Molecular Basis for the Selective Inhibition of Histone Deacetylase 6 by a Mercaptoacetamide Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.8b00487

Mercaptoacetamide histone deacetylase inhibitors are neuroprotective agents that do not exhibit the genotoxicity associated with more commonly used hydroxamate inhibitors. Here, we present the crystal structure of a selective mercaptoacetamide complexed w

Full text of DOI:10.1021/acsmedchemlett.8b00487

Note
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Molecular Basis for the Selective Inhibition of Histone Deacetylase 6
by a Mercaptoacetamide Inhibitor
Nicholas J. Porter,† Sida Shen,‡,§ Cyril Barinka,∥ Alan P. Kozikowski,⊥
and David W. Christianson*,†
†Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania
19104-6323, United States
‡Department of Medicinal Chemistry and Pharmacognosy, Drug Discovery Program, University of Illinois at Chicago, Chicago,
Illinois 60612, United States
∥Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec,
Czech Republic
⊥StarWise Therapeutics LLC, 505 South Rosa Road, Madison, Wisconsin 53719, United States
S
* Supporting Information
ABSTRACT: Mercaptoacetamide histone deacetylase inhib-
itors are neuroprotective agents that do not exhibit the
genotoxicity associated with more commonly used hydroxamate
inhibitors. Here, we present the crystal structure of a selective
mercaptoacetamide complexed with the C-terminal catalytic
domain of HDAC6. When compared with the structure of a
mercaptoacetamide bound to the class I isozyme HDAC8,
different interactions are observed with the conserved tandem
histidine pair in the active site. These differences likely
contribute to the selectivity for inhibition of HDAC6, an
important target for cancer chemotherapy and the treatment of neurodegenerative disease.
KEYWORDS: Protein crystallography, enzyme inhibitor, zinc-binding group, cancer chemotherapy
etal-dependent histone deacetylases (HDACs) catalyze
2 as the cytosolic tubulin deacetylase.16−18 Inhibition of
Mthe hydrolysis of acetyllysine residues in histone and HDAC6 suppresses microtubule dynamics, resulting in cell
cycle arrest and apoptosis.19 Additionally, HDAC6 inhibition
nonhistone protein substrates to regulate diverse cellular
processes.1−4 Phylogenetic analysis divides these enzymes
promotes survival and regeneration of neurons, suggesting that
selective HDAC6 inhibitors could be used in treating spinal
cord injury and neurodegenerative diseases.20,21 However,
structural and mechanistic conservation across HDAC
isozymes complicates the design of isozyme-selective inhib-
itors.
into the class I HDACs (1, 2, 3, and 8), the class II HDACs,
further subdivided into the class IIa isozymes (4, 5, 7, and 9)
and the class IIb isozymes (6 and 10), and the sole class IV
isozyme HDAC11.5 These enzymes adopt the α/β fold first
observed in the binuclear manganese metalloenzyme argi-
nase,6−8 so the protein fold of an HDAC is designated the
arginase-deacetylase fold.
Even so, inhibitors exhibiting better than 102−103-fold
selectivity for HDAC6 have been identified,22 and the first
crystal structures of HDAC6 catalytic domains enable a deeper
understanding of structure−activity relationships for selective
inhibitors.23,24 These structural studies indicate that the
molecular basis of selectivity relies on interactions between
sterically bulky capping groups and the surface of the enzyme,
particularly in the region of the L1 loop flanking the active site,
as well as interactions in the aromatic cleft of the substrate
binding groove.23−27
The active site metal ion of an HDAC, typically Zn2+ but
possibly Fe2+ in vivo,9 is required for catalysis. In the first step
of catalysis, the scissile carbonyl group of acetyllysine
coordinates to Zn2+ and accepts a hydrogen bond from a
nearby tyrosine;10,11 both interactions are required to polarize
the scissile carbonyl for nucleophilic attack by a metal-bound
water molecule, which in turn is activated by a histidine general
base.12,13
As epigenetic regulators of protein structure and function,
the HDACs currently serve as molecular targets in drug design
programs focusing on new strategies for the treatment of
cancer, neurological diseases, and immunological disor-
ders.14,15 In particular, the class IIb isozyme HDAC6 is a
prominent drug target due to the function of catalytic domain
To date, nearly all of the inhibitors that have been
structurally characterized in complex with HDAC6 bear
Received: October 16, 2018
Accepted: November 20, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00487
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ACS Medicinal Chemistry Letters
Note
hydroxamate Zn2+-binding groups. The genotoxicity associated
with the hydroxamate group, such as that of the classic HDAC
inhibitor suberoylanilide hydroxamic acid (SAHA,28 formu-
lated as the cancer chemotherapy drug Vorinostat29), argues
against the use of a hydroxamate-containing inhibitor as a long-
term therapy for diseases other than cancer.30,31 The chemical
basis of genotoxicity derives from the Lossen rearrangement
(Figure 1), which yields a reactive isocyanate intermediate
1). Full experimental details regarding the synthesis of MCA,34
IC50 determinations against HDAC6 and HDAC8, and the
crystal structure determination of the HDAC6−MCA complex
are outlined in the Supporting Information. Crystallographic
refinement converged smoothly to Rwork/Rfree = 0.190/0.226;
data collection and refinement statistics are recorded in Table
S1. The overall structure of the polypeptide chain in the
HDAC6−MCA complex is essentially identical to that in the
unliganded enzyme (root-mean-square deviation = 0.2 Å over
292 Cα atoms), indicating that no major structural rearrange-
ments are required for the binding of MCA.
An electron density map showing the bound inhibitor is
presented in Figure 2. The thiol group of MCA is presumably
ionized to the negatively charged thiolate and coordinates to
the active site Zn2+ ion such that the overall metal coordination
geometry is a slightly distorted tetrahedral, with ligand−Zn2+−
ligand angles ranging 94−127° across both monomers in the
asymmetric unit of the crystal; metal coordination geometry
deviates from ideal tetrahedral geometry by an average
deviation of 10
5° for ligand−Zn2+−ligand bond angles.
Figure 1. (a) Structures and selectivity data for SAHA43 and MCA
alongside (b) the mechanism for the Lossen rearrangement as
potentially catalyzed by the Zn2+ ion in the HDAC active site.
For monomers A and B, the Zn2+···S separations are 2.3 Å, the
C−S−Zn2+ angles are 114° and 120°, and the C−C−S−Zn2+
dihedral angles are 14° and 2°, respectively. Apart from the
cisoid C−C−S−Zn2+ dihedral angle, the Zn2+ coordination
geometry is ideal as outlined for thiolate−metal coordination
interactions in refined protein structures.35 In comparison, the
Zn2+-bound thiolate group of the cyclic depsipeptide Largazole
thiol exhibits similar coordination geometry except for a more
favorable C−C−S−Zn2+ dihedral angle of 92° in its complex
with HDAC8.36
capable of covalently modifying cellular components.30 This
undesirable chemistry motivates the search for HDAC
inhibitors with alternative zinc-binding groups.
Notably, inhibitors bearing mercaptoacetamide zinc-binding
groups are not genotoxic and exhibit superior neuroprotective
properties.32 Moreover, certain mercaptoacetamide inhibitors
exhibit nanomolar affinity and better than 103-fold selectivity
against HDAC6.33,34 Until now, the structural basis for
HDAC6 affinity and selectivity has not been defined.
Here, we present the 1.85 Å-resolution X-ray crystal
structure of the complex between the mercaptoacetamide
inhibitor N-(5-(5,6-dichloro-1H-indol-1-yl)pentyl)-2-mercap-
toacetamide (MCA, Figure 1)34 and HDAC6 catalytic domain
2 from Danio rerio (zebrafish). The active site of the zebrafish
ortholog is identical to that of human HDAC6 catalytic
domain 2, and the zebrafish ortholog is superior for X-ray
crystallographic studies (henceforth, HDAC6 catalytic domain
2 is simply referred to as “HDAC6”).23 MCA exhibits 240-fold
selectivity for the inhibition of HDAC6 over HDAC8 (Figure
The zinc-bound thiolate group of MCA accepts a hydrogen
bond from the side chain of H573 (Nε573···S separation = 3.1
Å), which in turn donates a hydrogen bond to D610 (Nδ573···
O610 separation = 2.8 Å), thus confirming that the side chain of
H573 is in the positively charged imidazolium state. H573 is
the first histidine in a tandem pair found in all HDAC active
sites. Similar interactions are observed in the crystal structure
of Schistosoma mansoni HDAC8 (SmHDAC8; 42% identity
with human HDAC8) complexed with a mercaptoacetamide
analog of SAHA, in which the Zn2+-bound thiolate group
accepts a hydrogen bond from H141 (average Nε141···S
separation for monomers A−D = 3.3 Å) (Figure 3).37
Figure 2. (a) Stereoview of polder omit map (4.0σ; green mesh) for MCA (orange) bound to HDAC6 (blue). Hydrogen bond and metal-
coordination interactions are shown as dashed and solid lines, respectively. The active site zinc ion is represented as a gray sphere. (b) Schematic
representation of active site interactions for MCA bound to HDAC6.
B
DOI: 10.1021/acsmedchemlett.8b00487
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Note
hydrogen bond to H143.38,39 Thus, the mercaptoacetamide
moiety is, in effect, a functional mimic of a hydroxamate group
in terms of its ability to make an identical constellation of
metal coordination and hydrogen bond interactions in the
HDAC6 active site. Even better, the mercaptoacetamide
moiety is not mutagenic since it is not subject to degradation
via the Lossen rearrangement.
Why, then, does the mercaptoacetamide exhibit different
binding interactions in the active site of SmHDAC8, lacking a
hydrogen bond with the second active site histidine in the
tandem pair? The mercaptoacetamide moiety clearly does not
serve as a functional mimic of a hydroxamate group in binding
to HDAC8. This difference likely contributes to weaker
inhibition of class I HDAC isozymes by mercaptoaceta-
mides.33,34 Structural comparisons of HDAC6, SmHDAC8,
and human HDAC8 reveal that the second histidine is in a
different electrostatic environment in each isozyme. In
SmHDAC8 and human HDAC8, this histidine (H142 in
SmHDAC8, H143 in human HDAC8) donates a hydrogen
bond to a negatively charged carboxylate side chain (D191 in
SmHDAC8, D183 in human HDAC8), which elevates the
histidine pKa.13 In contrast, the corresponding histidine in
HDAC6, H574, donates a hydrogen bond to the neutral
carboxamide side chain of N617, which would elevate the pKa
of H574 but not as much as would result if the hydrogen bond
were made with a negatively charged carboxylate. Accordingly,
H574 of HDAC6 is less basic than H142/H143 of
SmHDAC8/human HDAC8. Consequently, H142 of
SmHDAC8 and H143 of human HDAC8 are more likely to
be protonated than H574 of HDAC6 at a given pH.
Differences in the basicity of the second histidine in the
tandem pair also have implications for catalysis. In human
HDAC8, enzymological and structural studies indicate that the
second histidine, H143, functions as a single general base−
general acid.13 Although similar enzymological studies have
not yet been performed with HDAC6, deletion of the second
histidine by mutagenesis in the H574A variant enables the
crystallization and structure determination of an intact
enzyme−substrate complex as the tetrahedral intermediate in
the catalytic mechanism.23 This observation implies that the
general base functionality in the active site of H574A HDAC6
is preserved; formation of the tetrahedral intermediate requires
a sufficiently nucleophilic water molecule activated by Zn2+
coordination and a general base. Thus, the remaining active
site histidine, H573 in H574A HDAC6, might function as the
general base in this isozyme. H574 must serve as the general
acid since its deletion leads to the trapped intermediate. The
tetrahedral intermediate of amide hydrolysis cannot collapse
without a proton donor to the leaving amino group, so the
deletion of the general acid in H574A HDAC6 results in the
formation of the tetrahedral intermediate as a dead-end
complex.
Figure 3. Structures of (a) S. mansoni HDAC8 (dark red; PDB ID
4CQF) complexed with a mercaptoacetamide analog of SAHA
(yellow) and (b) the HDAC6−MCA complex (colors match Figure
2) showing interactions in the active site of each enzyme. Hydrogen
bond and metal-coordination interactions are shown as dashed and
solid lines, respectively.
In the HDAC6−MCA complex, the mercaptoacetamide
carbonyl oxygen accepts a hydrogen bond from the phenolic
hydroxyl group of Y745 (O745···O separation = 2.4 Å). This
interaction mimics the role of Y745 in polarizing the scissile
carbonyl group of acetyl-L-lysine.23 However, the carbonyl
oxygen of MCA is 2.0 Å away from the Zn2+ coordination site
ordinarily required for substrate binding, such that the Zn2+···
O separation is 3.4 Å. Since both metal coordination and
hydrogen bond interactions are required to activate the scissile
amide group for hydrolysis, the amide group of the
mercaptoacetamide is rendered chemically inert through its
binding geometry in the HDAC6 active site.
The mercaptoacetamide NH group donates a hydrogen
bond to Nε of H574 (Nε574···N separation = 3.2 and 3.0 Å in
monomers A and B, respectively), which requires that the side
chain of H574 is in the neutral imidazole form. This is the
second histidine in the tandem pair; intriguingly, the
corresponding interaction with the second histidine, H142,
in the mercaptoacetamide complex with SmHDAC8 is too
long for hydrogen bonding (Nε···N separation range of 3.5−
4.0 Å in monomers A−D). Structures of mercaptoacetamide
inhibitor complexes with HDAC6 and SmHDAC8 are
compared in Figure 3. The lack of a hydrogen bond with
H142 in the SmHDAC8−mercaptoacetamide complex may be
due to the side chain of H142 being protonated as the
positively charged imidazolium cation.
Other aspects of MCA binding to HDAC6 contribute to its
selectivity as well. The aliphatic linker packs into the aromatic
groove in the substrate binding cleft with distances of 3.7 and
3.5 Å to the phenyl rings of F583 and F643, respectively. The
capping group of the inhibitor is situated within the previously
characterized L1 loop pocket, an interaction that confers
HDAC6 selectivity.27 The chlorine atoms of the dichloroindole
capping group pack against the side chains of H463 and P464.
Additionally, the capping group of the inhibitor bound to
monomer A packs against R636, D638, and F642 of monomer
The role of the second histidine in the tandem pair as a
hydrogen bond acceptor, requiring a neutral imidazole side
chain, is similarly required for the binding of hydroxamate
inhibitors with bidentate Zn2+ coordination geometry. In
bidentate hydroxamate complexes with HDAC6, the hydrox-
amate NH group donates a hydrogen bond to H574.23,25,26
The same is true in human HDAC8−hydroxamate inhibitor
complexes, where the hydroxamate NH group donates a
C
DOI: 10.1021/acsmedchemlett.8b00487
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ACS Medicinal Chemistry Letters
Note
B. Meanwhile, the molecule bound to monomer B forms lattice
contacts against D460 and H462 of monomer A.
crystal structure of the HDAC6−MCA complex. All authors
interpreted results and prepared the manuscript.
It is worthwhile to note that thiol-containing drug
candidates such as MCA can be subject to oxidation chemistry
or reaction with an unintended electrophile in vivo, which
could compromise their inhibitory function. The thiol-
containing HDAC inhibitors Romidepsin and Largazole
evolved to exist as thioester and disulfide-linked prodrugs,
respectively.8 Romidepsin is activated by reduction of its
internal disulfide linkage to yield the active inhibitor
Romidepsin thiol, and Largazole is activated by hydrolysis of
its thioester linkage to yield Largazole thiol. That being said,
HDAC6 is localized in the cell cytosol,40 which is highly
reducing, so this would favor the free thiol form of such
inhibitors, including MCA. Moreover, there is precedent for
the efficacy of a thiol-functionalized drug as exemplified by
Captopril, which contains a thiol group targeting Zn2+
coordination in the active site of angiotensin converting
enzyme.41,42
In summary, the present study highlights a chemical
difference in the binding of mercaptoacetamides and
hydroxamates to HDAC6 and HDAC8, specifically with regard
to interactions with the tandem histidine pair in the active site.
While each class of inhibitor contains a functional group that
directly coordinates to Zn2+ (C−S− and N−O−, respectively),
interactions with nearby active site residues differ. When
bound to either enzyme, the hydroxamate N−O− group
interacts with both histidine side chains. However, mercaptoa-
cetamides exhibit different interactions in HDAC6 and
HDAC8, specifically with regard to the second histidine.
This highlights the importance of the tandem histidine pair in
each of these enzymes; differences in the basicity of the second
histidine residue can influence inhibitor binding and catalysis,
which in turn can be exploited to enhance inhibitor selectivity.
Funding
This research was supported by NIH grants GM49758 to
D.W.C. and NS079183 to A.P.K. N.J.P. received financial
support from the NIH through Chemistry−Biology Interface
Training Grant T32 GM071339. This work was in part
supported by the Czech Science Foundation (15−19640S),
the Czech Academy of Sciences (RVO: 86652036), and the
project “BIOCEV” (CZ.1.05/1.1.00/02.0109) from the Euro-
pean Regional Development Fund.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank B. Havlinova for excellent technical assistance.
Additionally, we thank Dr. R. Rajashankar and synchrotron
beamline staff at the Northeastern Collaborative Access Team
beamlines (supported by Grants P1 GM103403 and S10
RR02905) at the Advanced Photon Source (APS), a US
Department of Energy (DOE) Office of Science User Facility
operated for the DOE Office of Science by Argonne National
Laboratory (ANL) under contract DE-AC02-06CH11357.
ABBREVIATIONS
■
HDAC, histone deacetylase; MCA, N-(5-(5,6-dichloro-1H-
indol-1-yl)pentyl)-2-mercaptoacetamide; SAHA, suberoylani-
lide hydroxamic acid
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00487.
■
S
Materials and Methods; Table S1, data collection and
refinement statistics (PDF)
Accession Codes
The atomic coordinates and crystallographic structure factors
of the HDAC6−MCA complex have been deposited in the
Protein Data Bank (www.rcsb.org) with accession code 6MR5.
AUTHOR INFORMATION
Corresponding Author
*Tel: 215-898-5714. E-mail: chris@sas.upenn.edu.
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ORCID
Sida Shen: 0000-0002-0295-2545
Cyril Barinka: 0000-0003-2751-3060
David W. Christianson: 0000-0002-0194-5212
Present Address
§Department of Chemistry, Northwestern University, Evan-
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DOI: 10.1021/acsmedchemlett.8b00487
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