Conformational States of E7010 Is Complemented by Microclusters of

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Computational Biochemistry

Conformational states of E7010 is complemented by micro-clusters of water inside the #,#-Tubulin core Sarmistha Majumdar, Debadrita Basu, and Shubhra Ghosh Dastidar J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00538 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Journal of Chemical Information and Modeling

Conformational states of E7010 is complemented by micro-clusters of water inside the α,β-Tubulin core Sarmistha Majumdar, Debadrita Basu and Shubhra Ghosh Dastidar* Bioinformatics Centre, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700054 *Address for correspondence [email protected] Tel: +91-33-2569-3332 (O) +91-98301-63990 (M)

ACS Paragon Plus Environment

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Abstract: The α,β-tubulin is the building block of the microtubules, which gets associated with and dissociated from the microtubular architecture complying with the dynamic instability of the microtubules. This dynamics has direct relation with the spindle formation by the microtubules and cell division kinetics. E7010 is one of the promising ligands of α,β-tubulin protein that binds at the core of this protein and can diminish the protein’s ability to fit to a growing microtubule; thus frustrates the cell division. Although x-ray crystallography has reported a specific binding conformation of E7010 in PDB, molecular dynamics (MD) simulations have revealed two other conformational states of the ligand capable to bind to tubulin with stabilities close to that state reported in PDB. To rationalize this quasi degeneracy of ligand binding modes, MD simulations have further revealed that the understanding of the mechanism of E7010-tubulin binding remains incomplete unless the role of water molecules to bridge this interaction is taken into consideration; a very critical insight that was not visible from the PDB structure. Further, these water molecules differ from the standard examples of ‘bridging’ waters which generally exists as isolated water molecules between receptor and ligand. In the present case the water molecules sandwiched between ligand and protein, sequestered from the bulk solvent, integrate with each other by HBonds network forming a group, which appear as micro clusters of water. The structural packing with the ligand binding pocket and the bridging of interactions between protein and ligand take place through such clusters. The presence of this micro-cluster of water is not just cosmetic, instead they have crucial impact on the ligand binding thermodynamics. Only with the explicit consideration of these water clusters in the binding energy calculations (MMGBSA), the stability of the native mode of ligand binding reported in PDB is rationalized. At the same time the two other binding modes are elucidated to be quasi degenerate with the native state and that indicates the further possibility in gaining more


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entropic stabilization of the complex. The role of such ‘bridging’ water clusters to enhance the protein-ligand interaction will be insightful for designing next generation prospective compounds in the field of cancer therapeutics.

Key words: Tubulin, E7010, molecular dynamics, bridging water, micro-cluster, MMGBSA

Introduction: The α,β-tubulin heterodimers serve as the constitutional unit of the ‘microtubules’ which grow and elongate to form the mitotic spindles during the cell division. Strategic perturbation of the tubulin subunits would aid in arresting the cell cycle of uncontrollably/abnormally dividing cells. Hence the α,β-tubulin protein has emerged as a potential target of the anticancer drugs over the past few decades.1-3 The structure of microtubules formed by tubulin is understood in multiple tiers. First, α,β-tubulin dimers are thought to join in series from head to tail, i.e. along their longitudinal axis of growth, forming the protofilament. The protofilaments laterally associate in a helical fashion to build the wall of the hollow tube-like microtubules (Figure 1a). It has been pointed out by various structural studies

4-6

that in a

protofilament the relative orientation between the two adjacent dimers can differ which play an important role in the formation of microtubules. This variation in the structural orientation of tubulin dimers originate from the slight differences (3° to 5°) in the angle (θ) of orientation between the α and the β tubulin subunits (Figure 1b-c). The differences in angles between the subunits accumulate as they grow to form a protofilament yielding either a structure which is compatible with microtubules or a curved protofilament which misfits into a microtubule architecture, thus creating the states of assembly and disassembly that occur simultaneously



in a mode of dynamic equilibrium. This angle between the tubulin subunits could be biased by introducing different types of small molecules which results either in the disruption of the longitudinal interactions or in the lateral interactions of the tubulin polymers or in some cases both; ultimately disturbing the overall kinetics of the cell cycle.

Figure 1: (a) A schematic representation of the structure of microtubules (MT). The interactions between two subunits are distinguished as ‘longitudinal’ (along the direction of growth of MT) and ‘lateral’ (along the plane perpendicular to the direction of growth of MT). (b) The change in orientations between α and β, is measured as θ. (c) The θ of each dimer can cumulatively determine the geometry of the polymers, i.e. the protofilaments to grow as ‘straight’ (solid lines) or ‘curved’ (dashed lines).

Several such small molecules have been reported so far in literatures which are capable of establishing a therapeutic control over the cell divisions.7-9 These agents are broadly classified into two sub-groups; (i) microtubule destabilizing agents , e.g. Colchicine, TN16, Vinblastine, E7010 etc. and (ii) the microtubule stabilizing agents, e.g. taxol, epothilone etc. The destabilizing agents are known to bias the population distribution of the microtubules towards the depolymerized state of the microtubule structure thus decreasing the formation of


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long spindle fibres, whereas the stabilizing agents shift the population of microtubules towards a hyper-stabilized state in which case the spindle fibre is not able to depolymerize and separate the sister chromatids. In both cases, the cell division process gets halted. The ligand binding sites are predominantly present on the β-subunit of the heterodimer and each site or pocket is identified by the name of one of their binding ligands, e.g.vinca binding pocket10, taxol binding pocket11 (iii) Colchicine binding pocket

12

(iv) laulimalide/peloruside

binding pocket13 and (v) maytansine binding pocket14 ; the locations of these pockets have been shown in Figure 2.

Figure 2: The surface representation of the structure of α,β-tubulin. Different ligands, Colchicine, Taxol, Vinca, Peloruside, Maytansine have been shown at their corresponding binding site; the figure has been prepared by superimposing the structures of the complex of each ligand with tubulin (PDB IDs: 4O2B, 1JFF, 1Z2B, 4O4J, 4TV8). The buried ligands have been made visible by making the protein surface partially transparent. The Colchicine binding site is highlighted with dashed outline which is relevant for the present report. The structure of E7010 (magenta stick) has been shown separately that binds to the Colchicine binding site.



Among all the tubulin binding agents, some of the compounds are still under clinical trials; e.g. Patupilone, Vinflunine, Tasidotin etc.15-17 In recent times, some noticeable progress against many tumor cell lines has been made by the sulfonamide class of tubulin binding agents18-22 which are highly potent to disrupt the structure of the microtubules; amongst which

the

E7010

(N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-

methoxybenzenesulfonamide) is considered as one of the most potent inhibitory agents. The E7010, which occupies the Colchicine binding pocket of the tubulin protein (Figure 2), arrests the cell cycle at the G2/M phase.23,

24

It is an orally active compound which has

reached the phase II clinical trials and has been found to be effective against many of the multi-drug resistant cancer cell lines.23, 25, 26 For such a promising and successful inhibitor, it is very important to understand the mechanism of inhibition so that the ligand could be explored further for any refinement and to discover methods to overcome the limitations with which its usage is confronted. Dorléans et al. who resolved the structure of the E7010 complexed with tubulin (PDB ID: 3HKC) using X-ray crystallography, revealed that E7010 occupies the Colchicine binding site. A mentionable specific interaction that specifies the orientation of the ligand in the binding pocket is extracted from the crystal structure that shows the hydroxyaminophenyl group of E7010 (Figure 2) forming a polar interaction with the side-chain of Y β202 of tubulin protein.27 However, the same binding pocket is also found to be capable of accommodating several other ligands having diverse structures and topologies, e.g. Colchicine, TN16, T138067 etc.27, 28

Binding of TN16 and Colchicine inside the tubulin protein is well studied along with their

thermodynamic properties. It was already reported from some preliminary investigations that the ligands in the Colchicine binding site show flexible interactions.29 Detailed investigations on the multiple possible binding modes were reported earlier on Colchicine and TN16.30, 31


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But for E7010 detailed characterization of its conformational states are yet to be reported. This might be important for designing newer derivatives of E7010 in future as the E7010resistance (e.g. P388/4.0r-M clone) has developed a serious concern in recent times.29 Using Molecular Dynamics (MD) simulation, hereby an attempt has been made to extract the atomistic level details of the dynamics of E7010 binding which is expected to provide novel paradigms for better understanding of the mechanism of microtubule inhibition and might help to design better derivatives of the ligand.

Methods: Modelling and simulations: Initially the structural coordinates of the tubulin-E7010 complex was obtained from Protein Data Bank (PDB), using the code 3HKC27, resolved at 3.80 Å resolution. From the dimer of dimer (α,β) structure (3HKC) of tubulin protein complex; one dimeric unit of α and β (chain A had residues 2 to437 and chain B had residues 2-438 in PDB) along with the E7010, GTP, GDP and two Mg2+ ions were extracted. The apo structure of the tubulin dimer was also obtained from the PDB, using the accession code 3HKB.27 Modeller 9.1032 was used to model the regions whose coordinates were missing in the PDB. Then the structure of the entire complex was processed in the CHARMM-GUI web server33; and the CHARMM36 parameter set to represent the system.34-36 The E7010 ligand was parameterized using CHARMM General Force Field (CGenFF).37 Each system was solvated into a cubic water box where the TIP3P water model38 was used, ensuring minimum ~8.5 Å thickness of water layer everywhere. Mg2+ and Cl- ions were added into the water box to neutralize the charges as well as to maintain 0.15 M ionic concentration. Each system, apo and complex, contained ~175000 atoms. Three independent set of simulations (starting with three different seed numbers) for the complex and one set of simulation for the apo were performed using NAMD



2.9

39

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package as a simulation engine. Systems were initially minimized using the adopted

basis Newton-Raphson method (ABNR) and then heated to 300 K followed by equilibration. Each of the independent trajectories of the complex, as listed in Table 1, was simulated for a total time of 120 ns with an integration time step of 1fs at 300K under NPT conditions. The apo system was simulated for 100 ns. Short ranged non-bonded interactions were truncated at 12 Å and long ranged electrostatic interactions were computed using particle mesh Ewald (PME) technique.40 The pressure and temperature were controlled using Nosé–Hoover thermostat/barostat41,

42

algorithm. All the figures were prepared using either VMD43 or

PyMOL44.

Table 1. List of MD simulation trajectories

No.

Trajectory Names

Description of the systems

Simulation length (ns)

1

TE1

E7010 complexed with α,β tubulin heterodimer

120

2

TE2


120

3

TE3


120

4

Apo

α,β tubulin heterodimer

100

Total

460

Binding Energy Calculation: The binding energy (ΔEbinding) was calculated using the MM-GBSA protocol45 adapting the GBSW method for computing the

implicit solvation energy.46,

47

Two separate set of

calculations for ΔEbinding were performed, (i) IMPSOL: deleting all the water molecules from the trajectories and then splitting the trajectories into receptor and ligand to calculate the


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binding energy using equation 1, and (ii) MXDSOL: considering explicit water molecules selected at each time frame, within a cut off of 7 Å from the ligand using the equation 2. The water molecules became a part of the receptor protein when the complex trajectory was split into an uncomplexed unit thus ensuring that the same number of water molecules was selected in the complexed and uncomplexed states. ∆𝐸𝑏𝑖𝑛𝑑𝑖𝑛𝑔 = 𝐸𝑐𝑜𝑚 ―(𝐸𝑟𝑒𝑐 + 𝐸𝑙𝑖𝑔) ∆𝐸𝑏𝑖𝑛𝑑𝑖𝑛𝑔 = 𝐸𝑐𝑜𝑚 + 𝑤𝑎𝑡𝑒𝑟 ―(𝐸𝑟𝑒𝑐 + 𝑤𝑎𝑡𝑒𝑟 + 𝐸𝑙𝑖𝑔)

(1) (2)

Results: Dynamics of E7010: Trajectory 1 (TE1): The stability of the complex over the trajectory was assessed by computing the root mean square deviation (RMSD) of the Cα atoms. The system was overall stable as there were no major conformational changes; the Cα RMSD of the protein was within the range of 3 to 4.5 Å (Supplementary Informations (SI) Figure S1a). The RMSD of the E7010 inside the binding pocket was mostly between 6-7 Å which was computed after aligning the protein backbone and the trajectory average RMSD was ~ 5.3 Å, which indicated a substantial change in the binding orientation of the ligand. The Residue averaged root mean square fluctuations (RMSF) of each residue was found within the reasonable range, i.e. it did not indicate any appreciable structural fluctuations (SI Figure S2). The crystal structure of tubulin-E7010 complex (PDB ID: 3HKC), did not resolve any structural water molecules at a resolution of 3.8 Å; hence in the starting model no structural water molecule was present inside the cavities of the tubulin protein. But interestingly during the dynamics some water molecules soaked into the intramolecular spaces particularly around the E7010 inside the Colchicine binding cavity. The soaking of water molecules into the core of the tubulin dimer,



particularly into the Colchicine binding site, was reported earlier by Majumdar et al., for tubulin-TN16 complex and the present observations are in agreement with it.31

Figure 3: A 2D representation of the interactions between E7010 and the tubulin β-subunit observed from the structure available in PDB (3HKC), obtained from LIGPLOT48. The dashed lines indicate the interactions.

Ligplot analysis has revealed mainly the nonpolar interactions (e.g. V315, L255, I378, V238, L242, A316, C241, A250 and L248 of β-subunit of the tubulin protein) with the ligand and some polar interactions of K352, N258 and K254 as showed in the Figure 3. In the crystal structure, the –OH of E7010 had HBond with side chain -OH of the Y202 of β-subunit (shown in Figure 4a). [The ligand binds inside the β-subunit of the tubulin protein, eventually all residues and their indices of the protein as described in rest of this article correspond to the β-subunit of the tubulin].


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Figure 4: (a) Polar interactions between E7010 and the binding pocket residues as observed in X-ray crystal structure (PDBID: 3HKC). Three representative snapshots taken from three independent trajectories: (b) TE1 (c) TE2 and (d) TE3; showing the water mediated polar interactions between the protein and the ligand. The E7010 has been shown in magenta and the tubulin has been shown in green cartoon except the T7-loop shown in orange. The interacting sidechain atoms of tubulin, some atoms of E7010 and water atoms are shown in standard colouring scheme: N (blue), O (red), S (yellow), H (white). The Hbonds are indicated by dashed line where the numbers indicate their length in angstrom (Å).



Figure 5: Conformational rearrangement of the E7010 during the course of MD simulation. The –OH group of the E7010 (PDB conformation shown in mageenta stick) switched towards the sight where it can easily make Hbonds directly or through water with the backbone of V238; changed conformation of E7010 is shown in yellow stick.

In the starting structure, the interaction was direct, between the Y202 and the ligand, although the interacting residues of the tubulin altered and the interactions gradually optimized as the simulation progressed (Figure 5). For example, interaction with hydrophobic side chain of V238 was replaced by the polar side chain of Y202 (Figure 5) to form H-Bond and that continued to remain stable until the water molecules soaked into the cavity to solvate these interactions and occupied the space between them. Once the water molecules entered into the binding pocket, the options for polar interactions to get stabilized increased. This possibility redistributed the interactions between the ligand and the receptor. For example, one water molecule took the position between Y202 and –OH of E7010 and thus bridged them through Hbonds at both sides of the water, as shown in Figure 4b. This allowed a larger separation between the E7010 and Y202 retaining the stability of the complex which allowed Y202 to form more interactions elsewhere. It was observed that the largest possible separation between Y202 and –OH of E7010 was ~7Å and the empty space it created was filled up by few water molecules. These water molecules sandwiched between the E7010 and several


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polar residues N167, E200 and Y202, as shown in Figure 6, form an assembly of water molecules, which were effectively disconnected from the bulk as they are trapped. As shown in Figure 6, they form an Hbond network among themselves which appears as a group or cluster of water molecules. Since the number of water forming such a cluster is few compared to the bulk water, these clusters are referred as micro-clusters henceforth. Analogous to a single water molecule that sometime bridge the interactions between a protein and a ligand, this micro cluster serves the same except the fact that the singe water is now replaced by a group of water. As shown in Figure 6 in this system the micro water cluster to bridge the interactions of E7010 with a group of residues include, N167, E200, Y202 and V238.

Figure 6: (a-c) The water molecules confined between the protein (green) and the E7010 (magenta) which are forming a microcluster which is effectively disconnected from the bulk water. These water clusters bridging the interactions between the ligand and the protein. The blue dashed lines are shown to measure the distance between the –OH of E7010 and the –OH of Y202, which has been referred in the text for discussion. The representative snapshots shown in a,b and c are taken from TE1, TE2 and TE3 trajectories respectively.

The other Hbond bridging the E7010-protein interactions was between the N1 of E7010 (Figure 2) and the D251 through water molecule (Figure 4b) and this was possible because the larger separation between Y202 and E7010 (as mentioned above) allowed proper



positioning of the N1 near D251. Since the E7010 switched its orientations often, the hydrophobic interactions inside the binding pocket adjusted accordingly over the trajectory, which involved the residues especially L248, V238, L255, A250 and M259 (Figure 7).29

Figure 7: The hydrophobic packing around the E7010 (magenta stick) inside the αβ-tubulin (green cartoon) cavity. The hydrophobic side chains of relevant amino acids are shown in spheres.

Trajectory 2 (TE2): The average Cα RMSD of the system was 2.5 Å (SI Figure S1a). The RMSD of the ligand within the binding pocket was mostly between 3-4 Å and the trajectory averaged RMSD was ~ 3.5 Å (SI Figure S1b), which indicated a little to moderate change in the binding orientation of the ligand. The RMSF of the residues were observed within the range of 0.5 to 6 Å, i.e. it did not indicate any appreciable structural fluctuations (SI Figure S2). During the course of MD, water molecules soaked into the macromolecule and some of them also entered into the ligand binding site, similar to the events witnessed in TE1. In the crystal structure, i.e. at the starting conformation, the –OH group of E7010 had polar interactions with the Y202. But after first couple of nanoseconds, the conformation of E7010 started to alter and the –OH group of the ligand (i.e. O3-H1 in Figure 2) was then directly involved in forming the H-bond with the backbone O of V238 as shown in Figure 4c. This


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interaction, which was already found to be very stable in the TE1 trajectory, remained almost preserved in TE2 too, throughout the last 115 ns. A water mediated bridging interaction was formed between the –NH group (H14/N1) of E7010 (as in Figure 2) and the backbone O of A317 which was stable for last 45ns of the dynamics. A representative snapshot of the trajectory has been shown in Figure 4c. The motion of the T7 loop is comparatively less as observed in TE1, which has been reflected in the RMSF plot extracted from each of the trajectories (Figure S3). Due to the deviation of the ligand from the Y202 site towards the V238 of the H7 helix, no significant changes of the binding pocket residues (which mostly involved the residues like L242, M259, V238, L251, L255, I378 etc.) was observed. Trajectory 3 (TE3): The average Cα RMSD of the system was 3 Å (Figure S1a). The RMSD of the ligand within the binding pocket was mostly between 1.5-2.5 Å (Figure S1b) and the trajectory averaged RMSD was ~ 2 Å, which indicated a little adjustment of the orientation of the ligand, keeping the crystallographically detected orientation mostly preserved, which fact would be evident from the Figure 4a and 4d. Further, the PDB structure was resolved at 3.8Å which indicates a small but appreciable uncertainty of the atomic coordinates in the starting structure. Therefore during the simulation the positions of the atoms are expected to adjust little bit and given this, the trajectory average RMSD of 2 Å could be accepted as a good agreement with the PDB structures. The RMSF of all the residues were within the range of 0.5 to 6 Å, i.e. within the reasonable range of structural fluctuations (SI Figure S2). The events of soaking of water molecules into the tubulin core were also found to be consistent in TE3. In this trajectory it was observed that the T7-loop moved significantly (Figure 4) which started around 60 ns and was correlated with the reorientation of the ligand inside the pocket which required more space inside the pocket to change orientations. The –OH group of the E7010 shifted from Y202 site towards the H8 helix (Figure S4a). As a consequence, the hydrophobic packing of the binding pocket is changed which involved mostly the residues



along the H8 helix, T7 loop and the S8-9 sheets e.g. V238, L248, A250, L252, K254, L255, N258, N372, T314, A316, I370 etc. (Figure S4b). Initially, few of the water mediated Hbonds are formed between the –OH group (O3-H1) of the E7010 and the -OH group of the Y202 similar to the interactions observed in TE1 trajectory, although the orientation of the ligand was significantly different compared to the E7010 in TE1 trajectory (Figure 4d). This interaction was stable until ~65 ns, after which the ligand changed its orientation and the same O3H1 of the E7010 now directly formed H-bond with the backbone O atom of the D251 (a representative snapshot was shown in Figure 8a). After initial 25 ns of the dynamics, a second water molecule started to form another bridging interaction with the H14 of E7010 and the backbone O of V238, as witnessed in TE2. This interaction was stable for the next 35 ns (Figure 8b). The orientation of the pyridine ring of the E7010, which contains a nitrogen group (N1 in Figure 8b) remained just beside the H14 when integrated into the binding pocket. Both the H14 and N1 were prone to make H-bonds and therefore this interacting hotspot constantly got involved with 2-3 water molecules forming H-bond bridges with the protein surface (representative snapshots are shown in Figure 8). This water mediated interactions between the H14/N1 of E7010 and the V238 remained almost preserved entirely throughout 120 ns by replacing ~20-25 water molecules. In the following section the hydration property of the binding pocket of the apo tubulin dimer is analysed and the protein ligand contact formation over the time is discussed in details considering all the three trajectories.


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Figure 8: Representative snapshot taken from TE3 showing the water bridging the interactions between the E7010 (magenta) and the binding pocket of the tubulin (T7 loop in orange, the rest in green). Dashed lines represent the H-bonds (red) and the corressponding bond lengths are in Å unit. (a) Water bridge between N1 of E7010 and O of the V238. (b) Water bridge between H14 of E7010 and O of the V238. Hydration property of the Apo Tubulin protein: To investigate the hydration property of the Colchicine binding pocket of the tubulin protein in absence of any ligand, a separate simulation of the apo-tubulin dimer was performed. The water molecules within the 3.5 Å of the residues which encompass the Colchicine binding pocket were selected as the water within the binding pocket. During the simulations the total number of water inside the pocket varied from ~8 to 22 in different snapshots and the average count was ~14 (SI Figure S5). Any instantaneous count of water molecules inside the pocket were dependent on the conformation of the binding pocket residues at that particular snapshot, which substantially varied from time to time.



Figure 9: The three routes of water entry and exchange inside the β-tubulin core (Colchicine binding domain) in presence of the E7010 are marked as Route 1, 2 and 3 respectively. The percentage of water molecules reaching the space between ligand and receptor through the different routes were 59%, 8% and 33% for Route 1, 2 and 3 respectively. The colouring scheme is same as Figure 8.

Water exchange routes: As it has already been stated, in the starting structures (3HKC) there was no water inside the E7010 binding pocket of tubulin. Later the water molecules got soaked into the protein, reached the binding pocket and filled up the space between the ligand and receptor. The water molecules followed three different routes to enter, get exchanged or replaced by another water molecule, which have been shown in Figure 9. The paths of the entry of these water molecules were initially tracked by visual inspection, by running the trajectory backward in VMD43 and that was repeated by choosing the various different time points in the trajectory. It was observed that the scope for water molecule to find a path from the surface to the core was very limited and only three such paths could be identified which are distinctly different from each other and which transport significant number of water molecules. The significance


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of these paths were confirmed by computing the passage of water molecules through these routes and that was counted by setting a contact criteria of 7Å between the water and a few residues existing on the path (as shown in Figure 9). Thus a statistics of the water passage through this path was obtained. Route-1 was identified as the space between the A256-V257 and the E200-Y201 amino acid residues (as shown in the Figure 9), the Route-2 has been marked just beside the K352 of S9 β-sheet and the Route-3 was in between the D357 and the P245 of the β-tubulin protein. The Route-1 was found to be the busiest, as the water molecules reaching the space between ligand and receptor took this route for 59% of the cases of such water transport, whereas they availed route 2 and route 3 for 8% and 33% of time respectively. Since the total number of such events are generally few, e.g. approximately 60 in every 100ns, the exact statistics of the routes may little bit vary in much longer simulations. Contact points formed between the E7010 and the protein with time: The interaction between protein and ligand significantly deviated from those seen in the crystal structure; and was also found to be different in different trajectories. So the relative significance of the interactions needs to be investigated. Hence the contact point analysis is performed between the ligand and the receptor with respect to time. In this case only the βsubunit of the tubulin heterodimer is considered as a receptor since the ligand binds inside the cavity of the β-subunit and did not have any direct interactions with the α-subunit. A contact is considered to exist if an atom of the ligand is within 6 Å of another atom of the protein. The lifetime of any contact between the E7010 and the protein was calculated as a fraction of the simulated time throughout which the contact persists. When the lifetime of these contact/interaction points were plotted considering all the three trajectories, most of the interactions were seen to be preserved. In TE1 it was observed that the B-ring of E7010 (Figure 10a) did not form any significant contact with the S9 β-sheet of the protein, while



most of the time it remains in contact with the T7-loop and H7-H8 helices, thus showing different behaviour with the TE2 and the TE3 trajectories (Figure 10b and 10c). The contact points which were formed between the A-B-ring of the E7010 ligand (as shown in Figure 10) and the side chains of the protein were found to be same in all the three trajectories, showing that the points of contact between the ligand and the protein in all the trajectories were similar, differing only in their corresponding life time. It is notable from the plot that when the E7010 is interacting with the S9 β-sheet (TE2 and TE3 of Figure 10b and 10c), the probability of any significant interactions existing between the ligand and the zone of the T7loop, H7 and H8 helices is significantly low (longitudinally marked dotted areas of TE1 as represented in Figure 10a).


Page 20 of 37

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Figure 10: (a-c) A contact map is generated between the ligand and the β-tubulin from the ensemble of conformations of TE1, TE2 and TE3 separately; for each trajectory 20000 snapshots were used. The red to blue colour scale is used to indicate ‘no’ interaction to maximum numbers of interactions. The vertical axis shows the atom indices of the E7010; the atoms of the aromatic rings of the E7010 are written as A, B and C. The horizontal axis shows the protein residue index, only those stretches relevant for the protein-ligand contact formation. The differences in the contact points are outlined by dashed (black) lines. (d-f) For each of the trajectories TE1-3, a representative conformation has been shown. The protein and the ligand are shown in green cartoon and cyan stick respectively. Important secondary structures of the protein are marked as; H7, H8, S8, S9, S10 and also mentioned on the top of the map.



Page 22 of 37

Dynamics of water molecules inside the E7010 binding pocket: The speciality of this particular protein-ligand complex system is that they formed microclusters of water during ligand binding, whose characteristics and importance are significantly different from the cases where only one water molecule is sufficient to bridge the interaction between ligand and receptor. Generally the structural water molecules are isolated from the direct contact of the bulk water molecules and thus their properties largely differ from the bulk water. But in this case structural water molecules are found to form micro clusters of water, i.e. each molecule is solvated by a few more water molecules in the cavity and thus can also show a partial fluidity within the limited movements. In order to analyse the dynamics of these water molecules around the E7010 binding site, water molecules which satisfied the distance cutoff criteria of 8 Å from both the ligand and the tubulin, were selected. First 5 ns of the production dynamics was eliminated from the quantitative analysis to allow the water molecules to settle down inside the binding pocket. The average lifetime of the H-bond bridges were calculated for each trajectory separately and are listed in Table 2. Although the average lifetime of the Protein-Water-Ligand Bridges were listed in picosecond order but they lasted in the order of nanosecond (shown in 4th column of the SI Table S1) since those polar interactions were continuously broken and reformed with time. Table 2: Average Lifetimes of the Protein-Water-Ligand Bridge: Around the same functional groups of the ligand and receptor both, multiple options of donor-acceptor atoms (separated by slash) are available, that results the possibility of the Hbond formation in multiple ways and yet at the same site of interactions. The lifetime of all types of Hbonds are separated by slash “/”. System

Receptor

Ligand (Residue-


Protein-Water-Ligandbridging Hbond lifetime (ps)

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(Residue –atom1/atom2) TE1

TE2

TE3

atom1/atom2)

(Hbond1/Hbond2/Hbond3 etc)

Y202-OH/HH

E70-O3/H1

6.4/21.0/5.0/11

V238-O

E70-O3/H1

14/8

T240-HN/OG1

E70-O3/H1

5/5/16.4/6

C241-HN/HG1

E70-O3/H15

14/6/7/5.5

C241-O

E70-N1

16

A250-HN/O

E70-O2/N1/H14

5/14/9.3

D251-O

E70-N1/H14

27/30.2

K254-HZ1/HZ2/HZ3

E70-O1/O2

16/8/10

L255-HN

E70-N1/H14

6.5/6.2

V355-HN/O

E70-O1/H15

9/5.5/6/9

V238-O

E70-H1

62

N249-HN/OD1/O

E70-O1

5/5/13

K254-HZ1/HZ2/HZ3

E70-O1

6/8/7

A317-O/HN

E70-N1/H14

10/36.5/6.0/6.2

K352-O

E70-N1/H14

5/5

T353-HN/O

E70-N1

5/7

Q133-OE1

E70-O3/H1

15/31.4

N167-OD1/HD21

E70-O3/H1

5/14.2/5/5

E200-OE1/OE2

E70-O3/H1

5.4/21.2/7/22.5

Y202-OH/HH

E70-O3/H1

8.3/11.6/6/10.2

V238-O

E70-N1/H14

10/10

C241-HN/HG1

E70-N1/H14

6.2/6/8.5/6

A250-O

E70-O3/H1

18/28

D251-HN

E70-O1/O2/O3

12/8/7

L252-HN

E70-O1/O2/O3

7.5/11/10.3

K254-HZ1/HZ2/HZ3

E70-H15/O1/H14

7.5/8.4/6

L255-HN

E70-O1/O2/O3

6/7/7



Page 24 of 37

Example of some of the residues which are significantly involved in water mediated H-bond bridges are D251, C241, V355, V238, A317 Q133, Y202, V238 etc. (shown in Table 2 and Figure S6). Standard lifetime of H-bond in bulk water is ~5 ps,31 calculated using TIP3P water model under similar simulation conditions. In TE1 system, two of the most important and stable H-bond bridges are the Y202-E7010 (~21ps) and D251-E7010 (~30ps) respectively (Table 2 and Figure 4a). In TE2 a noticeable bridging interaction is found in between V238 and E7010 whose average lifespan was ~62 ps (Table 2 and Figure 4c). This polar interaction was also found in the TE1 trajectory with an average lifetime of ~14 ps of life time average (Table 2). Another important interaction that was observed in TE2 was with A317 and E7010 with a lifetime of ~36 ps (Table 2, Figure 4c). In TE3, most of the above mentioned bridging interactions are repeated except few like Q133-E7010 and E200-E7010, with a bridging H-bond lifetime of ~31 ps and ~22 ps respectively (Table 2). In the following section the rotational relaxation time of these important bridging water molecules are analysed. Rotational Dynamics of water molecules: The rotational relaxation time of water molecules 49-51 were investigated using the following autocorrelation function Γ2: 𝛤2(𝑡) = < 𝑃2(µ(0) ∙ µ(𝑡)) >

(1)

Here µ(t) is the unit vector along the water dipole at time t and the angular brackets indicate the time average. P2 is the second-order Legendre polynomial: 𝑃2(𝑥) = (3𝑥2 ―1)/2

(2)

The rotational correlation of water molecules were calculated separately considering two different layers of water molecules which were defined as (i) bridging and (ii) interfacial.


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‘Bridging’ water means those water molecules which were located in the interactive zone between the ligand and the protein (Figure 4). The water molecules under the 6 Å cutoff distance from the ligand were followed by this category. Water molecules which occupied the space between the α and β-subunits and satisfied the 7 Å cutoff distance from both the subunits except the ‘bridging’ water, were selected under this category of the ‘interfacial water molecules’ (SI Figure S7). Figure 11, illustrates that the rotational correlation functions of the ‘interfacial’ water molecules fall rapidly with time compared to the ‘bridging’ water molecules. From a review of literature the differences between the biophysical properties of the bulk water and the first hydration layer water is already seen to be well established.52, 53 In this investigation the decay rate of the ‘bridging’ and the ‘interfacial’ water is found to be 1.0 to 0.6 and 1.0 to 0.5 in 100 ps respectively, which is significantly slower than the ‘bulk’ and ‘first hydration layer’ water molecules (Figure 11). The slowest rate of rotational decay of ‘bridging’ water molecules suggests that these water molecules are most stable in these specific locations between the protein surface and the ligand molecule. In the following section the effect of these water molecules on overall protein-ligand stability in terms of energy are analysed.

Figure 11: The decay of the rotational correlation function of water molecules in the bridging and interfacial layers around the tubulin heterodimer and E7010.



Page 26 of 37

Binding Energy of Tubulin-E7010 complex: The binding energy of E7010 ligand was calculated using the MMGBSA protocol.54 To compute the solvation free energy, the GBMV

46, 47, 55

module of CHARMM is used. As

mentioned in the methods section, the binding energy was calculated in two different ways, (i) computing the solvation exclusively using implicit model, i.e. the standard MMGBSA method, referred as IMPSOL henceforth (ii) considering the water molecules (which remain within the 7 Å of the E7010) present at the protein-ligand interface explicitly and computing the rest of the solvation implicitly, protocol of which is referred to as MXDSOL in the following text. The ensemble averaged binding energy of E7010 and its energy components are separately shown in Table 3. Table 3: Averaged Binding Energy Components (in kilocalories/mole) in Different Trajectories applying the standard Implicit Model (GBMV) of Water Solvation only. Standard deviations are indicated in parentheses. Energy components

TE1

TE2

TE3

∆Eelec

-15.5 (4.4)

-10.0 (4.6)

-3.1 (4.3)

∆Evdw

-37.6 (3.7)

-47.8 (4.8)

-43.4 (2.4)

∆Egb(polar)

40.6 (6.9)

30.1 (5.6)

29.0 (7.0)

∆Egb(nonpolar)

-4.3 (0.2)

-4.2 (0.2)

-4.3 (0.2)

∆Etot

-16.9 (7.2)

-31.8 (9.5)

-21.9 (7.4)

Using the IMPSOL strategy the binding energies were found to be ~ -17, -32 and -22 kcal/mol for TE1, TE2 and TE3 trajectories respectively (Table 3). As it was described in the previous sections, the conformations of the ligand was different in three different trajectories and therefore apparently the difference in the binding energies were acceptable, which


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indicated different stabilities for different binding topology of the ligand. Thus it indicated that the binding orientation of E7010 in TE2 (Figure 4c) was the most stable compared to the others. Using the MXDSOL strategy, the binding energies were found to be -41, -40 and -44 kcal/mol for TE1, TE2 and TE3 respectively (Table 4). The cumulative average of the binding energies, with the progress of time, obtained using IMPSOL, was found to be divergent whereas, the same for MXDSOL was found to be nearly converging (Figure 12). Since the plots represent a cumulative average of the binding energy, as the simulation progresses and increase the size of conformation ensemble, the average binding energy is likely to become more and more stabilizing, as the overall system was stable. But for IMPSOL method, the TE2 and TE3 gradually became lesser stable with time, which does not reflect the structurally observed fact. Whereas for MXDSOL method, except the slight rise for TE1 beyond 95ns, all the systems has gradually attained more and more stabilization. Therefore the results of the MXDSOL could be accepted as a better reflection of the binding energy over the IMPSOL method. As shown in Figure 12 they came within an energy width of ~ 4 Kcal/mol (Table 4). As it has been stated already, the conformations in TE3 closely resembled the ligand-binding conformations reported in PDB structure, the binding energy obtained from this trajectory, which has been the lowest among the three trajectories, could be considered as the reflection of the native state; therefore the binding energy results from MXDSOL are in agreement with the PDB. Table 4: Averaged Binding Energy Components (in kilocalories/mole) after considering the structural water molecules explicitly around 7 Å of the E7010. Standard deviations are indicated in parentheses. Energy components

TE1

TE2

TE3

∆Eelec

-37.0 (7.5)

-14.5 (6.1)

-30.4 (8.2)

∆Evdw

-43.5 (3.8)

-49.9 (2.9)

-46.2 (3.1)

∆Egb(polar)

43.6 (12.0)

28.2 (8.7)

36.4 (10.8)

∆Egb(nonpolar)

-4.1 (0.2)

-4.1 (0.2)

-4.0 (0.2)



Page 28 of 37

∆Etot

-40.9 (12.1)

-40.3 (9.9)

-44.2 (11.5)

Water(7Å)

~43

~28

~35

Figure 12: Cumulatively averaged E7010-tubulin binding energy (∆E) plotted as the function of simulation time for TE1-3, computed using MMGBSA protocol. Three solid lines in blue, red and yellow are representing the calculations using implicit solvation model only (IMPSOL). The dashed lines of same colour scheme are the results of the same calculation of (∆E) but after explicitly including the water molecules present within 7 Å around the ligand, in addition to the use of implicit model of solvation energy calculation (MXDSOL).

Discussion: The trajectory averaged RMSD of the ligands have revealed that the binding conformation of the ligand in the ensemble of structures of TE1 and TE2 are substantially different from the conformation that was observed in PDB, whereas the same in TE3 was within the close proximity of the PDB data, considering the 3.8Å resolution of the PDB data and the acceptable range of positional fluctuations in MD simulation. Therefore the results of the TE3 could be accepted to represent the conformational state that was reported in PDB and TE1 and TE3 represent two other possible ligand binding conformations which are energetically close and yet slightly lesser stable than TE3. It is known that the Colchicine binding pocket of the tubulin can accommodate ligands of distinctly different shapes, sizes and topologies


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which is evident from the reported structures of tubulin with various ligands.12, 27, 56, 57 Within the same pocket, different ligands can occupy a slightly and yet significantly different positions and orientations. Further, it is also understood that the T7-loop (Figure 4a) can occupy this binding pocket in absence of a ligand and takes part in controlling the dynamics of this pocket.[22] In a previous investigation the binding dynamics of the TN16 (an analogous to E7010) inside this Colchicine binding domain was observed, where some of the dynamic and the static water molecules were found to play a critical role in the binding.31 The binding mode of the E7010 largely varies from that of TN16 (see SI Figure S8). TN16 penetrates deeply into the pocket and occupy a shallow channel-like space which is in contrast to the E7010 binding and the Colchicines binding (SI Figure S8).27 As stated above, within this same binding pocket, the exact site of ligand binding can differ, the region where the Colchicine or E7010 bind, (Colchicine binding pocket) is predominantly comprised of amino acids with hydrophobic side chains (Figure 3). In fact E7010 explores a much wider region of the binding pocket than other ligands as it switches its conformations; this has been witnessed from three trajectories (Figure 4b-d). As it was observed that the conformations of the ligand is always complemented by a cluster or a group of water molecules which complemented the binding by ensuring the packing by filling up the empty space (Figure 6). These water molecules not only ensured the packing but some of them also bridged the interactions between the ligand and the receptor (Table 2, SI Figure S9, S10). Some of these bridging occurred through multiple water molecules, as shown in Figure 6. Therefore these water molecules are not solely responsible for “bridging” as the bridging water generally remains isolated from other water molecules and bridge the interactions between solutes. On the other hand these water molecules do not even have the characteristics of bulk water, as they are trapped inside the binding pocket. These water molecules form a micro cluster and provide solvation-stabilization to each other and also bridge the interactions between the



solutes. They get exchanged with the bulk water molecules and the three distinct routes of water exchange have been identified (Figure 9). The average lifetime of these ‘bridging water’ was investigated separately and found to be in the range of ~5-62 ps (Table 2), which are significantly higher than the average Hbond lifetime of bulk water31. However they did not show lifetime of ~250 ps or more which was evident from the bridging water of TN16.31 Interestingly under special consideration, these bridging water molecules were found to possess slowest rate in rotational dynamics (Figure 11). This implies the importance and special role of those water molecules in E7010 binding which has been clearly reflected in the binding energy calculation. The possibility of accommodating water inside the Colchicine binding pocket is also evidenced from several other crystal structures of tubulin protein, complexed with different types of ligands.58

56

A list of such PDB IDs of tubulin-ligand

complexes has been provided in SI Table S2. Considering these crystal structures, it is evident that the Colchicine binding pocket can accommodate ~3-8 water molecules in presence of different types of ligands. The variation in the number of structural water inside the Colchicine binding pocket depends upon the binding topologies of the ligands. The tubulin-E7010 complex (3HKC) structure resolved at 3.8 Å is considered to be very inferior to observe the presence of structural water molecules in the proteins’ cavity. For example in the crystal structure of tubulin-6NL complex (PDB ID: 5JVD), it was observed that the Colchicine binding pocket contains three structural water (SI Figure S11) which was resolved at 2.4 Å. In this tubulin-6NL complex (PDB ID: 5JVD) one water molecule was found in between the polar heterocyclic ring of the 6NL and the Y202β of the tubulin protein (SI Figure S11) and another water was located in between the V238β and the 6NL (SI Figure S11). These two water molecules were highly probable to bridge interactions between the ligand and the tubulin as the water molecules were situated within 5Å of 6NL and tubulin58 (shown in SI Figure S11).


Page 30 of 37

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The significance of these water molecules is best understood from the analysis of the binding energies of the ligands. As discussed earlier, the binding conformations of the ligands in TE1, TE2 and TE3 differed significantly (Table 3). Standard MMGBSA calculations (considering the solvation energy implicitly) have identified one of the binding mode as the best, which revealed that the TE2 was the most stable conformation of the E7010-tubulin complex in terms of energy compared to TE1 and TE3 (Table 3). Cumulative average of the binding energies plot showed that the binding energies of the TE2 and TE3 became gradually destabilizing. This disagrees with the observations that the ligand gradually optimized itself within the binding pocket and at least structurally adapted to a stable situation on these trajectories and there was no structural signature of the destabilization. It strongly suggested that some important component of the binding energy was missed out from the calculations which was necessary to be included which was most likely the special contributions of the packing water of the ligands or those micro clusters of the water (Figure 6) which required to be specially included in the calculations. So to test the importance of micro clusters of bridging water molecules in E7010 binding, the MMGBSA analysis was again carried out after explicitly including these water molecules in the binding energy calculations. Once this was done, immediately the cumulative average of the binding energies became stabilized with time (Figure 12) and also the three trajectories came to very close agreement, i.e. the binding energies ranged between -40 to -44 kcal/mole (Table 4). The cumulative average of binding energy plot signifies that the structural differences of the E7010 observed during the MD simulation, were compensated by the bridging water molecules, with the energy differences between the TE1, TE2 and TE3 getting reduced from ~10-13 Kcal/mol to ~1-4 Kcal/mol (Figure 12, Table 3-4). So it could be claimed that in dynamic situation E7010 follows the ‘quasi degenerate’ binding modes inside the tubulin protein with the help of the micro clusters of the dynamic water molecules. The number of water molecules can vary



from one trajectory to another when the selected ligand is around 7 Å i.e. ~28 to 43 (Table 4), although instead of the number the characteristics of the Colchicine domain binders are important,31 as these findings can be a step forward in future drug designing approach against the Colchicine binding pocket.59 Considering all the three independent trajectories (TE1, TE2, and TE3); the sites which were highly prone to formation of the water mediated interactions with the E7010 binding were reflected equally in all the three trajectories, e.g. the polar -OH of Y202 and the backbone polar groups of D251, V238, C241 etc. (Table 2, Figure 4). The lifetime of the H-bond bridges was observed to be significantly high around some of the amino acid residues, like ~62 ps with V238-E7010 (Table 3, in TE2), ~30 ps with D251E7010 (Table 3, in TE1) and many more presented in Table 2. Instead of static water molecules, the water inside the Colchicine binding cavity was mobile as they were found to be continuously exchanging their positions by making and breaking of the H-bonds. The lifetimes of these water molecules were quite significant as compared to the bulk water which was in the order of 5 ps only.31 So a single static structure (crystal structure) fell short to describe all these binding features of E7010. Figure 10 quantitatively explores two major differences in the pattern of the binding interactions of the E7010 with the β-tubulin subunit. Considering the total conformational space sampled by the ligand, the E7010 could either significantly form contact with the region of the T7 loop and the H7-H8 helices (Figure 10a,d) or could orient to form maximum interaction with the S9 sheet of the β-subunit (Figure 10b-c,e-f).

Conclusions: The E7010 is capable to access multiple orientations inside the binding pocket of α, β tubulin, a subset of which is in agreement with the conformation reported in PDB. Such feature is likely due to the binding modes of E7010 which are energetically similar; among them the


Page 32 of 37

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little bit more stable state is the conformation seen in PDB structure that may be termed as ‘native’ for reference. Fewer water molecules are found to be interestingly associated with the ligand binding pocket at the tubulin-E7010 interface, inside the ligand binding pocket. These water molecules forming a micro-cluster inside the protein’s cavity, although are sequestered from bulk water, they are found to escape through the different channels or pores to get exchanged with the bulk water in the nanosecond timescale. As the size of the Colchicine binding pocket is quite larger than most of the ligands reported so far, it is clear that these water micro-clusters favour the binding of the E7010 in terms of structural packing inside the binding pocket and aid the bridging interactions between the ligand and the binding pocket residues. The binding energy calculation reveals that the different binding modes of E7010 are energetically comparable through the thermodynamic contributions of the bridging water clusters, which revealed the thermodynamic significance of these water clusters. Interestingly, such features are elucidated only if these water molecules are explicitly considered in the MMGBSA calculations; the necessity of adopting such a protocol was understood by benchmarking the methods with and without explicit water clusters in MMGBSA and matching the trend of the stabilities along the trajectories. Since the different conformational states of the complex become energetically comparable, the chance of interconversion becomes quasi-allowed which most likely contributes to the entropic stabilization of the system. Thus, this investigation quantitatively reveals the vitality of the structural water molecules in E7010-tubulin binding whose idea is anticipated to play a significant role in future designing of tubulin inhibitors.

Supplementary Informations: The Supplementary Informations (SI) contain Table S1, S2 and Figure S1 to S11.



Acknowledgement: SGD acknowledges the funding support from SERB, DST, Government of India (sanction no. EMR/2016/000617). SM has been supported through CoE-DBT, Government of India and also by the funding from Bose Institute and DB receives funding support from DBT, Government of India.

References: 1. Jordan, M. A.; Wilson, L., Microtubules as a target for anticancer drugs. Nature reviews. Cancer 2004, 4, 253-265. 2. Stanton, R. A.; Gernert, K. M.; Nettles, J. H.; Aneja, R., Drugs that target dynamic microtubules: a new molecular perspective. Medicinal research reviews 2011, 31, 443-481. 3. Nogales, E.; Wolf, S. G.; Downing, K. H., Structure of the alpha beta tubulin dimer by electron crystallography. Nature 1998, 391, 199-203. 4. Krebs, A.; Goldie, K. N.; Hoenger, A., Structural rearrangements in tubulin following microtubule formation. EMBO reports 2005, 6, 227-232. 5. Brouhard, G. J.; Rice, L. M., The contribution of alphabeta-tubulin curvature to microtubule dynamics. The Journal of cell biology 2014, 207, 323-334. 6. Kirschner, M. W.; Williams, R. C.; Weingarten, M.; Gerhart, J. C., Microtubules from mammalian brain: some properties of their depolymerization products and a proposed mechanism of assembly and disassembly. Proceedings of the National Academy of Sciences of the United States of America 1974, 71, 1159-1163. 7. Gupta, K.; Panda, D., Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity. Biochemistry 2002, 41, 1302913038. 8. Dumontet, C.; Jordan, M. A., Microtubule-binding agents: a dynamic field of cancer therapeutics. Nature reviews. Drug discovery 2010, 9, 790-803. 9. Guha, S.; Bhattacharyya, B., The colchicine-tubulin interaction: A review. Curr Sci India 1997, 73, 351-358. 10. Gigant, B.; Wang, C.; Ravelli, R. B.; Roussi, F.; Steinmetz, M. O.; Curmi, P. A.; Sobel, A.; Knossow, M., Structural basis for the regulation of tubulin by vinblastine. Nature 2005, 435, 519-522. 11. Lowe, J.; Li, H.; Downing, K. H.; Nogales, E., Refined structure of alpha beta-tubulin at 3.5 A resolution. Journal of molecular biology 2001, 313, 1045-1057. 12. Ravelli, R. B.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.; Knossow, M., Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 2004, 428, 198-202. 13. Prota, A. E.; Bargsten, K.; Northcote, P. T.; Marsh, M.; Altmann, K. H.; Miller, J. H.; Diaz, J. F.; Steinmetz, M. O., Structural basis of microtubule stabilization by laulimalide and peloruside A. Angewandte Chemie 2014, 53, 1621-1625. 14. Prota, A. E.; Bargsten, K.; Diaz, J. F.; Marsh, M.; Cuevas, C.; Liniger, M.; Neuhaus, C.; Andreu, J. M.; Altmann, K. H.; Steinmetz, M. O., A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs. Proceedings of the National Academy of Sciences of the United States of America 2014, 111, 13817-13821. 15. Carlson, R. O., New tubulin targeting agents currently in clinical development. Expert opinion on investigational drugs 2008, 17, 707-722.


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