because microtubules are the backbone of neurons, it is notsurprising that MTAs would cause neuropathy, particularly in theperipheral nerves that have the longest axons (9). Nerve degenerationoften persists well beyond the treatment period, and some patients nev-er fully recover from the damage (10). There is a pressing need for im-

proved, less toxic MTAs, which would enable sustained treatments,combine well with other therapies, and reduce neuropathy.

MTAs function largely by altering the dynamics and stability ofmicrotubules. Microtubules are polymers of ab-tubulin heterodimers,which associate head-to-tail to form protofilaments and then laterallyto form a tube. Current MTAs such as paclitaxel stabilize these poly-mers, whereas MTAs such as vinblastine and vinorelbine destabilizethem. More specifically, vinca alkaloids destabilize microtubules by in-troducing a wedge at the interface between two longitudinally alignedtubulin dimers, thus inhibiting the interdimer curved-to-straighttransition that is necessary to build the microtubule lattice. Micro-tubules polymerize by the addition of tubulin to the microtubule end,which is a tapered, gently curved, and flattened-out polymer structure(11). As the microtubule grows, the protofilaments straighten (12),forming lateral bonds with adjacent protofilaments within the micro-tubule lattice. Abruptly, the polymer switches from growth to shrinkagein a “catastrophe” (13–15). As the microtubule shrinks, individual pro-tofilaments lose their lateral contacts and curve outward steeply (1).Sporadically, the rapidly shrinking microtubule will switch back to growthin a “rescue” event. Understanding the mechanism of MTAs hascontributed substantially to the fundamental principles of microtubuledynamics (16, 17). An MTA that changes microtubule dynamics differ-ently from others, even subtly, could prove more effective at arresting cellsin mitosis without disrupting microtubule function in nondividing cells.

ManyMTAs are natural products or chemicallymodified analogs ofnatural products (3). Diazonamide A (DZA) is a natural product orig-inally isolated from themarine spongeDiazona angulata (18, 19). DZAwas initially modified into the synthetic analog AB-5, which showedencouraging antitumor activity in xenograftmodels (20). Here, we pres-entDZ-2384, an optimized synthetic derivative ofAB-5,which is in pre-clinical development. Relative to earlier diazonamide analogs, itssynthesis is simplified and commercially scalable (21). DZ-2384 showspotent antitumor activity in multiple cancer models but causes negligibleside effects (including neuropathy) at effective plasma concentrations.We demonstrate that DZ-2384 binds to the vinca alkaloid site of tubulin

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in a distinctive way that unexpectedly translates into superior antitumorefficacy and safety. Using x-ray crystallography and electronmicroscopy(EM),we show thatDZ-2384 changes the curvature between two tubulindimers, causing a pronounced straightening of protofilaments. In livecells, DZ-2384 increases the rescue frequency and has a less destabilizingeffect on microtubules than vinorelbine yet is sufficiently potent to dis-rupt mitotic spindle formation. We observed similar effects in a recon-stituted system using purified tubulin. We propose that structuraldifferences betweenDZ-2384 and vinca alkaloids correlate with amilderimpact on interphase microtubules and the absence of peripheral neu-ropathy at effective plasma concentrations. Our results demonstrate thata classic therapeutic site on tubulin can be targeted in a different way,resulting in a potent yet less toxic candidate MTA for chemotherapy.

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RESULTSDZ-2384 has potent antitumor activity in multipledisease modelsTo establish the in vivo antitumor efficacy of DZ-2384, we tested italong with vinorelbine in two subcutaneous xenograft models, namely,theMIAPaCa-2model of pancreatic ductal adenocarcinoma (PDACorPDA) and the HT-29 model of colon cancer. DZ-2384 was highly ef-fective in bothmodels (Fig. 1A and table S1). DZ-2384 caused completetumor regression in the MIA-PaCa-2 model and, at 9 mg/m2, all micewere tumor-free ~3 months after treatment (Fig. 1A). Vinorelbine wasalso active in bothmodels, but at higher doses and for a shorter duration(Fig. 1B and table S1). Table 1 summarizes the minimum effective dose(MED) for both DZ-2384 and vinorelbine in theMIA PaCa-2 and HT-29 models.

We also tested the efficacy of DZ-2384 in an experimental modelof metastatic triple-negative breast cancer (MDA-MB-231-LM2),which recapitulates the later stages of the metastatic cascade (22).We injected MDA-MB-231-LM2 cells into the tail vein of severe com-bined immunodeficient (SCID)/beige mice, allowed lung metastatic le-sions to develop, and then treated the mice with DZ-2384. DZ-2384caused regression of all lung metastases starting at the lowest dosetested (Fig. 1C and fig. S1A) and significantly increased the survivalof all DZ-2384–treated groups (P < 0.0001; Fig. 1D). Some animalswere tumor-free up to 106 days after initial treatment (2 of 8 miceat 18 mg/m2 and 1 of 5 mice at 4.5 mg/m2).

Finally,we testedDZ-2384 in theRS4;11model of adult Philadelphiachromosome–negative acute lymphocytic leukemia (ALL). DZ-2384reduced the number of tumor cells in circulation on day 23 (P =0.0028) and increased survival (P = 0.0024) at doses as low as 0.7 mg/m2

(fig. S1, B and C). In each of the xenograft models described above, themice experienced less than 10% body weight loss at all DZ-2384 andvinorelbine doses tested (fig. S2, A to F). Together, these results demon-strate that DZ-2384 is potent against tumors in human xenograftmodels of several different tumor types.

DZ-2384 enhances gemcitabine activity in two mousemodels of pancreatic cancerOur results in the MIA PaCa-2 model were encouraging because pa-tients with PDA continue to have very low survival rates. Treatmentoptions are limited, but the MTA nanoparticle albumin-bound(nab)–paclitaxel was recently approved for use in combination withgemcitabine in the metastatic setting, indicating that MTAs can im-prove the outcome of PDA (23). We sought to determine whetherDZ-2384 is effective in two rigorous in vivomodels of pancreatic cancer,


either alone or in combination with gemcitabine. In a patient-derivedxenograft (PDX) model, we propagated tumors in SCID/beige mice toobtain a large cohort of tumor-bearing animals for treatment. Whereaseither gemcitabine or DZ-2384 used as single agents had insignificanteffects on tumor growth, the combination of DZ-2384 and gemcitabine(DZ-2384/gemcitabine) caused significant (P = 0.0159) tumor regres-sion (Fig. 1E). Themice lost 11% of their bodyweight after the first doseof DZ-2384 and DZ-2384/gemcitabine but then quickly recovered (fig.S2G). One animal inexplicably died in the DZ-2384–treated group onday 29, whereas viability was 100% in the DZ-2384/gemcitabine–treated group for the duration of the experiment.

Next, we tested DZ-2384 and gemcitabine in a genetically engineeredmouse model (GEMM) of PDA, specifically KIC [p48(Cre); LSL-Kras(G12D); Cdkn2a(f/f)] mice that express Rgs16::GFP as a fluorescentmarker to assess tumor burden (24). Mice were treated with single-agentgemcitabine or with DZ-2384/gemcitabine from postnatal day 15 (P15)until they are sacrificed on P29 (Fig. 1F). Tumor burden was quantifiedby green fluorescent protein (GFP) intensity of micrographs taken fromthe five brightest fields of dissected pancreata (24). The absence of ob-served GFP in several micrographs from mice treated with DZ-2384/gemcitabine relative to untreated mice or mice treated with single-agentgemcitabine indicates fewer PDA initiation sites and a higher frequencyof mice with negligible or greatly reduced tumor burden (Fig. 1G). Theresponse rate (reduction in GFP fluorescence intensity) more thandoubledwith theDZ-2384 andgemcitabine combination relative to gem-citabine alone. Mild hair loss, diarrhea, and hindlimb paralysis were ob-served in weanling mice with DZ-2384/gemcitabine but not withgemcitabine alone (table S2).DZ-2384–related toxicitywas age-dependentin that DZ-2384 doses of 30mg/m2 were tolerated when administered onP23 (nomortality; table S2) but not on P16 (table S3).We did not observeweight loss, but growth delay was experienced by DZ-2384/gemcitabine–treatedweanlingmice during the experiment (figs. S2HandS3). Together,these results provide a rationale for the combined use of DZ-2384 andgemcitabine in the treatment of pancreatic cancer.

DZ-2384 has a wide safety margin compared to vinorelbineTo establish the safety and tolerability of DZ-2384 and vinorelbine inmice, we conducted toxicity studies to determine the no observable ad-verse effect level (NOAEL; dose at which animals showed minimalweight loss and no bone marrow toxicity) (tables S4 and S5 for DZ-2384 and vinorelbine, respectively). Then, the safety margin wasdetermined on the basis of compound exposure. For this, we measuredthe systemic concentrations of each compound by liquid chromatogra-phy–tandemmass spectrometry analysis in plasma over time at selecteddoses representing theirMEDs (determined in theMia PaCa-2 andHT-29 models) and NOAELs. The results are presented in Table 1. Com-paring compound exposure (plasma AUC) at the MED to exposure atNOAEL yields a safety margin for DZ-2384 of 24.4 to 53.5. The safetymargins for vinorelbine are 0.7 to 1.0 in the samemodels. We concludethat DZ-2384 has a much wider safety margin than vinorelbine.

DZ-2384 lacks neurotoxicity at effective dosesDamage to peripheral neurons often limits the use ofMTAs in the clinic(25, 26). Sprague-Dawley rats treated with DZ-2384 were evaluated forevidence of peripheral neuropathy by measuring nerve conduction ve-locity (NCV) and onset latency in the caudal nerve, the distal digitalnerve, and the motor branch of the tibial nerve. These nerves includesensory axons (distal digital), motor axons (tibial), and mixed sensory,motor, and autonomic fibers (caudal). A decrease in NCV or an in-

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DZ-2384 + GemP < 0.0001

GemUnt

GFP

at

P 29

Ctrl

P = 0.0237

3/46

(7%)

8/46

(17%)

9/31

(29%)

6/31

(19%)

0/11

(0%)

11/11

(100%)

P < 0.0001

P < 0.0001

Median:

19/28

(68%)

1/28

(3.6%)

P = 0.0021

10

10

10

10

10

10

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10

G

Dosing schedule: Pancreatic Rgs16::GFP; KIC model

Sac

Age (days):

G G G GG GD D

29272522201815

Days after tumor cell implantation

Me

an tu

mo

r vo

lum

e (m

m3

)

P = 0.0039

P = 0.0018

A

F

MIA PaCa-2 vinorelbine

20 40 60 80 1000

500

1000

1500

Days after tumor cell implantation

Me

an

tum

or v

ol u

me

(mm

3)

P = 0.0129

9 mg/m2

15 mg/m2 (1 tf)

30 mg/m2 (2 tfs)

45 mg/m2 (2 tfs)

Vehicle (1tf)

B

20 40 60 80 1000

500

1000

15001.13 mg/m 2

Vehicle

2.25 mg/m 2 (5 tfs)

4.5 mg/m 2 (4 tfs)

9 mg/m 2 (6 tfs)

MIA PaCa-2 DZ-2384

P = 0.0019

P = 0.0018

Day 28

Vehicle DZ-2384 (18 mg/m )2

DZ-2384 (9 mg/m )2 DZ-2384 (4.5 mg/m )2

MDA-MB-231-LM25.0

4.0

3.0

2.0

1.0

Radiance(photons s–1 cm–2 sr–1)

P = 0.0260

MDA-MB-231-LM2

DZ-2384

Days after randomization

Surv

ival

(%)

Vehicle (n = 8)

4.5 mg/m (n = 5)

9 mg/m (n = 8)

18 mg/m (n = 8)

0 20 40 60 80 1000

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Vehicle (n = 5)

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DZ-2384 (n = 4)

Gem + DZ-2384 (n = 5)

Gem

DZ-2384

E

0 20 40 60 80 1000

20

40

60

80

100

P = 0.0159

Pancreatic Rgs16::GFP; KIC model

Fig. 1. DZ-2384 has potent antitumor activity as a single agent and in combination with gemcitabine. (A and B) Mean volume ofMIA PaCa-2 tumors after 4 weeks of weeklybolus intravenous administrations (indicated by arrows) of vehicle, DZ-2384 (A), or vinorelbine (B). Error bars representmeans ± SEM, n = 6. Tf, number of tumor-free animals on day95. P values were determined on day 53. (C) In vivo bioluminescent imaging of luciferase-expressing MDA-MB-231-LM2 breast cancer metastases in the lungs is presented as apseudocolor heatmapof photon flux onday 28 after treatment initiation. (D) Kaplan-Meier curves representing survival ofmicewithMDA-MB-231-LM2breast cancermetastases tothe lung. P values <0.0001 for all treated mice were calculated using the log-rank (Mantel-Cox) test relative to vehicle-treated mice. (E) Mean tumor volume of a human pancreaticcancer PDX after treatmentwith gemcitabine (Gem) (100mg/m2) or DZ-2384 (36mg/m2) alone or in combination according to the schedule indicated by arrows. Error bars representmeans ± SEM, n = 5. P values were determined on day 92. (F) Treatment schedule for the Rgs16::GFP;KIC pancreatic model: DZ-2384 (7.5 and 30mg/m2 for the first and secondinjection on P16 and P23, respectively) and gemcitabine (37.5 mg/m2 three times weekly as indicated). (G) Maximum tumor burden (GFP) signal of untreated (Unt; n = 46),gemcitabine-treated (n = 31), or gemcitabine + DZ-2384–treated (n = 28) groups of mice on P29 is represented quantitatively. Each aligned vertical dot column represents onepancreas, and dots are the first to fifth brightest nonoverlapping pancreas fields; the median is in red. GFP expression in Rgs16::GFP; non-KIC control (Ctrl) mice is represented byone gray line per mouse. Top and bottom dashed horizontal lines mark the 95th and 1st percentile, respectively, of all untreated animal GFP values. Numbers below the 1stpercentile represent the mildly or nontumorigenic pancreatic fields, and comparison with untreated animals represents the response rate (indicated below the bottom dashedline). All P values except in (D) were determined by Student’s unpaired two-tailed t test with Welch’s correction; the color matches the comparison group.

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crease in onset latency is evidence of impaired neuronal function.Animals were exposed to either DZ-2384 or docetaxel, an MTA thatis known to cause peripheral neuropathy. Each compound wasdelivered onceweekly over a 4-week period at doses up to and includingthe maximum tolerated dose (MTD) as determined by death rate or>20% body weight loss. The MTDs for DZ-2384 and docetaxel were30 and 120 mg/m2, respectively (fig. S4). NCV and onset latency wererecorded before treatment, 2 days after the fourth dose (day 24), and3 weeks after the fourth dose (day 44).

As expected, docetaxel at 120 mg/m2 caused a decrease in NCV incaudal and digital nerves and an increase in onset latency in tibial nerveson day 24 (Fig. 2, A toC).After the 22-day recovery period, therewas noimprovement in either caudal or digital NCV; however, there was aslight improvement in tibial motor latency. Histology of the docetaxel-treated animals on day 44 showed incomplete recovery of the damageddorsal root ganglia (Fig. 2D) and peripheral (sciatic) nerves (Fig. 2E),indicated by the presence of degenerating neurons with condensednuclei and digestion chambers filled with myelin debris.

DZ-2384 was tested in rats at 12 mg/m2 (more than two timeshigher than its MED in mouse xenograft models) and at 30 mg/m2

(MTD). On day 24, normal NCV and onset latency were observed inrats exposed to DZ-2384 at 12 mg/m2 relative to age-matched controls(Fig. 2, A to C). Histology performed on day 44 showed no changes inthe lumbar dorsal root ganglia or in the sciatic nerve (Fig. 2, D and E).At 30 mg/m2, the tibial nerve was unaffected; however, a slowing ofthe caudal and distal digital NCV was observed, although the decreasewas smaller than that for docetaxel. At this dose level, the NCV re-mained slow on day 44 (Fig. 2, A to C). At the end of the recoveryperiod, we did not find histological evidence of neurotoxicity in thedorsal root ganglia or sciatic nerve (Fig. 2, D and E), suggesting a sub-stantial degree of recovery, although the distal axons were notexamined. Histopathology data are summarized in table S6.

To relate DZ-2384 neurotoxicity in rats to exposure (AUC) levelsthat are effective in mouse tumor models, we measured the pharmaco-


kinetics of DZ-2384 in Sprague-Dawley rats. At 12 mg/m2, where noneuropathy was seen, the rat AUC was 4371 ng⋅hour/ml, which is 13-to 28-fold higher than plasma levels that are effective in mouse xenograftmodels (Table 1). These data indicate that DZ-2384 does not cause neu-ropathy at doses that are highly effective in multiple cancer models.

Functional genomics points to an antimitotic mechanismof actionIdentifying the cellular target of diazonamides has been chal-lenging. One candidate is the mitochondrial enzyme ornithined-aminotransferase, which was identified by binding interactions(27), whereas other studies have implicated microtubules (28). Tointerrogate the mechanism of action without bias, we performed agenome-wide RNA interference screen in H1299 cells to uncovergenes that confer either sensitivity or resistance on DZ-2384. Halfmaximal growth inhibitory concentration (IC50) values for DZ-2384 werecalculated with each test small interfering RNA (siRNA) pool relativeto nontargeting siRNA controls. We identified 631 sensitizer genesand 179 resistor genes, defined as siRNA pools that shifted the IC50

for DZ-2384 by >2 SD from the mean of all data points (fig. S5A). Todetermine the major pathways that conferred sensitivity or resistanceand hence the potential mechanism of action, we performed pathwayanalysis of the hits. Of the top 10 most highly represented pathways, 6were related to cell cycle, mitosis, and microtubules (fig. S5B). To con-firmmitotic arrest and apoptosis, we treated H1299 cells with DZ-2384for 48 hours andmeasured theirDNAcontent and caspase-3 activation.DZ-2384–treated cells accumulated in the G2-M phase of the cell cyclestarting at 4 to 8 hours (fig. S5, C to F) and began to undergo apoptosisat 16 hours after treatment as demonstrated by procaspase-3 cleavage(fig. S5G). Combined with the results of the RNA interference screen,these results suggested that DZ-2384 acts as an antimitotic agent (5).

DZ-2384 binds to the vinca alkaloid site of tubulinTo investigate whether DZ-2384 binds to tubulin directly, we usedsurface plasmon resonance (SPR) to detect binding between biotiny-lated DZ-2384, which was immobilized to streptavidin-coated sensors,and purified tubulin dimers. We observed dose-dependent increasesin the SPR signal when tubulin was titrated in single-cycle kineticsmode (Fig. 3A). As negative controls, neither actin nor bovine serumalbumin (BSA) bound to biotinylated DZ-2384 surfaces, nor did tubulinbind to surfaces coated with streptavidin only or with a biotinylatedinactive diastereomer of DZ-2384 (DZ-2384D; fig. S6). As expected,preincubating tubulin with DZ-2384 inhibited its binding to bioti-nylated DZ-2384 surfaces (Fig. 3B), but similar incubations withDZ-2384D did not (Fig. 3C). To determine whether DZ-2384 bindsto other MTA-tubulin binding sites in solution, we evaluated bindingto the biotinylated DZ-2384 surface in the presence of vinorelbine.Preincubating tubulin with increasing concentrations of vinorel-bine blocked its binding to biotinylated DZ-2384 surfaces in adose-responsive manner (Fig. 3D), suggesting that DZ-2384 bindsat or near the vinca binding site.

To gain a more detailed view of the DZ-2384–tubulin binding site,we used a protein complex consisting of twoab-tubulin dimers (T2), thestathmin-like protein RB3 (R), and tubulin tyrosine ligase (TTL; the fullcomplex is denoted as T2R-TTL) (16, 29), and the structure of ligand-bound DZ-2384 (DZ-2384–T2R-TTL) was solved to a resolution of2.4 Å using x-ray crystallography. In the structure, DZ-2384 binds witha distinct binding mode (see the next section) to tubulin at the classicvinca alkaloid site, which lies at the longitudinal interface between

Table 1. DZ-2384 has a wide therapeutic window compared to vinorel-bine in mice.

Compound
Tumormodel
D(

MED*
NOAEL†
Safetymargin

atNOAEL‡

osemg/m2)

Exposure§

[AUC0–a(ng⋅hour/

ml)]

D(
osemg/m2)
Exposure§

[AUC0–a(ng⋅hour/

ml)]

DZ-2384 P
MIAaCa-2
1.13
157
36
8398
53.5

HT-29
4.5 344 24.4
Vinorelbine P
MIAaCa-2
30
2309 30 2309
1

HT-29
45 3380 0.7
*Minimum dose resulting in statistically significant antitumor activity relativeto vehicle control. Calculated using mean tumor volume for MIA PaCa-2 andoverall survival for HT-29 models.†No observable adverse effect level (NOAEL): Maximum dose at which noadverse clinical signs, no body weight loss, and no effects on blood cellcounts, biochemistry, and bone marrow were observed.‡Safety margin defined as the ratio between NOAEL and MED.§Pharmacokinetic exposure measured by circulating plasma concentration(area under the concentration-time curve, AUC).

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the b subunit of one tubulin (b1 of T2R-TTL) and the a subunit of thetubulin above it (a2 of T2R-TTL) (Fig. 3, E and F, and fig. S7). Overall,these results establishDZ-2384 as anMTA that binds to the vinca site ontubulin. To determine the consequences ofDZ-2384 biotinylation for tu-bulin binding, we modeled our biotin-linker DZ-2384 derivativeonto the DZ-2384–T2R-TTL complex (fig. S8). Because no structuralrearrangements of the tubulin molecules are required to accommo-date biotin-linker DZ-2384, and no clashes between the biotin-linkermoiety and tubulin were observed, we conclude that the biotinyla-tion of DZ-2384 does not affect binding to tubulin.

Our DZ-2384–tubulin structure also allowed us to explore thedifference between DZ-2384 and its inactive diastereomer DZ-2384D.The structure of DZ-2384D was modeled into the binding pocket on tu-bulin by keeping the core interaction stationary and changing only thestereocenters of the indoline moiety. The model revealed that a large hy-drophobic contact is missing in the diastereomeric ligand, namely, thecontact formed by the fluorinated indoline moiety of DZ-2384 with loop


T7, helix H10, and strand S9 of a2-tubulin (Fig. 3, F and G). This missingcontact provides a structural explanation for the lack of tubulin binding bythe diastereomer in the competitive SPR binding assay (Fig. 3, B and C).

DZ-2384 causes a change in curvature oftubulin protofilamentsGiven that DZ-2384 has a wider safety margin than vinorelbine, weexplored how DZ-2384 differs from other vinca alkaloid bindingcompounds. We began with a closer investigation of the DZ-2384–T2R-TTL crystal structure described above. The DZ-2384 binding siteis shaped by (i) the hydrophobic and polar residues of loop T7, helixH10, and strand S9 of a2-tubulin; (ii) the N-terminal residues of bothhelices H6 and H7; and (iii) the loops T5 and H6-H7 of b1-tubulin. Inthe DZ-2384–tubulin complex, the hydrophobic interactions with thesecondary structural elements of a- and b-tubulin described above arecomplemented by direct and water-mediated hydrogen bonds tomain-chain atoms of both tubulin subunits (Fig. 3E). Moreover, the

Caudal nerve conduction velocity

Vehicle 12 30120

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20

40

60

Dose (mg/m )2

Vel

ocity

(m/s

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DZ-2384 Docetaxel

P < 0.0001

P < 0.0001P =

0.0252

P = 0.0015

Tibial onset latency

Vehicle 12 30120

0

1

2

3

Dose (mg/m )2

Late

ncy

(ms)

DZ-2384 Docetaxel

Digital nerve conduction velocity

Vehicle 12 30120

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20

30

40

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ocity

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Dose (mg/m )2

DZ-2384 Docetaxel

P = 0.0008

P = 0.0063

P < 0.0001

P = 0.0002

A B C

D

E

P = 0.0043

Scia

tic

ner

veD

ors

al ro

ot

gan

glia

Vehicle DZ-2384

Vehicle DZ-2384 Docetaxel

Docetaxel

Fig. 2. DZ-2384has no effects onNCV, latency, andhistopathology at effective antitumor concentrations. (A toC) Electrophysiological parameters weremeasured on caudal(A), digital (B), and tibial (C) nerves. Blackbars representmeasurements takenonday24after fourweekly administrations ofDZ-2384ordocetaxel at thedoses indicated (n=12).Whitebars represent measurements taken on day 44, allowing a 22-day recovery as indicated (n = 5). Error bars represent SD. P values were determined using an unpaired nonparametricStudent’s t test. Each P value indicated is compared to vehicle bars of the corresponding color. (D and E) Histopathology of dorsal root ganglia (D) and sciatic nerve (E) tissue sectionstaken at necropsy on day 44. Arrows denote degenerating neurons with vacuolated cytoplasm (black) and condensed nuclei (blue). Black arrows in the sciatic nerve denote degen-erating axons. Green arrows in the dorsal root ganglia and sciatic nerve denote digestion chambers. Scale bars, 50 mm.

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two oxazole rings form a p-stacking interaction with the aromatic sidechain of Tyr224 and the nucleotide of b1-tubulin.

To understand how DZ-2384 differs from other vinca alkaloids, wesuperimposed atomic models of tubulin in complex with DZ-2384 and


vinblastine (Fig. 3H) (17). The superposition shows that DZ-2384 andvinblastine exploit the same hydrophobic pocket of a2-tubulin withtheir corresponding indoline moieties, and bothmolecules are involvedin the same p-stacking interaction (bottom dashed circle in Fig. 3H). In

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Fig. 3. DZ-2384 binds to the vincadomain of tubulin. (A) Specific,dose-dependent binding of tubulin di-mers to DZ-2384 as assessed by SPR insingle-cycle mode. Buffer (red), BSA(green), actin (blue), and tubulin (black)were titrated (0 to 10 mM; twofold dilu-tion series) over biotinylated DZ-2384[200 resonance units (RUs) immobi-lized]; under similar conditions, tubulinwas titrated over biotinylated DZ-2384D surfaces (pink). (B to D) Multi-cycle SPR experiments in which theinteraction between tubulin (3 mM) andDZ-2384 (biotin-immobilized) wastested in the presence of nonbiotinyl-atedDZ-2384 (B), DZ-2384D (C), and vi-norelbine (D) competitors at theconcentrations indicated. (E) Closeupview of the interaction between DZ-2384 (yellow sticks) and tubulin (grayandwhite ribbon). Interacting residuesof a- and b-tubulin are shown in grayand white stick representation and arelabeled. Hydrogen bonds are high-lighted as dashed black lines. Sec-ondary structural elements are labeledin blue. GDP, guanosine diphosphate.(F) View into the binding site turnedby 90°. The ligand DZ-2384 is in yellowsurface representation. The samedisplay settings as in (E) are applied.(G) Surface representation of the in-active DZ-2384D diastereomer aftermodeling it into the DZ-2384 bindingsite. The hydrophobic pocket that in-teracts with the indoline moiety ofDZ-2384 is devoid of any interactionin the case of the inactive compound(dashedblack circle). (H) Superpositionof the DZ-2384 (yellow) and vinblas-tine (VLB) [blue; PDB ID 4EB6 (60)]crystal structures. The top dashedcircle indicates the hydrophobicpocket in a-tubulin that is occupiedby the indoline moieties of bothcompounds in their complexes withtubulin. The bottom dashed circlehighlights the bulky catharanthinemoiety of vinblastine that is missingin DZ-2384.

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other words, both molecules share a similar space at the interdimerinterface. The exception is the bulkymoiety from the catharanthine nu-cleus in vinblastine, which is common in vinca alkaloids (30). This cath-aranthine nucleus is missing in DZ-2384. As a consequence, theintertubulin dimer interface in theDZ-2384–T2R-TTL complex ismorecompact compared to both the apo–T2R-TTL and vinblastine–T2R-TTL complexes (Fig. 4A). The compactness in turn causes an overallchange in the relative orientation of the two tubulin dimers in theDZ-2384–T2R-TTL complex compared to the corresponding orienta-tion observed for the vinblastine–T2R-TTL [Protein Data Bank (PDB)ID 5J2T] and the apo–T2R-TTL (PDB ID4I55) complexes (Fig. 4A). Toquantitatively evaluate this change, we analyzed helical superassembliesof different T2R-TTL complexes as described previously (31). We esti-mated radii and pitches of 210, 180, and 250 Å and 250, 340, and −80 Åfor apo–T2R-TTL, vinblastine–T2R-TTL, and DZ-2384–T2R-TTL, re-spectively (Fig. 4B). On the basis of the increased radii of DZ-2384–T2R-TTL, we hypothesize that DZ-2384 can unbend curved protofilamentsto bring them conformationally closer to the straight protofilaments ofmicrotubule shafts.

Vinca alkaloids stabilize curved, ring-like oligomers of tubulin athigh concentrations (17). To determine whether DZ-2384 changesthe pitch and curvature of these oligomers, we polymerized tubulin inthe presence of DZ-2384 or vinblastine. Electronmicrographs of crude-ly purified tubulin illustrate the decrease in pitch of protofilamentsformed in the presence of DZ-2384 compared to that of vinblastine(Fig. 4C). To measure the curvature of such curved protofilaments, wepolymerized highly purified tubulin with either DZ-2384 or vinblastine,mounted the polymers on EM grids, and measured them using customsoftware (Fig. 4D). Because the lengths of protofilaments induced by eachcompound were equivalent (fig. S9), we infer that they have a similar ori-entationon the grid surface. Therefore, the curvaturemeasured in thiswayis representative of the actual radius of the tubulin-compound structures.As predicted by the crystal structure, the DZ-2384–induced protofila-ments were straighter than those obtained with vinblastine [lower valuesfor curvature (k); Fig. 4E]. Together, the x-ray crystallography and EMdemonstrate that DZ-2384 differs from vinblastine by changing the pitchand curvature of protofilaments.

Given that many of our experiments have used vinorelbine ratherthan vinblastine, wemodeled the structure of vinorelbine into the vincabinding site. In our model, the catharanthine nuclei of vinorelbine andvinblastine heavily overlap (fig. S10); thus, we do not expect a differencein the change in protofilament curvature between these two agents. Wenote that vinblastine has two additional major hydrogen bonds to tu-bulin compared to vinorelbine, providing an explanation for the re-ported lower affinity of vinorelbine compared to vinblastine (32).

DZ-2384 has a milder effect on interphase and mitoticmicrotubule networks than vinorelbineAlthough DZ-2384 induces mitotic arrest and triggers apoptosis, itswider safetymargin suggests that it might havemilder effects onmicro-tubules in cells that are not actively undergoingmitosis. To test this idea,we used DZ-2384 and vinorelbine on a variety of cultured cells and as-sayed the integrityof theirmicrotubulenetworks.H1299 cellswere treatedwith various concentrations of DZ-2384, DZ-2384D, vinorelbine, ordocetaxel (Fig. 5A). In a microtubule sedimentation assay, a decrease inmicrotubule polymer mass was observed with increasing DZ-2384 con-centrations (but not with DZ-2384D), like with vinorelbine. However,despite having equivalent sensitivity to DZ-2384 and vinorelbine (IC50

values of 12 and 15 nM, respectively), H1299 cells treatedwithDZ-2384


maintained microtubule polymer mass at higher concentrations thanthose treated with vinorelbine. These differences indicate that DZ-2384 has a milder effect on the microtubule cytoskeleton in interphasecells. As a control, docetaxel increased microtubule polymer mass, asexpected (3).

Next, we examined the effects ofDZ-2384 onmicrotubules in active-ly dividing cells directly by confocal microscopy. HeLa cells were syn-chronized in late G2 with RO-3306 (a reversible cyclin-dependentkinase 1 inhibitor) (33) and released in the presence of equipotent con-centrations ofDZ-2384 (4 nM)or vinorelbine (11 nM), representing theIC80 of each compound in a 3-day cell viability assay. Synchronized cellsreleased in the absence of compound show that microtubules form theappropriate spindle on which chromosomes are aligned in metaphase.DZ-2384–treated cells (Fig. 5B) show the presence of mostly bipolarspindles with stunted microtubules and visibly misaligned chromo-somes. Similar results were obtained in H1299 cells (fig. S11). At equi-potent concentrations, vinorelbine-treated cells lacked any detectablespindlemicrotubules in both cell lines (Fig. 5B and fig. S11). These find-ings suggest that although DZ-2384 has milder effects on mitoticspindles, these effects are still sufficient to interfere with cell division.

Similarly, unsynchronized U-2 OS osteosarcoma cells in interphasewere treated with equipotent concentrations (3-day IC80) of DZ-2384and vinorelbine. In DZ-2384–treated cells, microtubules were present,although theywere less dense at the cell periphery (Fig. 5C). In vinorelbine-treated cells, intact microtubules were difficult to observe, consistentwith the loss of polymer mass observed in the pelleting assay above.

Finally, the effect of the two compounds on primary rat cortical neu-rons was assessed after 1 hour of treatment with 0.3 to 100 nM of eachcompound. Neurons treated with DZ-2384 up to 100 nM showedminimal qualitative differences compared to control-treated neuronsin terms of axonal morphology, width, or intensity of tubulin staining(Fig. 5D and fig. S12). However, vinorelbine treatment produced thin-ner and/or fragmented axons starting at 0.3 nM and an overall reduc-tion in tubulin staining intensity (Fig. 5D and fig. S12). Collectively,these results indicate that DZ-2384 has a milder effect on both inter-phase and mitotic microtubule networks than vinorelbine.

DZ-2384 has a distinct impact on microtubuledynamic instabilityThe milder impact of DZ-2384 on microtubules in cells suggests thatDZ-2384 causes distinct changes in the dynamic instability of micro-tubules. To determine how DZ-2384 changes dynamic instability, we vi-sualized growing ends of microtubules in U-2 OS cells stably expressingan EB3-mCherry fusion protein, which serves as a marker for growingends (34). The cells were treated with DZ-2384 and vinorelbine at equi-potent (IC80) concentrations. In untreated cells, microtubule growth ori-ginates from the centrosome and radiates outward (movie S1). DZ-2384treatment decreased the number of growing microtubules and the fre-quency with which they originate from the centrosome (movie S2), asexpected for a microtubule destabilizing agent. Vinorelbine treatment,however, had a more marked effect, vastly decreasing the number ofgrowing microtubules (movie S3).

To more precisely measure the effect of DZ-2384 on dynamic in-stability, we imagedLLC-PK1 cells expressingGFP–a-tubulin (35), whichallowed us to measure the growth rates, shrinkage rates, catastrophe(change from pause or growth to shrinkage) frequencies, and rescue(change from shrinkage to growth or pause) frequencies of dynamicmicrotubules. We compared the effects of DZ-2384 and vinorelbine,each at IC80 concentrations. Both DZ-2384 and vinorelbine reduced

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the microtubule growth rate and dynamicity equally (Table 2), andneither compound changed the shrinkage rate. DZ-2384 was distin-guished from vinorelbine by an increased rescue frequency relative tocontrol cells. Microtubules in vinorelbine-treated cells also spent a higherproportion of time in a paused state and less (3×) time in a growth statecompared to those in DZ-2384–treated or control cells. Overall, theseresults explain the greater integrity of the microtubule network in cellstreated with DZ-2384 compared to vinorelbine. Although DZ-2384may cause microtubules to grow more slowly, the increase in rescueevents compensates and helps tomaintain tubulin in the polymeric state.However, duringmitosis, DZ-2384’s decreased growth rate is sufficient todisrupt the mitotic spindle, sending cells into growth arrest.

DZ-2384 increases microtubule rescues in areconstituted systemCells contain dozens of microtubule-associated proteins (MAPs) thatregulate microtubule dynamics, including rescue frequency and growth


rate. Therefore, we cannot conclude from cell-based studies whetherDZ-2384 affects dynamic instability directly through interactions withtubulin or indirectly through MAPs. To determine whether the changein rescue frequency that we observed in DZ-2384–treated cells alsooccurs in a purified system in the absence of MAPs, we reconstitutedmicrotubule growth in vitro using the single molecule assay for micro-tubule dynamics (36). Microtubule seeds stabilized by guanosine-5′-[(a,b)-methyleno]triphosphate were adhered to the surface of a flowchamber, and fluorescently labeled tubulin was introduced (Fig. 6A).We generated kymographs of growing microtubules in the presence ofincreasing concentrations of either DZ-2384 or vinorelbine (Fig. 6, B toD) andmeasuredmicrotubule dynamics. Consistent with the growth in-hibition seen in LLC-PK1 cells, DZ-2384 strongly affected microtubuledynamics; at 100 nMDZ-2384, the growth and shrinkage rates decreasedby about twofold (Fig. 6, E and F) relative to untreatedmicrotubules, andcatastrophe frequencies were slightly increased (Fig. 6G). Comparablegrowth and shrinkage rate decreases were observed with vinorelbine

BA

CDZ-2384

Vinblastine

20 nm

Average curvature (µm )–1

0 20 40 60 800

5

10

15

20

25

)%( yc

neu

qerF

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DZ-2384P = 0.0002

β1Tub α2Tub

β2Tubα1Tub

DZ-2384– T2R-TTL

Vinblastine–T2R-TTL (5J2T)

Apo–T2R-TTL (4I55)

~210

Apo–T2R-TTL Vinblastine–T

2R-TTL DZ-2384 –T2R-TTL

~180 ~250

–80 250 340

DZ-2384

Vinblastine

20 nm

D E

100 nm

100 nm

Fig. 4. DZ-2384 affects tubulin dimer curvature. (A) Ribbon diagram outliningthe proposed difference in relative orientation between ab-tubulin dimers inapo–T2R-TTL [aquamarine ribbon; PDB ID 4I55 (16)] compared to T2R-TTL

complexed with vinblastine [gray ribbon; PDB ID 5J2T (61)] or DZ-2384 (blue ribbon). (B) Variation of the relative orientation of tubulin heterodimers in the different T2R-TTL complexes displayed in (A). Shown are models of helical oligomers obtained from the repetition of the indicated T2R-TTL complexes. The resulting helices are viewed along(bottom) and perpendicular (top) to their axes. Values for radii and pitches are given in black. (C and D) Electron micrographs of negatively stained MAP-rich (C) or purified (D)tubulin oligomers formed with 10 mM DZ-2384 (top) or 10 mM vinblastine (bottom). Curves used to quantitate oligomer curvature are shown in aquamarine (D). (E) Histogramof average curvature values for each condition [DZ-2384 (red) and vinblastine (blue)] with a bin of 5 mm−1 (n = 159 for DZ-2384 and n = 192 for vinblastine). P value wascalculated using Welch’s t test.

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and were consistent with previous reports(37, 38). We attribute the differences be-tween the single-molecule assay and in-cellmeasurements to the absence of MAPs inour reconstituted system. Strikingly, howev-er, DZ-2384 increased the rescue frequencyin the reconstituted system to twice the rateseenwith vinorelbine, consistent with the in-creased rescue frequency observed in cells (P=0.0236 at 50 nM drug treatment; Fig. 6H).Collectively, these results indicate that DZ-2384 can directly increase the rescue fre-quency of dynamic microtubules, both inpurified tubulin systems and in cells, sug-gesting that the changes to dynamic in-stability are related to the straightening ofprotofilaments induced by DZ-2384.

DISCUSSIONMTAs are part of the standard of caretreatments for awide range of cancer indica-tions; however, there is still a need for ef-fective but safer agents to combine withnext-generation targeted therapies, includingimmunotherapies. A key feature of DZ-2384is an improved safety margin (more than 24-fold versus 0.7- to 1.0-fold for vinorelbine)when comparing weight loss, clinical effects,bonemarrow toxicity, andmore than 13-foldconsidering its neurotoxicity profile. We have

Wieczorek et al., Sci. Transl. Med. 8, 365ra159 (2016)

A

B Control DZ-2384 Vinorelbine

C

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al n

euro

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OS)

Mit

oti

c (H

eLa)

10 µm10 µm

Fig. 5. DZ-2384 affects mitotic microtubules butminimally affects interphase and neuronal micro-tubules. (A) Western blot analysis of microtubulepolymers (a-tubulin) isolated fromH1299 cells treatedwith a dose range of DZ-2384, the inactive analog DZ-2384D, vinorelbine, or docetaxel for 4 hours. Ponceaustaining shows equal protein loading. DMSO,dimethylsulfoxide. (B) HeLa cells were synchronized with RO-3306 and then washed and released in DMSO (left)or at the IC80 of DZ-2384 (4 nM) (middle) and vinorel-bine (11 nM) (right) for 1 hour before fixation. Sampleswere immunostained with an antibody to a-tubulin(green) and phalloidin (red) and with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Images were acquiredby confocal microscopy with a 40× objective. Insetsshowan enlargement of single cells (bottom). (C) Non-synchronized U-2 osteosarcoma (OS) cells treated for2 hours with the IC80 of each compound (300 nMeach) and then immunostained with an a-tubulin an-tibody (green). (D) Treatment of E18 rat primary corti-cal neurons with 10 nM DZ-2384 or vinorelbine for1 hour (IC50 values for DZ2384 and vinorelbine were43 and 68 nM, respectively). Cells are immunostainedwith an a-tubulin antibody (green), and nuclei arestained with DAPI (blue).

16 November 2016 9 of 14



demonstrated thatDZ-2384has strong single-agent antitumor activity inmodels of pancreatic, colon, ALL, andmetastatic breast cancer. The cri-ticisms of xenograft models derived from cell lines are that they are notrepresentative of patient tumors, theymay respond differently to drugs,and themice in which tumors are grown lack immune surveillance.We


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have addressed these criticisms by demonstrating that DZ-2384 is po-tent inmultiple xenograftmodels, including a pancreatic PDXmodel, aswell as a Rgs16::GFP;KICGEMMmodel of pancreatic cancer in which themice are immunocompetent. The results from thepancreaticmodels showthat the DZ-2384/gemcitabine combination was more effective than gem-citabine alone, the clinical standard of care for PDA. This observation isexciting because the DZ-2384/gemcitabine combination reduced tumorinitiation and progression with a superior response rate (68%) com-pared to the nab-paclitaxel/gemcitabine combination (53%) in the sameRgs16::GFP;KIC model (24). Although this latter combination is ap-proved for the treatment ofmetastatic PDA, nab-paclitaxel suffers froma relatively high burden of peripheral neuropathy (39). Collectively,these results highlight DZ-2384’s promise as a drug candidate forPDA and demonstrates its potential for activity in a wide range of otherindications.

MTA-induced peripheral neuropathy has been linked directly toeffects on axonal microtubules (40). Given that the integrity of axonalmicrotubules is essential for transport processes and maintaining neu-romuscular junctions, it is not surprising that MTAs affect the longestnerves of the body, those innervating the feet and hands (41). DZ-2384demonstrates no electrophysiological ormicroscopic signs of peripheralneuropathy at circulatingDZ-2384 blood concentrations at least 13 timeshigher than effective antitumor concentrations in mice. However, be-cause neurotoxicity is seen at theDZ-2384 dose of 30mg/m2, a potentialfor clinical toxicity remains at high doses. Nevertheless, in culturedprimary rat neurons, the integrity of axonal microtubules was main-tained in the presence of DZ-2384 compared to that in the presenceof vinorelbine, suggesting a milder impact on nerve axons. Therefore,we propose that DZ-2384’s improved safety margin is linked to themanner in which DZ-2384 binds to tubulin and the impact it has onmicrotubule integrity and dynamics.

Our crystal structure of the DZ-2384–tubulin complex and our EMdata showed a pronounced straightening in the pitch and curvature ofmicrotubule protofilaments relative to vinblastine-tubulin complexes(17). Microtubule curvature, more specifically the curvature of tubulindimers and protofilaments, has emerged as the central theme in ourcurrent understanding of microtubule dynamic instability (11). GTP-tubulin dimers begin in the curved state, with an intradimer angle of12° according to available crystal structures (31, 42, 43). In solution, atubulin dimer is expected to adopt a range of conformations, withcurved species predominating (44). Sometime during polymerization,the tubulin dimer straightens as it incorporates into the microtubulelattice (12), only to return to the curved state during depolymerizationwhen the protofilaments curve outward as “ram’s horns” (1). It is nowapparent that cells control their microtubules by using MAPs that bindselectively to specific curvatures (11). SomeMAPs bind to curved tubu-lin (43, 45), some bind to straight lattices (46), and others can force tu-bulin to adopt curved conformations (47).

Microtubule curvature is also an important theme in our currentunderstanding of the mechanism of action of MTAs. Colchicine, forexample, is known to trap individual tubulin dimers in the curved state(48). Vinca alkaloids bind to the longitudinal interface between two tu-bulin dimers, forcing the interface to adopt a curved conformation(17, 49). If the vinca alkaloid binds to this interface at the growing end ofamicrotubule, the affected tubulin dimers will be unable to straighten, anecessary step for their full incorporation into the microtubule lattice(11, 50). Thus, vinca alkaloids block the interdimer curved-to-straighttransitions that must occur during microtubule growth, which may ex-plain why microtubule growth is slowed at low drug concentrations

Table 2. DZ-2384 has different effects from vinorelbine on micro-tubule dynamics in LLC-PK1 cells. Measurements represent the means ±SD of four independent experiments; each experiment represents at leastthree to five different cells for each condition. Catastrophe and rescuefrequencies are presented on an overall basis (not per microtubule). Pvalues were determined by an unpaired Student’s t test with Welch’scorrection relative to DMSO control-treated cells. Statistically significantvalues are marked in bold.

Dynamicparameters

DMSO
DZ-2384*
(P)
Vinorelbine*
(P)

Growth

Rate (mm/min) 1
2.0 ± 0.3 8.3 ± 1.6(0.0164)
7.8. ± 1.5(0.0091)

Distance (mm)
2.8 ± 0.1 2.4 ± 0.3(0.0631)
2.1 ± 0.2(0.0036)

Duration (s) 1
4.8 ± 0.6 19.0 ± 6.2(0.2666)
17.5 ± 5.8(0.4242)

Shrink

Rate (mm/min) 2
8.8 ± 3.4 27.7 ± 2.2(0.6081)
24.6 ± 3.5(0.1242)

Distance (mm)
5.3 ± 0.9 4.7 ± 0.9(0.3899)
6.4 ± 1.4(0.2345)

Duration (s) 1
1.4 ± 2.6 10.6 ± 1.3(0.6087)
17.6 ± 6.6(0.1570)

Dynamicity(mm/min)

1
2.8 ± 0.5 10.4 ± 1.4(0.0335)
10.2 ± 1.3(0.0165)

%Time per phase

Growth 4
1.1 ± 11.0 42.1 ± 13.1(0.9109)
13.1 ± 7.3(0.0074)

Shrink 2
8.6 ± 5.5 25.2 ± 1.4(0.3079)
38.6 ± 8.6(0.1068)

Pause 3
0.3 ± 6.5 32.7 ± 12.1(0.7422)
48.4 ± 8.1(0.0141)

Frequencies

Rescue (s−1)
0.023 ±0.007
0.046 ± 0.005(0.0024)

0.017 ± 0.002(0.1846)

Rescue (mm−1)
0.051 ±0.01
0.10 ± 0.008(0.0019)

0.043 ± 0.006(0.3547)

Catastrophe (s−1)
0.025 ±0.005
0.018 ± 0.003(0.0619)

0.027 ± 0.003(0.5360)

Catastrophe (mm−1) 0
.25 ± 0.1 0.27 ± 0.1(0.8116)
0.97 ± 0.4(0.0248)

Number ofmicrotubules

127
107 73
*Drug concentrations tested represent the IC80 of growth inhibition basedon a 3-day viability assay and were 24 nM for DZ 23484 and 11 nM forvinorelbine.

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P = 0.024

P = 0.041

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DZ-2384Vinorelbine

Fig. 6. DZ-2384 and vinorelbine affect mi-crotubule dynamics differently in vitro.(A) Schematic of the in vitro assay for observingmicrotubule dynamics. The seed microtubule(red) is adhered to a cover glass surface byanti-rhodamine antibodies (dark blue). Thedynamic microtubule (green) extends fromthe seed microtubule. Excitation by total in-ternal reflection (TIR) lasers (light blue witharrows) allows for the observation of individ-ual microtubules in the evanescent field (yel-low fade). (B to D) Kymographs (distanceversus time plot) depicting a dynamic micro-tubule (green) growing from a stabilized seed(magenta) in a standard polymerization bufferwith DMSO [1% (v/v)] (B), DZ-2384 (200 nM)(C), or vinorelbine (200 nM) (D). (E) Micro-tubule growth rates plotted against increasingconcentrations of DZ-2384 (blue squares) orvinorelbine (red circles). (F) Post-catastropheshrinkage rates plotted against increasingconcentrations of DZ-2384 (blue squares) orvinorelbine (red circles). (G) Cumulative frequen-cy distribution of the time until catastrophe inthe presence of DZ-2384 (200 nM) (bluesquares), vinorelbine (200 nM) (red circles), orin 1% (v/v) DMSO (black diamonds). Solid linesare fits to the gamma distribution, as describedin (62). (H) Column plot of rescue frequencies atincreasing concentrations of DZ-2384 (bluebars) or vinorelbine (red bars). Error bars in (E)and (F) represent SD. Data in (E) and (G) contain74 to 193 measurements from three differentexperiments. Data in (F) and (H) contain 75 to192 measurements from three different ex-periments. P values were calculated usingWelch’s t test.

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(51). At high concentrations, vinca alkaloids can stabilize curved tubulinoligomers in solution (52, 53). With sufficient vinca activity, thesecurved oligomers may dominate, effectively sequestering tubulin intoa polymerization-incompetent state. As microtubules undergocatastrophe and depolymerize, their tubulin becomes unavailable forincorporation, and in a short time, the entiremicrotubule cytoskeletonis depolymerized (54).

It is interesting to compare DZ-2384 to other vinca alkaloids in lightof the discussion above. Both DZ-2384 and vinorelbine slow down themicrotubule growth rate in vitro and in tissue culture cells. However,unlike vinorelbine, DZ-2384 increased the microtubule rescue frequen-cy. This change inmicrotubule dynamics is distinct relative to other de-polymerizing MTAs such as maytansine (55), cryptophycin 1 (56),dolastatin 10 (57), eribulin (58), and vincristine (59).More frequent res-cues should help to rebuild microtubule polymers, which may explainwhy the microtubule cytoskeleton persists in DZ-2384–treated cells.Our comparison of rescue frequencies and other parameters under-reports the difference between the DZ-2384–treated and vinorelbine-treated cells. By definition, any measurement of catastrophes and rescuesignores the microtubules that are paused or that have depolymerizedcompletely. Vinorelbine-treated cells contain fewer dynamic microtu-bules that could be included in the analysis than DZ-2384–treated orDMSO-treated cells (Table 2). DZ-2384’s effect on rescue frequencymay result from the straightening of the interdimer interface and the de-crease in the pitch of curvature. Because DZ-2384 straightens this bend,the probability that a rescue event occursmight increase. Decreases in thepitch may also facilitate lateral contacts between protofilaments duringrescue events. In this way, the structural difference between DZ-2384and other vinca alkaloids can explain the functional difference in rescuefrequencies.

Together, our results demonstrate that a classicMTAbinding site ontubulin can be targeted in a distinct way that results in a therapeuticagent with an unexpected lack of treatment-induced neuropathy at effec-tive plasma concentrations. Our results provide a framework for therational design of MTAs that deliberately alter microtubule curvature.Such changes to MTAs could confer improved pharmacological proper-ties and broaden the scope of use of this important class of chemotherapies.

er 16, 201

MATERIALS AND METHODSMaterials and methods are described in the Supplementary Materials.

6

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/8/365/365ra159/DC1Materials and MethodsFig. S1. Antitumor efficacy of DZ-2384 in metastatic breast and adult ALL models.Fig. S2. Body weight in murine xenografts after administration of DZ-2384, vinorelbine, or DZ-2384 and gemcitabine.Fig. S3. Body weights of weanling control and Rgs16::GFP;KIC mice treated with DZ-2384 andgemcitabine on P29.Fig. S4. Body weight change in rats administered DZ-2384 or docetaxel weekly for 4 weeks.Fig. S5. Functional genomic screen and cell cycle regulation as the DZ-2384 mechanism ofaction.Fig. S6. DZ-2384 and inactive DZ-2384D compound structures.Fig. S7. X-ray crystal structure of T2R-TTL in complex with DZ-2384.Fig. S8. Model of biotinylated DZ-2384 binding to tubulin in the context of T2R-TTL.Fig. S9. Tubulin oligomer lengths measured during curvature analysis.Fig. S10. Modeled comparison of vinblastine and vinorelbine binding to tubulin.Fig. S11. The effects of DZ-2384 and vinorelbine on mitotic H1299 cells.Fig. S12. Axon width of E18 rat cortical neurons treated with DZ-2384 and vinorelbine.Fig. S13. Preparation of an extended, multipurpose diazonamide side chain harboring a biotin tag.


Fig. S14. Conjugation of extended side chain 4 with diastereoisomeric diazonamide corestructures.Table S1. Survival of DZ-2384–treated and vinorelbine-treated mice bearing HT-29 tumors.Table S2. Summary of clinical observations in Rgs::GFP;KIC mice administered DZ-2384 andgemcitabine.Table S3. Summary of lethality in Rgs16::GFP;KIC mice and littermates administered DZ-2384.Table S4. Summary of the effects of DZ-2384 on mouse bone marrow.Table S5. Summary of the effects of vinorelbine on mouse bone marrow.Table S6. Microscopic changes in dorsal root ganglia and sciatic nerves of rats treated with DZ-2384 and docetaxel.Table S7. X-ray crystallography data collection and refinement statistics.Movie S1. Microtubule growing ends in U-2OS cells: vehicle control.Movie S2. The effect of treatment on microtubule growing ends in U-2OS cells: DZ-2384.Movie S3. The effect of treatment on microtubule growing ends in U-2OS cells: vinorelbine.References (63–77)

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Acknowledgments: We thank R. Ohi (Vanderbilt University) for the GFP–a-tubulin–expressingLLC-PK1 cells, A. Bird (MPI Dortmund) for the U-2 OS cells expressing EB3-mCherry, andJ. Massagué (Memorial Sloan Kettering) for the MDA-MB-231-LM2 cells. We thank J. Haruna forneuropathology, R. Jacobs for bone marrow pathology, J. Ouellette for electron microscopeexpertise, D. Ethier for fluorescence-activated cell sorting expertise, E. Küster-Schöck forconfocal microscopy expertise, and O. Cleaver for access to the fluorescent dissectionmicroscope. Funding: This work was funded by research grants to G.C.S. and P.G.H. fromDiazon Pharmaceuticals Inc., Montreal, QC, Canada. Research grant support was provided byGénome Quebec and the Quebec Consortium for Drug Discovery (CQDM) (to G.C.S.), bythe Canadian Institutes for Health Research (CIHR) (MOP-137055 to G.J.B., MOP-6192 andMOP-106530 to G.C.S., and MOP-119401 to P.M.S.), by the Swiss National Science Foundation(31003A_166608 to M.O.S.), and by the National Cancer Institute (NCI_CA192381 to T.M.W.),and G.Z. was supported by the Fonds de recherche du Québec–Santé clinician-scientist salaryaward. G.J.B. is a CIHR New Investigator. Author contributions: M.W., J.T., C.B., A.E.P., Y.R.,M.A.H., J.C.A., O.O., G.Z., P.G.H., M.O.S., T.M.W., R.A.B., P.M.S., G.C.S., G.J.B., and A.R. designedresearch studies; M.W., J.T., C.B., A.E.P., S.C., Y.R., C.G., M.A.H., J.C.A., O.O., C.R., N.O., H.D., M.G.A.,


and A.H. conducted experiments; M.W., J.T., C.B., A.E.P., S.C., Y.R., C.G., M.A.H., J.C.A., O.O.,C.R., N.O., A.B., M.G.A., M.O.S., T.M.W., G.C.S., G.J.B., and A.R. analyzed data; and M.W., J.T., C.B.,A.E.P., M.A.H., J.C.A., M.O.S., T.M.W., P.G.H., G.C.S., G.J.B., and A.R. wrote or edited themanuscript. Competing interests: Diazon Pharmaceuticals Inc. holds the rights to the PatentCooperation Treaty publication no. WO2009/134938 that covers DZ-2384. None of the authorsof this manuscript are named inventors on this patent. G.C.S., A.R., and P.H. are founders andshareholders in Diazon Pharmaceuticals Inc. R.A.B. and T.M.W. are founders of TuevolTherapeutics Inc. and are inventors on patent applications (62/067,304; 62/067,276;62/232,901; and 62/232,922) held or submitted by the UT Southwestern Medical Centerthat cover the use of the GEMM rapid in vivo assay (Rgs16::GFP;KIC model) for screeningtherapeutics for pancreatic ductal adenocarcinoma. Data and materials availability:Coordinates have been deposited in the Protein Data Bank (PDB) under accession no. 5LOV(DZ-2384–tubulin complex).

Submitted 11 May 2016Accepted 28 September 2016Published 16 November 201610.1126/scitranslmed.aag1093

Citation: M. Wieczorek, J. Tcherkezian, C. Bernier, A. E. Prota, S. Chaaban, Y. Rolland,C. Godbout, M. A. Hancock, J. C. Arezzo, O. Ocal, C. Rocha, N. Olieric, A. Hall, H. Ding,A. Bramoullé, M. G. Annis, G. Zogopoulos, P. G. Harran, T. M. Wilkie, R. A. Brekken,P. M. Siegel, M. O. Steinmetz, G. C. Shore, G. J. Brouhard, A. Roulston, The syntheticdiazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics withoutneurotoxicity. Sci. Transl. Med. 8, 365ra159 (2016).

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10.1126/scitranslmed.aag1093] (365), 365ra159. [doi:8Science Translational Medicine

Shore, Gary J. Brouhard and Anne Roulston (November 16, 2016) Rolf A. Brekken, Peter M. Siegel, Michel O. Steinmetz, Gordon C. Annis, George Zogopoulos, Patrick G. Harran, Thomas M. Wilkie,Olieric, Anita Hall, Hui Ding, Alexandre Bramoullé, Matthew G. Hancock, Joseph C. Arezzo, Ozhan Ocal, Cecilia Rocha, NatachaProta, Sami Chaaban, Yannève Rolland, Claude Godbout, Mark A. Michal Wieczorek, Joseph Tcherkezian, Cynthia Bernier, Andrea E.microtubule curvature and dynamics without neurotoxicityThe synthetic diazonamide DZ-2384 has distinct effects on

Editor's Summary

protofilaments, which may explain its unusual effects on microtubule dynamics and decreased toxicity.DZ-2384 binds at the vinca site but induces a distinctive change in the curvature of growing tubulin models. By analyzing the structure of tubulin with different compounds, the authors determined thatnetwork in non-dividing cells and in primary neurons at effective doses and is much safer in mouse DZ-2384 is very effective at targeting cancer cells by inhibiting mitosis, it preserves the microtubulecalled DZ-2384, which may offer a safer alternative to the vincas. The authors found that although

. identified a druget alin normal undividing cells including neurons, resulting in toxicity. Wieczorek mitosis, are commonly used for the treatment of cancer. Unfortunately, they also damage microtubules

Drugs such as vinca alkaloids, which target tubulin and interfere with microtubule function inThrowing a curve ball to cancer

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