1521-0111/89/2/233–242$25.00 http://dx.doi.org/10.1124/mol.115.100149MOLECULAR PHARMACOLOGY Mol Pharmacol 89:233–242, February 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Structural Insights into the Pharmacophore of Vinca DomainInhibitors of Microtubules s

Yuxi Wang, Frederick W. Benz, Yangping Wu, Qisheng Wang, Yunfeng Chen,Xiangzheng Chen, Huiyan Li, Yonghui Zhang, Rundong Zhang, and Jinliang YangState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative InnovationCenter for Biotherapy, Chengdu, China (Yu.W., Ya.W., Y.C., X.C., H.L., R.Z., J.Y.); Department of Pharmacology and Toxicology,University of Louisville School of Medicine, Health Sciences Center, Louisville, Kentucky (F.W.B.); Shanghai Institute of AppliedPhysics, Chinese Academy of Sciences, Shanghai, China (Q.W.); and Pharmacology and Pharmaceutical Sciences School ofMedicine, Tsinghua University and Collaborative Innovation Center for Biotherapy, Beijing, China (Y.Z.)

Received June 8, 2015; accepted December 7, 2015

ABSTRACTAntibody-drug conjugates (ADCs) have achieved great successin cancer therapy in recent years. Some peptidyl microtubuleinhibitors consisting of natural and unnatural amino acids, suchas monomethyl auristatin E (MMAE) and F (MMAF), are ex-tremely cytotoxic and have been used as a payload in ADCs.However, their precise molecular interaction with tubulin andmicrotubules remains unclear. We determined the crystal struc-tures of tubulin in complex with three ultra-potent peptidyl micro-tubule inhibitors [MMAE, taltobulin (HTI- 286), and tubulysinM] at2.5 Å. Our data showed that the three peptides bound to thevinca domain and shared a common and key pharmacophorecontaining two consecutive hydrophobic groups (Val, Ile-likeside chain). These groups protruded in opposite directions intohydrophobic pockets on the tubulin b and a subunits. Nitrogen

and oxygen atoms from the same backbone formed hydrogenbonds with Asn329 from the a subunit and Asp179 from the bsubunit in a direction normal to the surface formed by theaforementioned hydrophobic groups. In addition, our crystalstructure data indicated that tubulysin M bound to the b subunitalone, providing a structural explanation for its higher affinity.We also compared the conformations of two representativestructurally different vinca domain compounds, ustiloxin D andvinblastine, with those of the aforementioned peptidyl ligands,and found that they shared a similar pharmacophore. Ourfindings lay a foundation for the rational design of novel vincadomain ligands and may facilitate the development of microtu-bule inhibitors with high specificity, affinity, and efficiency aspayloads for ADCs in cancer therapy.

IntroductionMicrotubules—highly dynamic structures of the cytoskele-

ton composed of tubulin—play crucial roles in cell mitosis,growth, and movement (Etienne-Manneville and Hall, 2002;Akhmanova and Steinmetz, 2008). The biologic functions ofmicrotubules are mainly regulated by polymerization anddepolymerization of the a- and b-tubulin heterodimers(Etienne-Manneville, 2010). Microtubules and tubulin havebeen important cancer chemotherapy targets for decades, andmicrotubule-targeting agents (MTAs) can efficiently result inmitotic arrest and cell death (Cragg and Newman, 2004;Dumontet and Jordan, 2010). To date, several dozen MTAshave been discovered and developed, such as paclitaxel

(Komlodi-Pasztor et al., 2012), vinblastine (Carlson andOcean, 2011), and epothilone (Roque et al., 2013). Amongthem, paclitaxel and vinblastine represent a very successfulclass of anticancer drugs widely used clinically for thetreatment of leukemias, lymphomas, and some solid malig-nancies (Muldoon et al., 2007; Risinger et al., 2009).MTAs can bind to tubulin directly, causing conformational

changes and thus interfering with microtubule structure anddynamics. MTAs are divided into two general categories:microtubule-stabilizing and -destabilizing agents (Hartleyet al., 2012). Microtubule-stabilizing agents, represented bytaxol-like compounds, bind to the lateral side of tubulin,stabilizing the M-loop of b-tubulin, which establishes thelateral contacts of tubulin in microtubules (Prota et al.,2013a). Microtubule-destabilizing agents bind to the longitu-dinal interface between the a/b-tubulin subunits, causingconformational shifts of consecutive a-, b-tubulin hetero-dimers from a straight to a curved form, facilitating microtu-bule disassembly (Ravelli et al., 2004; Gigant et al., 2005).Based upon the specific interface to which they bind,

Work in the authors’ laboratory was supported by research funding from theNational Major Scientific and Technological Special Project for “SignificantNew Drug Development” [Grant 2012ZX09103301-030] and the NationalNatural Science Foundation of China [Grant 81372822].

dx.doi.org/10.1124/mol.115.100149.s This article has supplemental material available at molpharm.

aspetjournals.org.

ABBREVIATIONS: ADC, antibody-drug conjugate; AMPPCP, b,g-methyleneadenosine 59-triphosphate disodium salt; G-PEM, general tubulinbuffer; HP, hydrophobic pocket; HTI-286, taltobulin; MMAE, monomethyl auristatin E; MMAF, monomethyl auristatin F; MTA, microtubule-targetingagent; PDB, Protein Data Bank; RB3-SLD, stathmin-like domain of RB3; T2-R-TTL, consisting of a/b-tubulin, the stathmin-like protein RB3, andtubulin tyrosine ligase; TTL, tubulin tyrosine ligase.

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microtubule-destabilizing agents can be further subdividedinto a colchicine-like group that binds to the a/b interfacewithin one tubulin heterodimer (intradimer), termed thecolchicine site, or into a vinblastine-like group that binds tothe a/b interface between two tubulin heterodimers (inter-dimer), named the vinca domain. Colchicine site ligands binddeeply into the small hydrophobic pocket formed primarily byb-tubulin and are, in general, structurally small, makingfurther modifications of this type of ligand more problematic.In contrast, vinca domain ligands bind to the interface formedby two tubulin heterodimers and are, in general, structurallylarger. This feature provides for not only more structuraldiversity, but also the opportunity for higher affinity fortubulin and greater cytotoxicity. Some MTAs, such as HTI-286, monomethyl auristatin E (MMAE), and tubulysin M (Fig. 1),are extremely cytotoxic, with in vitro IC50 values in thenanomole to picomole range (typically 100- to 1000-fold lowerthan vinblastine and taxol). When used alone, these agentsare unsuitable clinically because of their narrow therapeuticwindow. However, they have recently been used successfullyin clinical applications as payloads on antibody-drug conju-gates (ADCs) to increase their target specificity. For example,brentuximab vedotin (Adcetris), an ADC with MMAE as thepayload, was the first Food and Drug Administration–approved ADC for Hodgkin disease in 2011(Klute et al., 2014).Structural studies of the binding of vinca domain ligands

with tubulin have facilitated our understanding of theirmechanism of interaction and have provided the basis forrational drug design. Efforts in this direction began in 2005,when the crystal structure of vinblastine bound to tubulinwas determined at 4.1 Å. Subsequently, the conformation ofseveral additional vinca domain ligands bound to tubulin havebeen studied, such as soblidotin (Cormier et al., 2008) andustiloxin D (Ranaivoson et al., 2012).With the exception ofustiloxin D at 2.7-Å resolution, all of the others were

determined at intermediate resolutions (.3.5 Å), whichcannot provide sufficient structural detail critical for ratio-nal drugdesign. MMAE, the payload of Adcetris, HTI-286(Poruchynsky et al., 2004), and tubulysins (Cohen et al.,2014), are vinca domain ligands of linear tri- or tetrapeptidescomposed of diverse unnatural amino acids with ultra-potentcytotoxicity. The details of how these agents bind to tubulinand whether their binding modes share common featuresamong themselves or with other structurally different vincadomain ligands remain unclear.We determined the crystal structures of the three afore-

mentioned agents bound to tubulin at a high resolution(� 2.5 Å). Our data revealed that these three MTAs all bindto the vinca domain and share a common and key pharmaco-phore. In addition, we observed tubulysin M bound to the bsubunit alone, suggesting a rationale for its higher affinity fortubulin, and our structural analysis provided a molecularmechanism for it. Finally, we found that two other representa-tive MTAs with diverse structure targeting the vinca domain,ustiloxin D and vinblastine, shared a similar pharmacophoreidentified earlier. These findings provide the possibility forstructure-based rational design of novel microtubule inhibitorstargeting the vinca domain with high specificity, affinity, andefficiency as payloads for ADC cancer therapy.

Materials and MethodsPorcine brain tubulin was purchased from Cytoskeleton, Inc

(Denver, CO). The clone of stathmin-like domain of RB3 (RB3-SLD)was a gift from Dr. Benoît Gigant (Centre National de la RechercheScientifique, Paris, France). The plasmid of tubulin tyrosine ligase(TTL)was also a kind gift fromDr.MichelO. Steinmetz (Paul ScherrerInstitute, Switzerland). Tubulysin M and HTI-286 were purchasedfrom Levena Biopharma Co., Ltd (Nanjing, China). MMAE wasprovided by Yi Qun Biotechnology Co., Ltd (Shanghai, China).

Fig. 1. Chemical structures of the compounds in this study.

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Vinblastine was obtained from Selleck Chemicals (Houston, TX).Antiprotease cocktail was purchased from Sangon Biotech (Shanghai,China). Bis-tris propane, imidazole, tyrosine, dithiothreitol, andb,g-methyleneadenosine 59-triphosphate disodium salt (AMPPCP) werepurchased from Sigma-Aldrich (St. Louis, MO). b-Mercaptoethanolwas obtained from XiYa Reagent (Chengdu, China).

Protein Expression and Purification. RB3-SLDwas expressedin Escherichia coli, purified by anion-exchange chromatography andgel filtration, concentrated to 10 mg/ml, and stored at 280°C(Charbaut et al., 2001; Dorléans et al., 2009). Protein production ofTTL was also carried out using E. coli according to the procedurereported by Prota et al. (2013a). After expression in E. coli, the proteinwas purified by affinity chromatography and gel filtration, concen-trated to 20 mg/ml, and stored at 280°C. The purity of RB3 and TTLwas assessed by SDS-PAGE. Porcine brain tubulin was supplied at10 mg/ml in general tubulin buffer (G-PEM) [general tubulin buffer:80 mM PIPES (pH 6.9), 2 mMMgCl2, 0.5 mM EGTA, and 1 mM GTP]as a frozen liquid and preserved at 280°C until use.

Protein Crystallization and Crystal Soaking. Crystals of T2-R-TTL (consisting of a/b-tubulin, the stathmin-like protein RB3, andtubulin tyrosine ligase) were obtained by vapor diffusion using thesitting-drop method according to the provided protocols (Prota et al.,2013a,b, 2014). The protein solution consisting of 82 ml of tubulin(10mg/ml), 9.9ml of TTL (20mg/ml), and 8.1ml of RB3 (10mg/ml), witha molar ratio of tubulin:RB3:TTL of 2:1.3:1.2, was incubated on icewith 1 mM AMPPCP, 5 mM tyrosine, and 10 mM dithiothreitol, andthen concentrated to 20 mg/ml at 4°C. Crystallization drops contained1 ml of the T2-R-TTL protein solution and 1 ml of the precipitantsolution consisting of 6% poly(ethylene glycol) 4000 Da, 5% glycerol,0.1MMES, 30mMCaCl2, and 30mMMgCl2 (pH 6.7) on a sitting plateand incubated at 20°C. Crystals appeared after 1 day and reached alength of 200–300 mm within 3–5 days.

For crystal soaking, all ligands were dissolved in dimethylsulfoxideat 30 mM. MMAE (0.1 ml), tubulysin M (0.1 ml), and HTI-286 (0.1 ml)were soaked into crystals for 24 hours at 20°C. The crystals weretransiently dipped into a cryoprotectant (30mMMgCl2, 30mMCaCl2,0.1M MES, and 20% glycerol, pH 6.7) and flash-cooled in liquidnitrogen for synchrotron X-ray diffraction data collection.

X-ray Data Collection, Structure Determination, Refine-ment, and Analysis. All X-ray diffraction data were collected atbeamline BL17U1 and BL19U1 at the Shanghai Synchrotron Radia-tion Facility (Shanghai Institute of Applied Physics, Chinese Academyof Sciences, Shanghai, China) at wavelengths of 0.97915 and 0.97853 Å.Data were indexed, integrated, and scaled using theHKL-2000 software(HKLResearch, Inc., Charlottesville, VA) (Otwinowski andMinor, 1997).The structure was solved by the molecular replacement method with anexisting ligand-free structure T2-R-TTL [Protein Data Bank (PDB) code:4I55] as our research model. The models were improved by cyclesof manual rebuilding in Coot (Emsley et al., 2010) and REFMACrefinement (Vagin et al., 2004) with the CCP4 software suite (Re-search Complex at Harwell, Didcot, UK) (Winn et al., 2011). The finaldata collection and refinement statistics are summarized in Table 1.The figures are drawn using PyMol software (http://www.pymol.org).

ResultsOverall Structure of the Three MTAs Bound to

Tubulin. Using soaked crystals of a protein complex (T2-R-TTL) consisting of a/b-tubulin, the stathmin-like protein RB3,and tubulin tyrosine ligase, we determined the structures ofHTI-286, MMAE, and tubulysin M (Fig. 1) bound to tubulin at2.5–2.55 Å (Fig. 2; Table 1). The difference electron densitymap calculated using the ligand-omitting protein modelclearly defined the envelope into which each of the threeligands could be fit unambiguously (Fig. 2, B and C). The overallstructure of tubulin in the three complexes superimposed well

with the previously determined ligand-free structure (PDBcode: 4I55), with root mean square deviation ranging from0.168 to 0.279 Å over 700 Ca atoms, indicating little effect ofthe binding of each ligand on tubulin’s overall structure.HTI-286 and MMAE bind to the vinca domain, which is

between tubulin subunits b1 and a2 (Fig. 2A). Surprisingly,tubulysin M bound not only to the vinca domain illustrated byTM1 but also to the tubulin b2 subunit alone illustrated byTM2 (Fig. 2A). When the b1 and b2 subunits were super-imposed, the structures of TM1 and TM2 superimposed aswell. The conformations of both were almost identical, withthe exception of small variations observed at the molecularridges pointing away from the tubulin b subunits (Supple-mental Fig. S1). These variations were due to the additionalrestrictions imposed on the ligand by the a2 subunit in thevinca domain. The similarity of the conformation of TM2to TM1 indicated that the bound conformation of both isdetermined predominantly by the b subunit, as the influenceof the a subunit was absent in the structure of TM2.Detailed Interactions of HTI-286 with Tubulin a and

b Revealed the Most Important Elements for Bindingto the Vinca Domain. In the vinca domain, HTI-286,MMAE, and tubulysin M are sandwiched between subunitsb1 and a2, and they structurally superimposed quite well(Figs. 3 and 4). HTI-286 is the smallest ligand of the three, andtherefore, its interactions with tubulin should display themost important elements for binding.On the a2 subunit, HTI-286mainly interacted with loopH7-

H8, helix H10, and strand S9 (Fig. 3A). Both hydrophobic andhydrogen bonding interactions were observed. Hydrophobicinteractions involved the tert-butyl group (C16–C19) of HTI-286 and the hydrophobic pocket (HPa) formed mainly byLeu248 from loop H7-H8, Pro325 and Val328 from H10, andVal353 and Ile355 from S9. In addition, Ile332 from H10 andPhe351 fromS9 extendHPa and contact themethyl groupC21(Fig. 3A). Two hydrogen bonding interactions with a2 wereobserved to be shared by all three ligands. The first involvesthe side chain of Asn329 on H10 directly with O6 and N8 ofHTI-286. The other is mediated by watermolecules H2O-1 andH2O-3 between the main chain elements of S9 and HTI-286(Fig. 3A).On the b1 subunit, HTI-286 interacted with loop T5, helix

H6, loop H6-H7, and helix H7 (Fig. 3B). The interactionsincluded the following hydrophobic contacts: (1) the isopropylside chain (C13–C15) of HTI-286 inserted into a hydrophobicpocket (HPb) formed by Val177 from loop T5, Tyr210 fromH6,and Tyr224 and Leu227 from H7; and (2) the benzyl group(C24–C29) and twomethyl groups (C22–C23) on the other sideof the ligand contact the C-terminus of loop T5. In addition,several hydrogen bonds contributed to the binding to the b1subunit (Fig. 3B). Asp179 from loop T5 hydrogen bonded N20directly and indirectly via water molecule H2O-3. Interest-ingly, this same N20 formed a hydrogen bond network withthe main chain N and O atoms of a2 via the same H2O-3(Fig. 3A). Asp179 also hydrogen bonded O9 via water mole-cule H2O-1 (Fig. 3B). An additional hydrogen bond networkexisted between O6 of HTI-286 and the side chain of Tyr210and the main chain of Pro222 via H2O-2.Additional Hydrogen Bonding Interactions between

MMAE and the Vinca Domain of Tubulin. The interac-tions between MMAE and tubulin b1 included virtually all ofthe components observed with HTI-286. However, because

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MMAE is a considerably larger molecule, there was theopportunity for additional favorable contacts, such as thoseobserved between the ligand and loop H6-H7, helix H7, loopS7-H9, and helix H1 (Fig. 3, D and E). The additionalinteractions were as follows: (1) O12 of MMAE formed ahydrogen bond with the main chain N atom from Tyr224; (2)O4 formed a hydrogen bond network with the main chain Natom from Gly225 (H7), the side chains of Thr223 (H6-H7),and Arg278 (S7-H9) via water; and (3) O1 formed a hydrogenbond with the side chain of Gln15 on H1.The interactions between MMAE and the tubulin a2 sub-

unit were quite similar to those observed for HTI-286. Thehydrogen bond interactions were almost identical (Fig. 3,A and C). The additional structure of MMAE (C6–C24) pointedout into the solvent and did not contribute substantially, withthe exception of an extra hydrophobic contact between thepyrrolidinyl ring of MMAE and loop H7-H8 containing Ala247and Leu248 (Fig. 3C). At the other end of MMAE (N22), themolecule did not extend as deeply into a2 as did HTI-286,where its phenyl group (C24–29) could interact with I332(Fig. 3, A vs. C).Tubulysin M Bound to the Vinca Domain of Tubulin.

Tubulysin M interacted with a2 in a manner almost identicalto HTI-286 and MMAE (Fig. 3F). More interesting was itsinteraction with the b1 subunit. Similar to MMAE, theinteraction contained almost all the components observed forHTI-286. The small difference was that the H-bond betweenAsp179 and the N19 atom, corresponding to N20 in HTI-286,was reduced due to the chiral conformation of the hexahy-dropyridine ring (Fig. 3G). In addition, because of its largerstructure, tubulysin M formed additional interactions withloop H6-H7, helix H7, loop S7-H9, and helix H1 (Fig. 3, G andH). Similar toMMAE, the additional interactions weremainlycharged but, unlike MMAE, its structure branches allowed fordifferent and possibly stronger interactions with subunit b1.The following interactions were observed (Fig. 3, G and H):

(1) N8 and O6 of tubulysin M formed hydrogen bonds with themain chainNatoms of Tyr224 andGly225 at theN terminus ofhelixH7; (2) the oxygen atomsO1a/O1b at one end of tubulysinM formed a salt bridge with the side chain of Arg278 (S7-H9);and (3) an additional observation was the hydrogen bondnetwork between tubulysin M directly with the side chain ofThr223(H6-H7), and indirectly via H2O-3 and H2O-4 withAsn228 (H7) and Gln15 (H1). These additional interactions,especially the salt bridge, may explain why tubulysin M canbind to subunit b alone (Fig. 2A).These Three Ligands Define a Key Fundamental

Pharmacophore for Binding to the Vinca Domain ofTubulin. In summary, the bound conformations of thesethree peptidyl ligands shared a common and key pharmaco-phore, two consecutive hydrophobic groups (Val, Ile-like sidechain) protruding in opposite directions into separate hydro-phobic pockets on the b1 and a2 subunits of tubulin (Fig. 4, Aand C). Additionally, in the direction normal to the surfaceformed by the aforementioned hydrophobic groups, atoms Nand O from the same backbone formed hydrogen bonds withAsn329 on a2 and Asp179 on b1 (Fig. 4B). These features areunique, as these two consecutive amino acid residues fitsnuggly into the narrowest channel between two tubulinsubunits in the vinca domain and use both backbone and sidechain elements to interact in all four orthogonal directionswith tubulin (Fig. 4).Two Structurally Different Vinca Domain Ligands,

Ustiloxin D and Vinblastine, Share the Same KeyInteracting Components. Several crystal structures ofvinca domain ligands have been reported with resolutions inthe range of 2.7–4.4 Å (Gigant et al., 2005; Cormier et al., 2008;Ranaivoson et al., 2012). Among those, the 2.7-Å structure ofthe circular molecule ustiloxin D (Fig. 1) has been determinedwhen ustiloxin Dwas bound to tubulin that had been partiallydigested with subtilisin. Superimposing tubulin b1 withbound ustiloxin D, we were surprised to find that removing

TABLE 1Data collection and refinement statistics

Ligand (PDB code)

HTI-286 (4ZI7) MMAE (4ZHQ) Tubulysin M (4ZOL) Vinblastine (5BMV)

Data collection statisticsX-ray source SSRF-BL17U1 SSRF-BL17U1 SSRF-BL17U1 SSRF-BL19U1Wavelength (Å) 0.97915 0.97915 0.97915 0.97853Resolution range (Å)a 50.0–2.50

(2.54–2.50)39.34–2.55 (2.59–2.55) 50.0–2.50 (2.54–2.50) 50.0–2.50

(2.54–2.50)Space group P212121 P212121 P212121 P212121Unit cell: ambic (Å) 105.31, 157.26, 181.78 104.94, 156.81, 182.47 105.13,154.82, 186.52 105.47, 157.43, 183.19Total reflections 734,243 723,140 771,643 709,659Unique reflections 103,939 (5093) 98,202 (4843) 105,685 (5207) 106,106 (5233)Redundancy 7.1 (6.6) 7.4 (7.3) 7.3 (7.3) 6.7 (6.8)Completeness (%) 99.8 (99.4) 99.9 (100) 99.9 (100) 100 (100)Mean I/sigma (I) 14.1 (2.0) 13.7 (2.1) 18.6 (2.53) 19.0 (2.75)Remerge 0.179 (0.90) 0.129 (0.93) 0.137 (0.884) 0.105 (0.776)

Refinement statisticsR-factor/R-free 0.184/0.239 0.185/0.2444 0.173/0.231 0.184/0.235RMSD bond length (Å) 0.0146 0.0134 0.0152 0.0138RMSD bond angle (°) 1.7415 1.6928 1.9217 1.727Ramachandran plot statisticsFavored regions (%) 95.6 95.3 95.1 95.2Additional allowed regions (%) 3.9 4.2 4.3 4.3Disallowed regions (%) 0.5 0.5 0.6 0.5

RMSD, root mean square deviation; SSRF, Shanghai Synchrotron Radiation Facility.a The highest resolution shell is shown in parenthesis.

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Fig. 2. Overview of a T2-R-TTL–ligand complex structure and the conformations of the bound ligands. (A) Overview of the T2-R-TTL–tubulysin Mcomplex. Tubulin (gray), RB3 (sage green), and TTL (pale green) are depicted in ribbon representation; tubulysin M (TM1 or TM2, orange), GDP (green),andGTP (red) are shown in sphere representation. (B) Omitmaps of the four ligands (contoured at 3s): HTI-286 (blue), MMAE (green), TM1 (orange), andTM2 (pink) are shown in stick representation. (C) The conformation ofMMAE inside the electron density omitmap determined at 2.55Å against tubulin bsubunit. (D) The conformation of soblidotin, which is very similar toMMAE in structure, inside the electron density omitmap determined at 3.8 Å againsttubulin b subunit (PDB code: 3E22). (E) Comparison of the conformations of MMAE and soblidotin with superimposition of tubulin b subunit. The curveddouble-ended arrows indicate the distance difference (in Å) of the corresponding atoms.

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the constricting group connecting C2 and C9, which circular-ized the molecule, revealed that the remaining portions werealmost identical to the overlapping structures of the threeligands reported here (Fig. 5, A–C). Specifically, an Ile-likeside group at position 3 made a hydrophobic contact with HPb,and N9 made a direct hydrogen bond with the side chain ofAsp179. In addition, ustiloxin D bound to a2 in two ways aswell: (1) its isopropyl group at position 6 fit into HPa; and (2)the amide (N7) and carboxyl (O5) of its main chain formedhydrogen bonds with O and N atoms of the side chain ofAsn329, respectively (Fig. 5B).Phomopsin A is another vinca domain ligand with its

structure bound to tubulin determined at 4.1 Å (Cormieret al., 2008). Due to this resolution limitation, we did notattempt to use its structure to make a superimposition. Wecan reasonably assume phomopsin A could adopt the sameconformation as ustiloxin D because a large part of phomopsinA’s structure is almost identical to that of ustiloxin D.

Vinblastine, a vinca domain ligand after which the domainis named, shared the least structural similarity with thecompounds described earlier. Its structure bound to tubulinhas been determined at 4.1 Å (Gigant et al., 2005) andsubsequently at 3.47 Å (Ranaivoson et al., 2012). Thisstructural resolution is inadequate to compare with thestructural models reported here. Therefore, to complete ourinvestigation of a pharmacophore important for vinca domainbinding, we determined the structure of the vinblastinecomplex with T2-R-TTL at 2.5 Å (Table 1). Superimposingthe structure we determined with the published 3.47 Åstructure (PDB code: 4EB6) indicated that our structure fit theelectron density envelope better (Supplemental Fig. S2).Superimposing the vinblastine structure on those of the otherthree ligands, as was done with ustiloxin D, led to theunanticipated revelation that, despite its structural disparity,all of the essential interactions identified with the aforemen-tioned compounds were maintained (Fig. 5, D–F). Specifically,

Fig. 3. Detailed interactions of HTI-286, MMAE, and tubulysin M with the tubulin vinca domain. HTI-286 interaction with the a2-domain (A) andb1-domain (B). MMAE interaction with the a2-domain (C) and b1-domain (D and E). Tubulysin M interaction with the a2-domain (F) and b1-domain(G and H). See text for details.

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the ethyl group from the D9-ring of the catharanthine portionfit intoHPb, and theN9 atom of the vindoline portion formed ahydrogen bond with Asp179 (Fig. 5E). In addition, the benzylA9-ring at the opposite side of the catharanthine portion fit intothe hydrophobic pocket HPa, and the N atom on the B9-ring andthe O atom from the side chain of the C9-ring contributed to thetwo hydrogen bonds with the Asn329 side chain.

DiscussionVinca domain ligands are a class of microtubule inhibitors

with great potential for cancer therapy as payloads for ADCsbecause of their high affinity for tubulin, resulting in good

clinical efficacy. Understanding the structure-activity rela-tionships controlling tubulin binding is critical to new drugdevelopment in this area. Although several crystal structuresof the complexes between MTAs and the vinca domain oftubulin have been determined, none were of sufficient resolu-tion to serve as guides for rational drug design. As a result, nogeneralized consensus on a pharmacophore for ligand bindingto the vinca domain of tubulin has been attained.In the present study, the structures of four ligands bound to

the vinca domain of tubulin were determined at atomicresolution (� 2.5 Å). Of these, only the structure of thevinblastine complex had been determined previously, but onlyat a resolution of 3.47 Å. HTI-286, MMAE, and tubulysin M

Fig. 4. Comparison of the interactions of HTI-286, MMAE,and tubulysin M with tubulin. When the structures ofthe three ligands bound to subunit b1 are superimposed,the three structures largely overlap. Two Val- or Ile-likehydrophobic side chains protrude from consecutive posi-tions of the ligand backbones and fit into hydrophobicpockets a and b, as seen in (A) and (C). Three hydrogenbond interactions with either D179.b1 or N329.a2 wereobserved as depicted in (B). These hydrophobic and hydro-gen bond interactions were found to be common to allligands. MTAs are shown in stick representation andcolored as in Fig. 2. Tubulin a2 and b1 are shown in surfacerepresentation.

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compose a unique class of vinca domain ligands in that theyare short peptidyl compounds. Soblidotin has a structuresimilar to MMAE (Fig. 2D; Supplemental Fig. S3), and is theonly compound in this class for which a crystal structurebound to tubulin was reported (Cormier et al., 2008) but onlyat a resolution of 3.8 Å (PDB code: 3E22; Fig. 2D). Soblidotindiffers from MMAE at three minor positions, which play littlerole in binding to tubulin (Supplemental Fig. S3). Althoughthe reported orientation of soblidotin in the vinca domain wascorrectly defined, its precise position between the tubulinsubunits is very likely incorrect. When we superimposed thetubulin b1 subunit, we found that many details of thepreviously determined conformation of soblidotin were differ-ent from MMAE determined at 2.5 Å. The most significantshift of about 5.5 Å occurred at the phenyl group at the tail endof the molecule (Fig. 2E). Because of this disparity, wedetermined that we could readily fit soblidotin into theelectron density envelope of MMAE, and therefore, weconsider the refitted conformation of soblidotin likely to bethe correct one (Supplemental Fig. S3).The phenomenon that tubulysin M binds not only to the

vinca domain, but also to the tubulin b2 subunit alone hasbeen observed previously for phomopsin A (Cormier et al.,2008). This suggests a rationale as to why both tubulysin Mand phomopsin A have higher affinity for tubulin than HTI-286 and MMAE in that the latter two agents require in-teraction with both subunits for binding, whereas the formerdo not. Thus, the unrequired but additional favorable binding

interactions of the former two agents with the a subunitshould translate into higher affinity for the vinca site.However, insight into the higher affinity of phomopsin A

Fig. 5. Comparison of the structures ofustiloxin D and vinblastine with HTI-286,MMAE, and tubulysin M in their tubulin-bound conformations. The ligand-boundtubulin b1s are superimposed, and theconformations of bound ligands arecompared. The three ligands, HTI-286,MMAE, and tubulysin M, are in the sameorientation as in Fig. 3 but shown in linerepresentation. (A–C) The tubulin-boundconformation of ustiloxin D. (D–F) Thetubulin-bound conformation of vinblastine(PDB code: 5BMV). Interestingly, bothustiloxin D and vinblastine contain thesame common functional elements identi-fied in Fig. 3 and summarized in Fig. 4.

Fig. 6. Pharmacophore of vinca domain ligands. The pharmacophorecontains two hydrophobic groups (large spheres I and II) and threehydrogen-bond-forming atoms (small spheres, blue for N atom, red forO atom). The two hydrophobic groups I and II interact with hydrophobicpockets b and a, respectively. The small gray spheres represent C atomsat the edges of the hydrophobic groups. The three hydrogen-bond-forming atoms interact with D179.b (one N atom) and N329.a (one N andone O atom). There are two areas (A and B) at the opposite sides of thepharmacophore where extra chemical groups can be added to makeadditional interactions to increase the affinity of the ligand.

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was not achieved in the previously determined crystal struc-ture due to inadequate resolution (4.1 Å). In contrast, ourcurrent study documents several additional favorable inter-actions observed for tubulysin M, including the energeticallyimportant salt bridge with Arg278 (Fig. 3H).By comparing the complexes for all of the structurally

distinct ligands bound to the vinca domain of tubulin, wewere able to define a fundamental pharmacophore for binding(Fig. 6). The pharmacophore consists of two hydrophobicregions, I and II, that interact with hydrophobic pockets onthe b and a subunits, respectively, and two hydrogen bondingregions that interact with Asp179.b and Asn329.a. In additionto these four components, two additional regions, A and B,were identified where ligands can make additional favorableinteractions for tighter binding. Specifically in space B, whereboth MMAE and tubulysin M project their additional struc-ture, there is opportunity for structural modifications toenhance binding.Although the conformation of phomopsin A bound to tubulin

was determined only at 4.1 Å (Cormier et al., 2008), it canserve as an example to illustrate our structural insights intothe pharmacophore of vinca domain ligands. Its structurecontains two portions—a macrocycle, which is similar toustiloxin D, and a dicarboxylate end. If only the macrocycleportion bound to the vinca domain, as does ustiloxin D, itsdissociation constant (Kd) should be similar to that of ustiloxinD (1.8 6 0.6 mM) (Ranaivoson et al., 2012). The 10-fold higheraffinity of phomopsinA (Kd, 0.1mM) (Ranaivoson et al., 2012)may be partly due to the additional dicarboxylate end that canproject into space B, allowing interaction with subunit b.Interestingly, phomopsin A, similar to tubulysin M, bound toboth the vinca domain and to the b subunit alone, whereasustiloxin D, similar to HTI-286 and MMAE, bound only to thevinca domain. This corroborates the observation that bindingto two sites in the T2-R crystal is consistent with higheraffinity of the ligand for the vinca domain of tubulin.The binding of each of the three vinca domain ligands causes

the two tubulin subunits to bend and twist slightly, as wasobserved previously for bound ustiloxin D (Ranaivoson et al.,2012). Aligning tubulin b1, the root mean square deviation ofthe Ca atoms of the a2 subunit in the complex with boundHTI-286, MMAE, and tubulysin M is 0.765, 0.863, and 2.20Å,respectively (over 440 Ca atoms). The bending and twisting ofthe subunits can also be seen clearly in Supplemental Fig. S4.The larger bending caused by tubulysin M is due to the largersize of the molecule between two tubulin subunits.In summary, we determined the structures of four vinca

domain ligands bound to tubulin at high resolution. Three ofthese structures have not been reported previously, and theother only at 3.47 Å. Together with the high-resolutionstructure for ustiloxin D, we analyzed their bound conforma-tions in the vinca domain of tubulin andmapped out a commonpharmacophore for the first time. We also observed thattubulysin M was able to bind to the tubulin b subunit aloneand provided the structural insight into its higher affinity.This study lays a foundation for the rational design of novelMTAswith high specificity, affinity, and efficiency as payloadsfor ADCs in cancer therapy.

Acknowledgments

The authors thank Dr. Benoît Gigant (Centre National de laRecherche Scientifique, Paris, France) and Dr. Michel O. Steinmetz

(Paul Scherrer Institute, Switzerland) for their kind offers of theplasmids of RB3-SLD and TTL; Qiang Li (Institute of Modern Physics,Chinese Academy of Sciences, Lanzhou, China) and John YunChungChen (UC Berkeley, Berkeley, CA) for improving this manuscript;Yuqin Yao, Qinhuai Lai, Lantu Gou, Hao Chen, Qiang Chen, andDan Su for their help in the experiments and data analysis. Finally,the authors acknowledge the technical expertise of Dr. Feng Yu andDr. Wenming Qin for training in the collection of X-ray data at thebeamline BL17U1 and BL19U1 at the Shanghai Synchrotron Radia-tion Facility (Shanghai, China).

Authorship Contributions

Participated in research design: Yang, R. Zhang, Y. Wang.Conducted experiments: Y. Wang, Wu, Q. Wang, Y. Chen, X. Chen,

Li, Y. Zhang.Performed data analysis: R. Zhang.Wrote or contributed to the writing of the manuscript: R. Zhang,

Y. Wang, Yang, Benz.

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Address correspondence to: Jinliang Yang, 3-17 Renmin South Road, StateKey Laboratory of Biotherapy and Cancer Center, West China Hospital,Sichuan University, and Collaborative Innovation Center for Biotherapy,Chengdu 610041, China. E-mail: jinliangyang@scu.edu.cn or Rundong Zhang,3-17 Renmin South Road, State Key Laboratory of Biotherapy and CancerCenter, West China Hospital, Sichuan University, and CollaborativeInnovation Center for Biotherapy, Chengdu 610041, China. E-mail:rundongzhang@scu.edu.cn

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