This section is adapted from the article to be submitted in the Journal of Chemical Information and Modeling. Nathalie Deschamps1, Carolina Dos Santos Passos2, Benjamin Grenier3, Pierre-Alain Carrupt1, Claudia Simões-Pires1 and Alessandra Nurisso1.
Affiliation
1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne Quai Ernest -Ansermet, 30, CH- 1211, Geneva 4, Switzerland.
2 Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, USA.
3 Centre Interdisciplinaire de Nanoscience de Marseille, Université de la Méditerranée, Faculté des Sciences de Luminy, Case913, 163 avenue de Luminy, 13288, Marseille, France.
Context
The HDAC6 protein structure used in the previous chapters was a homology model: the residues orientation may not be adequate. Also, docking calculations were performed using rigid receptors in an in vacuo environment. MDs simulations performed in this new chapter, using the recently released HDAC6’s crystallographic structure, would address some of these matters, providing flexibility and solvent-mediated effects that may be useful for fully understanding the selectivity profile of peculiar HDAC6-selective compounds lacking cap group according to the typical HDAC pharmacophoric scheme.
Contribution
Design of the study. Docking calculations and Molecular Dynamics simulations. Data analysis, article writing and proof-reading.
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Introduction
Human Histone Deacetylases (HDACs) are a family of enzymes known to deacetylate the lysine residues of histones involved in chromatin condensation 33, 34. Indeed, deacetylated histone lysines display a positive charge which leads histones to interact properly with the negatively charged DNA therefore compacting chromatin. Tighter DNA wrapping decreases accessibility for transcription factors, leading to gene transcriptional repression. The HDACs family has become, in the past decade, validated therapeutic targets because of the benefits their inhibition may provide in hypoacetylation conditions, typical of aging-related disorders and cancers 35, 36. This family of enzymes, composed of eighteen isoforms, is divided into four phylogenetic classes: class I (HDAC 1-3, 8), class IIa (4, 5, 7 and 9), class IIb (6 and 10), class III also called sirtuins, and class IV counting solely HDAC11. Among these enzymes, only sirtuins are NAD+-dependent proteins, whereas the remaining classes exhibit zinc-dependency 35, 37. HDACs display several tissue and cellular locations; in particular, HDAC6 is unique both by its domains organization and by its exclusively cytosolic localization, having non-histone substrates and controlling cell mobility and aggresome formation 33, 38, 39. Indeed, the interest on this isoform has recently been freshened up through its targeting combined to proteasome inhibition for hematological malignancies treatment 40-43. Nevertheless, the molecular mechanism of such a synergistic mode of action is not fully understood yet, but HDAC6 selectivity is mandatory.
Although there are several studies about it, reaching HDAC6 selectivity with a druggable compound remains a challenge due to the high homology within active sites of the HDAC family. There is thus a need to deepen the knowledge on this isoform at the molecular level. Today, HDAC catalytic inhibitors are built according to the classic HDAC pharmacophore scheme (Fig. 24) 44. It is composed of a zinc-binding group (e.g. hydroxamic acid), linked to a cap moiety (which size and lipophilicity may vary) by a hydrophobic chain (linker) 45. This scheme has been widely studied and used to design HDAC inhibitors, including those on the market 31,36,46. Interestingly, a large capping moiety was considered critical for HDAC6 selectivity so far through the exploration of a large channel rim of this isoform (Tubastatin A) predicted by homology modelling methods 47. Recently, Wagner and co-workers identified one of the most potent selective HDAC6 inhibitors reported in the literature. These compounds, lacking in capping groups, showed IC50 ranging from low micromolar to nanomolar values
48. This finding, among others 49, leads to reconsider the HDAC pharmacophore model reported so far, and raises several questions. How do these capless compounds interact with HDAC6 versus the other isoforms? Which are the structural features responsible for this selective profile? Taking advantage of the very recent structural information concerning human HDAC6 50, 51, computational-based structural insights able to explain this selective behavior are provided.
A
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B
Figure 24. (A) Structure of the HDAC6 selective inhibitors Tubastatin A 52, compound 180 48, and compound 181 48. The prototypical pharmacophoric scheme for HDAC inhibition is highlighted.
(B) The computational-based protocol adopted in this study is also reported.
Results & Discussion
Wagner and colleagues identified capless HDAC inhibitors able to selectively and potently decrease HDAC6-dependent deacetylation. In order to understand this enzymatic behavior, also proven at the cellular level, computational-based structural studies were here carried out first through molecular docking, to understand their possible binding mode, and then through molecular dynamics (MD) simulations, to understand the role of flexibility and solvated environment in binding and selectivity.
Two of the compounds synthetized by Wagner et al.48 were selected for further investigation: 180 (Wagner’s numbering 4), the most potent and selective HDAC6 inhibitor following the classical HDAC pharmacophoric scheme, and 181, the most potent and selective HDAC6 capless inhibitor (Wagner’s numbering 14, Fig. 24).
Molecular docking reveal structural features that may partially solve the selectivity puzzle The molecular docking protocol was first validated through the re-docking of the corresponding co-crystallized ligand within each protein structure. Docking calculations revealed the capability of the docking tool to retrieve the correct binding mode within each protein. Poses with Root Mean Square Deviation (RMSD) values lower than 1.5 Å with respect to the reference ligand were obtained, confirming the reliability of the adopted docking protocol (section 4.8, Table S24). Molecular docking results are summarized in Table 7.
Molecular docking Gold 5.2
PLP scoring function
Best docking poses comparisons
Systems selection for MD, RESP charges
Molecular Dynamics Simulations
Amber 12, 3 x 10 ns, non-bonded zinc model
Trajectories analysis
RMSD, RMSF, zinc chelation, H2O monitoring
Compounds 2
2
Isoforms
2 2
2, 4, 6, 8
2, 4, 6, 8
2, 4, 6, 8
2, 4, 6, 8
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Table 7. Summary of the molecular docking results for N-4 and N-14 conducted on HDAC isoform 2, 4, 6 and 8.
Isoform PDB id Ligand IC50
μMa
HDAC6 SI index
Best-ranked ligand ChemPLP score
Clustering % (cut-off 2Å)
HDAC2 4LXZ 180 ± 0.035 0.607 152 74.51 52
HDAC4 4CBY 180 > 33.33 > 8333 72.54 85
HDAC6 5EDU 180 ± 0.0001 0.004 - 86.14 63
HDAC8 1T64 180 ± 0.136 1.15 288 72.57 56
HDAC2 4LXZ 181 ± 0.165 1.79 60 51.40 92
HDAC4 4CBY 181 ± 0.091 21.8 727 41.56 98
HDAC6 5EDU 181 ± 0.006 0.03 - 52.30 100
HDAC8 1T64 181 ± 0.110 1.09 36 47.93 100
aData coming from Wagner et al. 48.
The classical HDAC inhibitor 180 was predicted having a monodentate binding mode through its hydroxamic acid functional group in all HDAC catalytic sites, with the exception of HDAC4 , in which 180 flipped when exploring HDAC4 catalytic pocket with its cap group occupying the zinc binding region (Fig. 25). These observations may explain the low activity detected toward HDAC4 with respect to the other isoforms. 180 was predicted to make three hydrogen bonds and π-stacking interactions within the HDAC6 catalytic pocket, which constituted the most favorable and stabilized environment, in comparison with its best-ranked pose into the other three isoform pockets (Table 8).
This gave structural clues for its high SI toward HDAC6. ChemPLP scores of the best-ranked compounds (180) were qualitatively consistent with the biological data retrieved in the literature 48.
The capless compound 181 was predicted to chelate the zinc ion in a bidentate fashion in all isoforms, with the exception of HDAC6 (Fig. 25). For this isoform, 181 was found to chelate the zinc ion through its carbonyl group only. These first observations prompted us to conduct a more detailed analysis of the interactions between 181 and the immediate environment of the zinc binding region of each HDAC catalytic pocket. In HDAC2, 4, and 8 isoforms, 181 was predicted to make two hydrogen bonds, involving a conserved tandem histidines that is known to be essential for both substrate binding and catalytic activity (Table 8) 17,50,53-55. In HDAC6, those two histidine residues were too far from the ligand to interact with. Nevertheless, 181 made a hydrogen bond with Gly619 (Fig. 25). An additional polar interaction between the ligand and a conserved tyrosine residue in HDAC2, 6, and 8 pockets (Tyr308, Tyr782, and Tyr306, respectively) was detected. HDAC4, having a histidine (His976) pointing outward the channel, prevented any hydrogen bonding with the ligand. This observation, already reported to be the key distinction between class I and class IIa HDACs 37,56, 57, may contribute to explain, at least in part, the lower activity of 181 on HDAC4.
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Figure 25. 180 and 181 best-ranked docking poses into HDAC2 (A), HDAC4 (B), HDAC6 (C) and HDAC8 (D) catalytic sites. 180 is shown as yellow sticks whereas 181 is shown as dark grey sticks and balls. Key residues involved in, either active site comparison or stabilizing the complex, are labeled in normal or bold policies and shown in lines or sticks, respectively. A molecular surface is drawn 6 Å around the docked ligands; the color code ranges from purple for hydrophilic areas, to green for hydrophobic ones. The zinc ion is shown as a light blue ball, and all chelating triads are displayed: (A) Asp181, Asp269 and His183, HDAC2 backbone is shown as light green ribbon, (B) Asp840, Asp934 and His842, HDAC4 backbone is shown as light violet ribbon, (C) Asp649, Asp742 and His651, HDAC6 backbone is shown as light blue ribbon, (D) Asp178, Asp267 and His180, HDAC8 backbone is shown as light yellow ribbon. Figures generated with MOE 2015.10.
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Table 8. Details about the interactions between 180, 181 and HDAC2, HDAC4, HDAC6, and HDAC8 best-ranked docking poses.
180 capped compound
Isoform Zinc distances H-bondsa Hydrophobic contacts
Isoform Zinc distances H-bondsa Hydrophobic contacts
a: hydrogen bonds (cut-off between 2 heteroatoms of 3 Å). b: pocket volumes calculated through the CASTp server 58.
c: hydroxamic acid.
Interestingly, hydrophobic contacts with conserved phenylalanine residues (Phe210/Phe155, Phe871/Phe812, Phe680/Phe620, and Phe210/Phe155 in HDAC2, 4, 6 and 8 respectively) were detected in all complexes. Interestingly, this Phe couple has already been reported to be gate-keepers for water access to HDAC catalytic pocket 59. It is worth to point out that only in HDAC6, the hydrophobic contracts between the 5-membered ring of 181 and Phe680/Phe620 are maximized through a face to face sandwich arrangement. T-shaped or parallel displaced hydrophobic interactions occur in the other isoforms (Fig. 25).
The fact that the best docking pose of 181 exactly overlaid the hydroxamic group and the phenyl ring of the capped ligand 180 highlights the importance of these hydrophobic interactions for HDAC6 potency and selectivity (Fig. 25). Moreover, the van der Waals interactions between the aromatic cycle and the hydrophobic wall characterized by Tyr782 and Leu749, not detected in the other complexes, may further contribute to 181-HDAC6 stability. According to the pocket volumes calculated through the CASTp server 58, HDAC6 has the most constricted active site among the 4 isoforms here considered (Table 8). This reduced pocket volume in HDAC6 seems to be particularly adapted to host a small molecule like 181, optimizing the interactions able to stabilize the complex 58. Nevertheless, this
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observation is in contrast with previous studies based on HDAC6 homology models, attributing the HDAC6-selectivity profile to bulky ligands like Tubastatin A (Fig. 24), interacting with a predicted wide channel 60. A dynamic behavior of the HDAC6 catalytic pocket allowing induced-fit movements is therefore suspected. There is thus a missing piece for explaining affinities and isoform-selectivity, and it may be related to missing water information and/or entropic phenomena 48. In order to unravel in more detail those aspects, MD simulations were conducted on the eight complexes.
Molecular dynamics simulations highlighted flexibility as the key player in HDAC ligand recognition
To explore the possible reasons of HDAC6 selectivity for compounds 180 and 181 with respect to other isoforms (2, 4 and 8), 10 ns molecular dynamics simulations were performed in triplicate for each complex obtained by docking. During the first 200 ps of simulation, pressure, temperature, energy, and density were plotted as a function of time. All these parameters resulted stable indicating a successful equilibration of all the systems (section 4.8, Fig. S16-S23). During the production phase, no important changes in backbone conformation was observed for all HDAC isoforms as indicated by the low RMSD values and standard deviations (section 4.8, Table S25). This confirmed that no unfolding phenomena happened during the simulations. Very low fluctuations were also observed for the two docked compounds, with RMSD values lower than 2 Å, suggesting a stable binding within the HDAC catalytic sites. As expected, higher fluctuations were detected for the cap compound 180 with respect to 181, according to its flexibility (Fig. 24). In order to identify the key interactions responsible of HDAC6 selectivity (Table 8), interactions between ligands and protein atoms were monitored as a function of time. First of all, zinc ion coordination was analyzed. Interestingly, for all complexes, a monodentate chelation was observed, despite the starting structural information coming from docking. The zinc-hydroxamate chelation mode is influenced by the protonation state of the hydroxamic acid 59. In this study, a protonated form was used. It is, indeed, considered to be the most abundant at pH 7.4 61, although deprotonated forms cannot be excluded, as the pKa may be influenced by the local protein environment.
A priori key interactions, previously predicted during the docking phase, both polar and apolar, were not all maintained during the MD phase. Indeed, significant fluctuations were detected through Root Mean Square Fluctuations (RMSF) at both loops and residues level. RMSF, distances and trajectories clustering were analyzed altogether to extract trends if any (section 4.8, Fig. 26, 28, S32 and S18). Two behavior types were thus detected among the eight monitored complexes: those that underwent punctual and/or loop motions away from the ligand and exhibiting one cluster each (180-HDAC2, 180-HDAC4, 180-HDAC6 and 181-HDAC4), and those in which significant fluctuations were detected around the ligand hence exhibiting one or more cluster(s) (181-HDAC2, 180-HDAC8, 181-HDAC8 and 181-HDAC6).
Within the 180-HDAC2 complex, the hydrogen bonds with Tyr308 and Gly154, formerly identified through docking, were not maintained along the 30 ns simulation, partially favoring the histidine couple (His145 and His 146 bound around 25% of the simulation, section 4.8, Fig. S24).
Despite these weak polar interactions, the compound 180 was found to keep its parallel-displaced hydrophobic contacts with Phe155 and Phe210. On the other way round, 180 was able to sustain more favorable contacts within HDAC6’s pocket, explaining, at least in part, its selectivity profile. Hydrogen bonds previously identified were maintained much longer than in HDAC2 (around 90% for Gly619, and 30% for Ser568 of the simulation time, section 4.8, Fig. S28), and one extra polar anchoring point was
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present, either through His611 or through His610 (about 66% of the simulation time, section 4.8, Fig.
S28). Moreover, a T-shaped hydrophobic interaction between Phe680 and the aryl linker part of 180 was seen stable over 90% of the simulation time, although those with Phe620 and Phe679 were not (Fig.
29). Whatever capped or capless structure, the interaction within HDAC4 were poor and fluctuating with respect to His802 which was detected to interact with 181 in a stable way, hence giving argument to explain its activity regarding 180 within HDAC4. Nonetheless, loop movements were seen that corresponded to the structural zinc-binding sub-domain (loop α1-α2 from His665 to Glu680 and loop α6-α7-β3-β4 from Leu733 to His766). These mass movements have already been reported to be involved in ligand accommodation (section 4.8, Fig. S26, S27 and S33) 20, 56,62.
Much more noticeable fluctuations were found for the four remaining complexes, confirming the suspected flexible behavior of HDAC6. Conversely to its complex with the capped ligand, the 181-HDAC2 complex exhibited 2 trajectory clusters, i.e. two sampled conformations. The most populated cluster (70.2% of the frames) displayed the closest conformation to the one coming from docking (section 4.8, Fig. S34). The second cluster (29.8% of the frames) showed a modified channel rim in which the loop Glu208 – Asp218 allowed to reduce the channel entrance, thus burying 181 inside, while opening an adjacent sub-pocket (section 4.8, Fig. S34). This loop was also reported in the literature for ligand accommodation 63, 64, 65, 66, 61. In this specific conformation, Pro211 only interacted with its neighboring residue Phe210 which pointed outward, thus opening the adjacent sub-pocket. These two residues networked with His146 in the most populated cluster, making a hydrophobic wall (section 5.8, Fig. S34). The adjacent sub-pocket configuration seemed to be more favorable for 181 stability, offering a more confined catalytic site. This may justify that 181 was such an active compound on HDAC2 despite its small size, as it allowed this ligand accommodation behavior to happen.
HDAC8 was several times reported in the literature to be highly flexible 19, 48,54,64, 66-72. This was also the case in this study, as demonstrated by the two and seventeen clusters obtained from the analysis of the MD trajectories of HDAC8 in complex with 180 and 181, respectively (Fig. 26). HDAC8 is known to convert from a wide-open catalytic pocket to a closed conformational state, showing a large cavity instead of a channel, an intermediate configuration with two adjacent channels (11 and 14 Å respectively), or a constricted 11 Å deep channel 19, 48,54, 64,66-72. When simulated in complex with the capped compound 180, HDAC8 mainly displayed the intermediate configuration (the most populated cluster representing 88% of the frames, Fig. 27). The second cluster displayed the closed state of HDAC8 catalytic pocket (Fig. 27). The two conformations are ruled by the specific motions of the loop Glu85 – Glu106 and the gate-keepers Tyr306 and Phe152. They were exactly those involved in the docking interactions predicted with 180. Consistently with this, they were also detected as highly flexible points through the RMSF plot (Fig. 26). Indeed, while the hydrogen bond with Gly151 was maintained half the simulation time, neither Tyr306 nor Asp101 sustained their polar contacts with the ligand. The T-shaped π interaction between the 180’s cap group and Tyr100 was also only transient (section 4.8, Fig. S30). Conversely, the interaction between 180 linker and Phe208 was rather stable along the simulation (around 66% of the time, section 4.8, Fig. S30); this residue is indeed known to play a key role in ligand accommodation 19, 48, 54, 64, 66-72. Although exhibiting the highest degree of flexibility, showing diverse conformational states summarized in seventeen clusters (from 18.9% to 1.6%), the 181-HDAC8 complex followed the same trend as the former one with the capped ligand (Fig. 26). Moreover, His142 and His143, which were formerly predicted as key polar contacts with the capless compound in docking, only made transient interactions in both capped and capless complexes (section 4.8, Fig. S30). This common behavior was consistent with Wagner et al. biological data
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regarding this isoform (Table 7). Besides, HDAC8 made optimized contacts with 181 only during 25.7% of the whole trajectory (mean contacts duration with His142, His143 and Phe152), while it was around 70% with HDAC6 (mean contacts duration with His611, Glu779 and Phe680). Again, this was consistent with the enzymatic data, further explaining the gap between HDAC8 and HDAC6 SI concerning the capless ligand.
Figure 26. Root Mean Square Fluctuation (RMSF) plots for HDAC8 complexes. RMSF were calculated for each residue (side chain atoms) characterizing the system. Significant motions with RMSF higher than 2 Å are highlighted through orange/grey arrows or bands for punctual or loop movements, respectively.
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Figure 27. Zoom on HDAC8 fluctuations: comparison between 180- and 181- complexes.
Average structure of the 2 most populated clusters from the 180- and 181-HDAC8 complexes are presented in rose (yellow balls and sticks for the ligand, rose ribbons and sticks for the protein) and grey (orange balls and sticks for the ligand, grey ribbon and sticks for the protein), respectively. The zinc ion is shown as a blue ball. (A) Zoom on the Glu85-Glu106 loop. This channel rim loop is known for both 11 Å channel opening/closing and for ligand accommodation 19, 48, 54,64,66-72. (B) Zoom on the gate-keepers Tyr306 and Phe152. They are known for interconversion between the conformational states of HDAC8 catalytic pocket 19,48, 54, 64,66-72. (C) Zoom on the Val104-Gln223 loop. This channel rim loop is also known for ligand accommodation 19,48, 54, 64,66-72. (D and E) A focus on the channel rim of N4- and N14-HDAC8 complexes (most populated clusters). A molecular surface is drawn 8 Å around the ligand allowing to see the influence of each loop lining the channel rim; the color code ranges from purple for hydrophilic areas, to green for the hydrophobic ones. Figures generated with MOE 2015.10.
Despite having only one trajectory cluster, the 181-HDAC6 complex showed significant RMSF at the loops lining the channel rim (Fig. 28 and 29). Similarly to the other complexes, the hydrogen
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bonds predicted by docking were not maintained during the 30 ns trajectories, favoring, in this case, an extra polar anchor between Glu779-OE2 and 181-NH, a residue located at the very bottom of the pocket (section 4.8, Fig. S29). Although the hydrophobic contacts with both Phe620 and Phe680 were present for 30% of the simulation time, they displayed significant RMSF (Fig. 28). Phe680 belongs to the small loop Phe679-Pro681, which is located at the channel rim. This loop was responsible for the channel
bonds predicted by docking were not maintained during the 30 ns trajectories, favoring, in this case, an extra polar anchor between Glu779-OE2 and 181-NH, a residue located at the very bottom of the pocket (section 4.8, Fig. S29). Although the hydrophobic contacts with both Phe620 and Phe680 were present for 30% of the simulation time, they displayed significant RMSF (Fig. 28). Phe680 belongs to the small loop Phe679-Pro681, which is located at the channel rim. This loop was responsible for the channel