Small Molecule Antagonists of the Interaction between the Histone Deacetylase 6 Zinc-Finger Domain and Ubiquitin

Article abstract of DOI:10.1021/acs.jmedchem.7b00933

Inhibitors of HDAC6 have attractive potential in numerous cancers. HDAC6 inhibitors to date target the catalytic domains, but targeting the unique zinc-finger ubiquitin-binding domain (Zf-UBD) of HDAC6 may be an attractive alternative strategy. We developed X-ray crystallography and biophysical assays to identify and characterize small molecules capable of binding to the Zf-UBD and competing with ubiquitin binding. Our results revealed two adjacent ligand-able pockets of HDAC6 Zf-UBD and the first functional ligands for this domain.

Full text of DOI:10.1021/acs.jmedchem.7b00933

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Brief Article
Small molecule antagonists of the interaction between the
histone deacetylase 6 zinc-finger domain and ubiquitin
Rachel J. Harding, Renato Ferreira de Freitas, Patrick Michael Collins, Ivan Franzoni,
Mani Ravichandran, Hui Ouyang, Kevin A. Juarez-Ornelas, Mark Lautens, Matthieu
Schapira, Frank von Delft, Vijayaratnam Santhakumar, and Cheryl H. Arrowsmith
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Oct 2017
Downloaded from http://pubs.acs.org on October 11, 2017
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Small molecule antagonists of the interaction between the histone
deacetylase 6 zinc-finger domain and ubiquitin
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Rachel J. Harding†, Renato Ferreira de Freitas†, Patrick Collins‡, Ivan Franzoni¶, Mani Ravi-
chandran†, Hui Ouyangᶲ, Kevin A. Juarez-Ornelas¶, Mark Lautens¶, Matthieu Schapira†#,
Frank von Delft‡§, Vjayaratnam Santhakumar†, Cheryl H. Arrowsmith†*
†Structural Genomics Consortium, University of Toronto, MaRS South Tower, Suite 700, 101 College Street, Toron-
to, ON M5G 1L7, Canada, ‡Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0QX,
U.K., ¶Department of Chemistry, Davenport Chemical Laboratories, 80 St. George St., University of Toronto, To-
ronto, Ontario, M5S 3H6 Canada, ᶲGrandPharma, Wuhan City Plaza, 23rd Floor, 160 Qiaokou Rd, Wuhan, Hubei,
China, #Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada,
§Structural Genomics Consortium, University of Oxford, Roosevelt Drive, Oxford OX11 9HP, U.K.
ABSTRACT: Inhibitors of HDAC6 have attractive potential in numerous cancers. HDAC6 inhibitors to date target the
catalytic domains, but targeting the unique zinc-finger ubiquitin-binding domain (Zf-UBD) of HDAC6 may be an attrac-
tive alternative strategy. We developed X-ray crystallography and biophysical assays to identify and characterize small
molecules capable of binding to the Zf-UBD and competing with ubiquitin binding. Our results revealed two adjacent
ligand-able
pockets
of
HDAC6
Zf-UBD
and
the
first
functional
ligands
for
this
domain.
INTRODUCTION Histone deacetylases (HDACs)
comprise a family of 18 enzymes with diverse roles in
mammalian cell homeostasis1,2. HDAC6 is distinct from
other zinc-dependent catalytic enzymes in this class,
with unique features including two functional catalytic
domains and a zinc-finger ubiquitin binding domain
(Zf-UBD) spanning resides 1109-12153–6. Localized to the
cytosol, HDAC6 has diverse roles in the function of the
cytoskeleton7,8, cell migration9, cell reprogramming and
pluripotency10,11 and is implicated in numerous patholo-
gies including cancer12, neurodegeneration13,14 and viral
infection15,16. HDAC6 can deacetylate many non-histone
proteins including α-tubulin8,15,17, HSP9018 and cortac-
tin19. HDAC6 knockout mouse studies show elevated
acetylation levels in more than 100 proteins in the liv-
er20,21 suggesting that HDAC6 may deacetylate a wide
range of substrates in vivo, reflecting its diverse cellular
roles. A deep cavity in the Zf-UBD of HDAC6 binds the
C-terminal RLRGG motif of free ubiquitin as well as C-
terminally unanchored polyubiquitin moieties with high
affinity4,5,22, leading to its activation23. This promotes the
autophagic clearance of ubiquitinated protein aggre-
gates in the cell by gathering and loading them on
dynein for aggresome formation and degradation24. The
Zf-UBD is also involved in the recruitment of HDAC6 to
vent deacetylation of tubulin, a modification required
for transport of misfolded proteins along microtubules
to the aggresome31. Used in combination with pro-
teasome inhibitors, HDAC6 catalytic inhibitors can
promote apoptosis in a number of different hematologi-
cal malignancies including multiple myeloma and lym-
phoma32–36. Challenges related to tedious chemical syn-
thesis37,38, low bioavailability39 and cytotoxicity for hy-
droxamate-based compounds40 have been reported for
some HDAC6 inhibitors. Targeting the recognition of
protein aggregates by the Zf-UBD41 rather than their
loading on microtubules as catalytic inhibitors do, could
be an alternative mechanism to antagonize aggresome
degradation.
In order to identify novel antagonists and ligands of
the HDAC6 Zf-UBD, we developed and optimized three
different biophysical binding assays as well as a crystal-
lographic method for screening ligands for this domain.
We then performed a high throughput screen of frag-
ments using X-ray crystallography as well as a virtual
screen using the crystal structure bound to the ubiquitin
C-terminal peptide. We initially identified four small
molecule ligands of HDAC6 Zf-UBD and characterized
them by surface plasmon resonance (SPR), fluorescence
polarization (FP) peptide displacement assay and iso-
thermal titration calorimetry (ITC). Preliminary struc-
ture–activity relationship (SAR) studies found com-
pounds of improved potency, able to displace a FITC-
labelled C-terminal ubiquitin RLRGG peptide, as well as
a compound which links the two ligand-able pockets
influenza viral fusion sites and subsequent infection25,26
.
Inhibitors of this domain could represent a novel mech-
anism to interrupt the viral life cycle.
Numerous specific and potent inhibitors of HDAC6
catalytic domains have been developed27–30 which pre-
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Table 1. Initial ligands identified by virtual and X-ray crystallography fragment screen.
1
2
3
Ligands
1
2
3
4
4
5
Cocrystal PDB codes 5KH9
5B8D
5KH3
5KH7
6
7
8
Structure
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10
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Identification
method
High throughput X-ray High throughput X-ray
crystallography
Virtual screen
Virtual screen
crystallography
Pocket bound
SPR (KD, µM)a
ITC (KD, µM)a
FP (IC50, µM)a
LEc (ITC)
Ubiquitin
N.B.b
N.B.b
N.I.c
Adjacent
N.B.b
N.B.b
N.I.c
Ubiquitin
60 ± 3
Ubiquitin
220 ± 20
210 ± 30
350 ± 50
0.28
40 ± 4
55 ± 11
-
-
0.40
aValues calculated as average of 3 independent measurements; No Binding; No Inhibition Ligand efficiency was calculated
b
b
c
using the equation LE = (1.37 × pKD)/HA; LE is expressed as kcal mol−1 atom−1
of HDAC6 Zf-UBD. Together our work comprises a key
resource to enable future discovery of potent and selec-
tive compounds targeting the HDAC6 Zf-UBD, and
probe the functional role of this domain in cells.
RLRGG peptide bound structure of HDAC6 Zf-UBD
(PDB - 3GV4). Cocrystal structures were solved with
HDAC6 Zf-UBD in complex with ligands 3 and 4 identi-
fied from the virtual screen (Table 1) to 1.6 Å and 1.7 Å
resolution for ligands 3 and 4 respectively. Both ligands
occupy the ubiquitin binding site. For all structures, the
position of each non-hydrogen atom of the soaked lig-
ands, pocket residues and water molecules were clear in
the electron density permitting precise modelling of the
ligand pocket (Supporting Information). Together
these ligands represent a diverse range of chemotypes.
RESULTS AND DISCUSSION The structure of HDAC6
Zf-UBD, residues 1109-1215, was previously solved in the
apo state (PDB ID: 3C5K) as well as in complex with the
RLRGG peptide (PDB ID: 3GV4). The apo structure re-
veals that this crystal form is not amenable to soaking as
the ubiquitin binding pocket is occluded by the C-
terminus of the adjacent HDAC6 molecule in the crystal
lattice. Using a C-terminally truncated version of this
construct, encompassing residues 1109-1213, we found an
alternate crystal form, grown using a seed mix of the
HDAC61109-1215 crystals. This new crystal form has the
same space group and <2.5 % deviation in unit cell di-
mensions compared to that of the HDAC61109-1215 crystals
allowing direct structure solution using the existing
3C5K structure and was also tolerant of 5 % (v/v) DMSO
for up to 3 h. The HDAC61109-1213 crystals differ in that
residues 1209-1213 are not resolved in the electron densi-
ty and the ubiquitin binding pocket is not occluded
making it accessible to a nearby solvent channel and
rendering this crystal form amenable to soaking with
small molecules.
Two juxtaposed pockets are bound by the ligands
Scheme 1. Synthesis of compounds 8 and 11a-f.a
Initial ligands of HDAC6 Zf-UBD were identified by
two methods (Table 1). The new crystal form and soak
conditions were used to conduct a high throughput X-
ray crystallography fragment screen of 1031 ligands at
the LabXChem pipeline based at the i04-1 beamline,
Diamond Light Source (U.K.)42,43, resulting in 571 da-
tasets with 15 map “events” including two fragment hits;
1 which occupies the C-terminal ubiquitin pocket, and 2
which binds in an adjacent pocket. Cocrystal structures
of these ligands were solved to 1.07 Å and 1.05 Å resolu-
tion for ligands 1 and 2 respectively. In parallel, a virtual
screen was conducted using the C-terminal ubiquitin
a Reagents and conditions (for synthesis of compounds 6
and 9, see Supporting Information): (a) MeI, K2CO3, DMF,
rt; (b) LiOH, THF/EtOH/H2O; (c) ROH, NaH, DMF, rt; (d)
Os4O, NaIO4, THF/ H2O, 0 °C; (e) NaClO2, NaH2PO4, t-
BuOH/2-methyl-2-butene,
rt.
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8
A
B
E
C
F
Table 2. Preliminary SAR ligands by FP assay.
Ligand
5
Structure
FP calculated IC50 (µM)a
60 ± 9
D
8
70 ± 10
9
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11a
270 ± 80
Figure 1: Structures of ligands binding HDAC6 Zf-UBD
show the malleability of the pocket. Ubiquitin binding and
adjacent pockets shown for HDAC6 Zf-UBD in complex
with A) RLRGG with adjacent pocket highlighted with red
circle - PDB: 3GV4, B) 1 – PDB: 5KH9, C) 2 - PDB: 5B8D, D)
3 - PDB: 5KH3, E) 4 - PDB: 5KH7 and F) 11b - PDB: 5PWB.
11b
11c
N.B.
N.B.
To characterize HDAC6 Zf-UBD ligand binding in
vitro, we devised three different biophysical assays (Fig-
ure 3). Surface plasmon resonance (SPR) and isothermal
titration calorimetry (ITC) protocols were optimized to
characterize binding kinetics of protein-ligand interac-
tions. To measure direct competition of ligands with
ubiquitin, we developed a fluorescence polarization (FP)
peptide displacement assay using FITC-labeled RLRGG
peptide. KD values for 3 and 4 were calculated at 60 and
220 µM respectively by SPR (Table 1). ITC assay con-
firmed the KD values determined by SPR for 3 and 4
(Table 1). Similarly, 3 and 4 were able to displace a
FITC-labeled RLRGG peptide from HDAC61109-1215 with
IC50 values of 55 and 350, respectively. Consistent with
the SPR results, these two carboxylate-containing com-
pounds show activity within the µM range and the more
potent of the two is 3. Compound 1 and 2 bind
HDAC61109-1215 but no KD/IC50 values could be determined
with any of the three biophysical methods, suggesting
that interactions made by the carboxylate group con-
tribute most significantly to potency within this group
of ligands.
11d
11e
140 ± 60
110 ± 40
180 ± 40
11f
aAverage of 3 independent measurements
screened. 3 and 4 both bind in the deep cavity which
normally accommodates the C-terminal ubiquitin di-
glycine motif (Figure 1D, E). For both of these com-
pounds the ring groups are stacked between Trp1182 and
Arg1155 forming π-stacking and cation-π interactions
whilst the carboxylate group occupies the same position
as that of the ubiquitin C-terminus. 1 forms the same π-
stacking interactions (Figure 1B), but lacking the car-
boxylate group, does not form any interactions deeper
in the pocket. A water molecule is found in each cocrys-
tal structure at the end of the ubiquitin binding cavity,
coordinating the ligand carboxylate group. 2 binds in a
cavity adjacent to the ubiquitin-binding pocket, the thi-
azole ring stacking on Trp1143 (Figure 1C). Interestingly,
Arg1155 occupies a similar position to that of the apo
structure in this cocrystal, whereas in all ligands in-
volved in π-stacking interactions, this residue swings
out to allow efficient stacking of the ring groups be-
tween Arg1155 with Trp1182 (Figure 2A). By doing so,
Arg1155 blocks the channel between the ubiquitin bind-
ing pocket and the adjacent pocket. Arg1155 is able to
move to accommodate the different ligands as they bind
in two distinct pockets, of HDAC6 Zf-UBD, which in
turn leads to variability in the shape and connectivity of
these two pockets.
To explore the potential of improving activity of the
fragments, we conducted a preliminary SAR study by
testing a limited number of close analogues of the most
active hit 3 (Table 2). Initially, we tested two commer-
B
A
Tyr1156
Arg1155
Trp1143
Trp1182
Tyr1189
Figure 2: A) Apo HDAC6 - 3C5K (green) modelled with
different Arg1155 poses of RLRGG bound - 3GV4 (mauve), 2
bound - 5B8D (magenta), 3 bound - 5KH3 (cyan) and 11b
bound - 5PWB (purple). B) Apo HDAC6 - 3C5K (green) is
shown with ligands 2 bound - 5B8D (magenta), 3 bound -
5KH3 (cyan) and 11b bound - 5PWB (purple).
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A
B
70
60
50
40
30
20
10
0
70
60
50
40
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20
10
0
CONCLUSION We have reported the first small mole-
1
2
3
4
5
6
7
8
cules known to bind the HDAC6 Zf-UBD, explored pre-
liminary SAR and identified a compound which can oc-
cupy both pockets of this domain. This demonstrates
the feasibility of improving the activity of the fragments.
We have also reported a robust crystal system to screen
ligands by X-ray crystallography and resolve high reso-
lution structures of HDAC61109-1213 in complex with lig-
ands. Three different biophysical methods (SPR, FP and
ITC) were optimized for ligand binding characterisation.
The optimized crystal system we developed led to the
solution of five high (< 1.7 Å) resolution cocrystal struc-
tures of HDAC61109-1213, revealing the precise binding po-
sitions of different compounds with varying affinities
(µM to mM). High quality crystallographic data proves
especially valuable when modelling the mobile Arg1155
which can block the channel between the ubiquitin and
adjacent pockets and such structural details of ligand
binding and the subsequent effects on pocket residue
positions will be critical if further fragments linking the
two pockets are to be designed and characterized to
increase the specificity and potency of the inhibitors.
HDAC6 Zf-UBD inhibitor development represents a
novel approach to dissecting the in vivo roles of HDAC6
in both normal cellular function as well as numerous
disease states. The optimized assays described in this
paper will lay the ground work for forthcoming research
to increase the potency of compounds beyond those
described in this paper by growing fragments, under-
standing structure activity relationships and assessing
specificity against a panel of other Zf-UBD proteins.
0
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Time (s)
[Compound]/(μM)
Molar ratio
Time (s)
2000
C
0
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-10
Figure 3: Exemplary assay data of the characterization of
ligand 3 by A) SPR, B) ITC and C) FP.
cially available analogues (5 and 8) with two different
heterocyclic cores, which have the potential of growing
into the adjacent pocket with appropriate substitutions
at the oxygen or nitrogen atom, by FP assay. Carboxylic
acid 8 was resynthesized in house in two steps from the
corresponding ethyl ester quinoxaline derivative 6 via
regioselective methylation followed by hydrolysis under
basic conditions (Scheme 1). Both compounds show
similar activity to 3. Our modelling indicated the phenyl
group of 5 could potentially reach the adjacent pocket
occupied by 2, but, we could not get a co-crystal struc-
ture of 5 with clear ligand electron density to prove this
hypothesis.
Encouraged by this result, we synthesized close ana-
logues of 5 with extended linkers to explore the poten-
tial of reaching the adjacent pocket with the substitu-
tions at the linker. Compounds 11a-f were prepared from
the chloroquinoxaline derivative 9. Deprotonation of
the desired alcohol with NaH followed by the addition
of 9 provided compounds 10a-f. The tethered double
bond functionality was converted into the desired car-
boxylic acid by Lemieux-Johnson oxidation followed by
Pinnick oxidation of the aldehyde intermediate
(Scheme 1). Compound 11a showed a moderate drop in
activity while the corresponding phenyl analogue 11b
lost activity completely. We were able to co-crystallise
HDAC6 Zf-UBD with 11a and solve the structure to 1.55
Å resolution (PDB – 5PWB). We were delighted to find
the pyridyl group occupied the adjacent pocket as pre-
dicted (Figures 1F and 2B). However, the pi-stacking of
the heterocyclic core with Arg1155 and Trp1182 is dis-
rupted. Arg1155 is not fully resolved in the electron den-
sity indicating flexibility of this residue in this binding
mode, which could explain the weaker affinity of 11a.
We have synthesized analogues of 11a with two hetero-
cycles (11c, 11d) and analogues with extended linkers
(11e, 11f) in order to identify more potent compounds
which could pick up additional interactions in the adja-
cent pocket while maintaining efficient pi stacking in
the peptide binding pocket. However, none of the com-
pounds showed improved affinity compared with the
original hit. We believe, the activity can be improved by
identifying a suitable linker and substitutions, which
will allow both heterocyclic ring and substitutions to
make optimal interactions in the peptide binding pocket
and adjacent pockets respectively.
EXPERIMENTAL SECTION
Compounds: All compounds detailed in this paper were as-
sessed for purity using high or ultra-performance liquid chro-
matography (HPLC/UPLC) and were shown to be >95% purity.
3-(4-methyl-3-oxo-3,4-dihydroquinoxalin-2-yl)propanoic acid
(8) Step 1: To a mixture of potassium carbonate (68 mg, 0.49
mmol, 1.2 equiv) and 6 (100 mg, 0.41 mmol, 1.0 equiv) in dry
DMF (0.2 M) was added MeI (33 µL, 0.53 mmol, 1.3 equiv) and
the reaction was stirred at room temperature for 16 h. The
resulting mixture was diluted with water and extracted with
ethyl acetate. The combined organic layers were dried over
MgSO4 and concentrated in vacuo. The crude mixture was
purified by flash chromatography (SiO2, hexanes/ethyl acetate
1:1) to afford 3-(3-oxo-3,4-dihydroquinoxalin-2-yl)propanoic
acid (7) as an off-white solid (94 mg, 90%). Step 2. To a mix-
ture of 7 (50 mg, 0.19 mmol, 1.0 equiv) in a 3:1:1 THF-EtOH-
H2O mixture (0.03 M) was added LiOH·H2O (18 mg, 0.77
mmol, 4.0 equiv) and the reaction was stirred at room temper-
ature till completion (TLC, 4 h). The resulting mixture was
diluted with water and concentrated in vacuo. The residue was
carefully acidified with aq. HCl 1N to pH 6-7. The resulting
solid was filtered, washed with water and dried to afford com-
pound 8 as a white solid (26 mg, 58%).
General Procedure for the Synthesis of Compounds 10a-f. To a
suspension of NaH (1.2 equiv) in DMF (0.2 M) was added the
desired alcohol (1.2-2.0 equiv). After 15 minutes stirring at
room temperature, a solution of 9 (1.0 equiv) in DMF was add-
ed and the reaction was stirred at room temperature till com-
pletion (TLC, 2-6 h). The reaction was diluted with ethyl ace-
tate and quenched with sat. aq. NH4Cl. The aqueous phase was
extracted with ethyl acetate, the combined organic layers dried
over MgSO4 and concentrated in vacuo. The crude mixture was
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purified by flash chromatography (SiO2, hexanes/ethyl acetate)
(PDB code 3GV4), resulting in the selection of 33 hit candi-
1
2
to afford the desired compound 10.
dates, 7 of which were confirmed biochemically. Two follow-up
rounds of hit expansion produced 3 and 4.
General Procedure for the Synthesis of Compounds 11a-f. Step
1) To a solution of 10 (1.0 equiv) and Et3NBnCl (0.1 equiv) in a
5:3 THF-H2O mixture (0.1 M), a small grain of OsO4 was added
at 0°C. NaIO4 (3.0 equiv) was added and the reaction vigorous-
ly stirred at 0°C till completion (TLC, 1-2 h). The reaction was
quenched with sat. aq. Na2SO3 and the aqueous phase extract-
ed with ethyl acetate. The combined organic layers were dried
over MgSO4 and the solvent evaporated in vacuo. The crude
mixture was purified by flash chromatography (SiO2, hex-
anes/ethyl acetate). Step 2) The aldehyde intermediate (1.0
equiv) was immediately dissolved in a 1:1 t-BuOH/2-methyl-2-
butene mixture (0.05 M). A freshly prepared aq. solution of
NaClO2 (10.0 equiv) and NaH2PO4 (5.5 equiv) was added and
the reaction vigorously stirred at room temperature overnight.
The resulting mixture was diluted with water and the organic
solvents were evaporated in vacuo. The residue was carefully
acidified with aq. HCl 1N to pH 6-7. The resulting solid was
filtered, washed with water and dried to afford compound 11.
Cloning, Protein Expression and Purification: DNAs en-
coding HDAC61109-1213 and HDAC61109-1215 were subcloned into a
modified pET28 vector encoding a thrombin cleavable (Gen-
Bank EF442785) N-terminal His6-tag (pET28-LIC) and
HDAC61109-1215 was also subcloned into a modified pET28 vector
encoding an N-terminal AviTag for in vivo biotinylation and a
Surface Plasmon Resonance (SPR): Studies were performed
using a Biacore T200 (GE Health Sciences). Approximately
5000 response units (RU) of biotinylated HDAC61109-1215 were
coupled onto one flow cell of a SA chip as per manufacturer’s
protocol, and an empty flow cell used for reference subtrac-
tion. 2-fold serial dilutions of compounds were prepared in 10
mM HEPES pH 7.4, 150 mM NaCl, 0.005 % (v/v) Tween-20, 1 %
(v/v) DMSO. KD determination experiments were performed
using single-cycle kinetics with 30 s contact time, 30 µL/min
flow rate at 20 °C. KD values were calculated using steady state
affinity fitting and the Biacore T200 Evaluation software.
Fluorescence Polarisation (FP) RLRGG Peptide Displace-
ment: All experiments were performed in 384-well black poly-
propylene PCR plates (Axygen) in 10 µL volume. Fluorescence
polarization (FP) was measured using a BioTek Synergy 4 (Bio-
Tek) at excitation and emission wavelengths were 485 nm and
528 nm, respectively. In each well, 9 µL compound solutions in
buffer containing 10 mM HEPES pH 7.4, 150 mM NaCl, 1 %
(v/v) DMSO were serially diluted. 1 µL 30 µM HDAC61109-1215 and
500 nM N-terminally FITC-labelled RLRGG were then added to
each well. Following 1 min centrifugation at 250 g, the assay
was incubated for 10 min before FP analysis. Previous assay
optimization with titration of protein concentration at 50 nM
peptide showed these conditions gave ~85 % maximum FP.
Isothermal Titration Calorimetry (ITC): HDAC61109-1215 was
diluted to 50 µM in 10 mM HEPES pH 7.4, 150 mM NaCl, 1 %
(v/v) DMSO. All compounds were diluted to 1 mM in the same
buffer. All ITC measurements were performed at 25 °C on a
Nano ITC (TA Instruments). A total of 25 injections, each of 2
µL, were delivered into a 0.167 mL sample cell at a 180-second
interval. The data were analyzed using Nano Analyze software
and fitted to a one-site binding model.
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C-terminal His6-tag (p28BIOH-LIC) using
a
ligation-
independent InFusion™ cloning kit (ClonTech) and verified by
DNA sequencing. Proteins were over-expressed in BL21 (DE3)
Codon Plus RIL E. coli (Agilent). Cultures were grown in M9
minimal media supplemented with 50 µM ZnSO4 and also 10
µg/mL biotin for HDAC61109-1215 p28BIOH-LIC expression. Ex-
pression cultures were induced using 0.5 mM IPTG overnight
at 15 °C. Proteins were purified using nickel-nitrilotriacetic acid
(Ni-NTA) agarose resin (Qiagen) and the tag was removed by
thrombin for HDAC61109-1213 pET28-LIC. Uncleaved proteins and
thrombin were removed by another pass with Ni-NTA resin.
Proteins were further purified using gel filtration (Superdex 75,
GE Healthcare). The final concentrations of purified proteins
were 5-10 mg/mL as measured by UV absorbance at 280 nm.
Crystallization: The apo crystal form of HDAC61109-1215 (PDB
ID: 3C5K) was previously reported4. These crystals can be used
to seed for the soaking amenable crystal form for fragment
screening. Diluting 1 µL crystal suspension 1:10,000 with moth-
er liquor and vortexing the sample vigorously yields a seed
mix. HDAC61109-1213 can be crystallized in 2 M Na formate, 0.1 M
Na acetate pH 4.6, 5 % ethylene glycol in 5:4:1 3.5 mg/mL pro-
tein, mother liquor and seed mix per drop. HDAC61109-1213 crys-
tals were soaked by adding 5 % (v/v) of a 200 mM or 400 mM
DMSO-solubilized stock of compounds 1-4 to the drop for 2
hours prior to mounting and cryo-cooling. Cocrystals of 11a
were grown using the same 5:4:1 ratio seeding protocol with 3.5
mg/mL HDAC61109-1213 pre-incubated with 5:1 molar ratio of the
11a ligand to concentrated HDAC6 protein.
ASSOCIATED CONTENT
SUPPORTING INFORMATION
The Supporting Information is available free of charge on
the ACS Publications website.
X-ray crystallography data collection and refinement statis-
tics as well as chemical synthesis information can be found
in: Supporting_Information.pdf
Authors will release atomic coordinates and experimental
data upon article publication.
HDAC6 Cocrystal Structure PDB ID Codes:
1 – 5KH9 2 – 5B8D 3 – 5KH3 4 – 5KH7 11b – 5WPB
AUTHOR INFORMATION
Corresponding Author
Cheryl H. Arrowsmith: email carrow@uhnresearch.ca
Author Contributions
Data Collection, Structure Determination and Refine-
ment: X-ray diffraction data for HDAC61109-1213 cocrystals with 3,
4 or 11a were collected at 100K at Rigaku FR-E Superbright
home source at a wavelength of 1.54178 Å. HDAC61109-1213 crystals
soaked during LabXChem pipeline were collected at 100K at
i04-1, Diamond Light Source (Harwell, U.K.) at a wavelength of
0.92819 Å, hit fragment datasets were identified using a
DIMPLE-PANDDA42 pipeline. All datasets were processed with
XDS44 and Aimless45. Models were refined with cycles of
COOT46, for model building and visualization, with REFMAC47,
for restrained refinement and validated with MOLPROBITY48.
Virtual Screening of Compounds: A diverse chemical library
of 1.28 million commercial compounds was docked to HDAC6
Experiments were performed by RJH, RF, PC, IF and JKA.
All authors contributed to writing the manuscript.
Funding Sources
The SGC is a registered charity (number 1097737) that re-
ceives funds from AbbVie, Bayer Pharma AG, Boehringer
Ingelheim, Canada Foundation for Innovation, Eshelman
Institute for Innovation, Genome Canada through Ontario
Genomics Institute, Innovative Medicines Initiative
(EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck
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Page 6 of 11
(11) Staszkiewicz, J.; Power, R. A.; Harkins, L. L.; Barnes, C. W.;
& Co., Novartis Pharma AG, Ontario Ministry of Economic
Development and Innovation, Pfizer, São Paulo Research
Foundation-FAPESP, Takeda, and the Wellcome Trust.
Additional funding from the Collaborative Research and
Training Experience grant (Aled Edwards 432008-2013)
from the Natural Sciences and Engineering Research Coun-
cil of Canada.
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Strickler, K. L.; Rim, J. S.; Bondioli, K. R.; Eilersten, K. J. Silenc-
ing Histone Deacetylase–Specific Isoforms Enhances Expres-
sion of Pluripotency Genes in Bovine Fibroblasts. Cell. Repro-
gramming 2013, 15 (5), 397–404.
(12) Aldana-Masangkay, G. I.; Sakamoto, K. M. The Role of
HDAC6 in Cancer. J. Biomed. Biotechnol. [Online] 2011, 2011,
875824. https://www.hindawi.com/journals/bmri/2011/875824/
(accessed Aug 28, 2017).
(13) Pandey, U. B.; Nie, Z.; Batlevi, Y.; McCray, B. A.; Ritson, G.
P.; Nedelsky, N. B.; Schwartz, S. L.; DiProspero, N. A.; Knight,
M. A.; Schuldiner, O.; Padmanabhan, R.; Hild, M.; Berry, D. L.;
Garza, D.; Hubbert, C. C.; Yao, T.-P.; Baehrecke, E. H.; Taylor,
J. P. HDAC6 Rescues Neurodegeneration and Provides an Es-
sential Link between Autophagy and the UPS. Nature 2007,
447 (7146), 859–863.
ACKNOWLEDGMENT
We thank Diamond Light Source for access to beamline
i04-1 and the XChem facility that contributed to the results
within this manuscript.
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ABBREVIATIONS
FP, fluorescence polarization; HDAC, histone deacetylase;
ITC, isothermal titration calorimetry; LE, ligand efficiency;
SPR, surface plasmon resonance; Zf-UBD, zinc-finger ubiq-
uitin binding domain
(14) Ganai, S. A. Small-Molecule Modulation of HDAC6 Activi-
ty: The Propitious Therapeutic Strategy to Vanquish Neuro-
degenerative Disorders. [Online early access]. Curr. Med.
Chem. 2017. DOI: 10.2174/0929867324666170209104030 Pub-
lished Online: Feb 8, 2017. http://www.eurekaselect.
com/149943/article (accessed Aug 28, 2017).
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