Rational Design of Suprastat: A Novel Selective Histone Deacetylase 6 Inhibitor with the Ability to Potentiate Immunotherapy in Melanoma Models

Article abstract of DOI:10.1021/acs.jmedchem.0c00567

Selective inhibition of histone deacetylase 6 (HDAC6) is being recognized as a therapeutic approach for cancers. In this study, we designed a new HDAC6 inhibitor, named Suprastat, using in silico simulations. X-ray crystallography and molecular dynamics simulations provide strong evidence to support the notion that the aminomethyl and hydroxyl groups in the capping group of Suprastat establish significant hydrogen bond interactions, either direct or water-mediated, with residues D460, N530, and S531, which play a vital role in regulating the deacetylase function of the enzyme and which are absent in other isoforms. In vitro characterization of Suprastat demonstrates subnanomolar HDAC6 inhibitory potency and a hundred- to a thousand-fold HDAC6 selectivity over the other HDAC isoforms. In vivo studies reveal that a combination of Suprastat and anti-PD1 immunotherapy enhances antitumor immune response, mediated by a decrease of protumoral M2 macrophages and increased infiltration of antitumor CD8+ effector and memory T-cells.

Full text of DOI:10.1021/acs.jmedchem.0c00567

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Article
Rational Design of Suprastat, a Novel Selective Histone Deacetylase 6
Inhibitor with the Ability to Potentiate Immunotherapy in Melanoma Models
Satish Noonepalle, Sida Shen, Jakub Ptacek, Maurício T Tavares, GuiPing
Zhang, Jan Stransky, Jiri Pavlicek, Glaucio M Ferreira, Melissa Hadley,
Guido Pelaez, Cyril Ba#inka, Alan P. Kozikowski, and Alejandro Villagra
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00567 • Publication Date (Web): 20 Aug 2020
Downloaded from pubs.acs.org on August 20, 2020
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Rational Design of Suprastat, a Novel Selective Histone Deacetylase 6
Inhibitor with the Ability to Potentiate Immunotherapy in Melanoma
Models
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a,†
b,†,‡
d
b, ⁋
Satish Noonepalle , Sida Shen , Jakub Ptáček , Maurício T. Tavares , Guiping
b
e
e
f
a
Zhang , Jan Stransky , Jiri Pavlicek , Glaucio M. Ferreira , Melissa Hadley , Guido
a
,d
,c
,a
Pelaez , Cyril Bařinka* , Alan P. Kozikowski* , Alejandro Villagra*
a
Department of Biochemistry and Molecular Medicine, The George Washington
University, Washington, District of Columbia 20052, United States
b
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,
University of Illinois at Chicago, Chicago, Illinois 60612, United States
c
Bright Minds Biosciences, Toronto, ON M5H 3V9, Canada
d
Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy
of Sciences, BIOCEV, Prumyslova 595, 252 50 Vestec, Czech Republic
e
Centre of Molecular Structure, Institute of Biotechnology of the Czech Academy of
Sciences, BIOCEV, Prumyslova 595, 252 50 Vestec, Czech Republic
f
Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo,
São Paulo, SP, 05508-000, Brazil
†
These authors contributed equally to this work.
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KEYWORDS: catalytic domain 2, hydrogen-bonding interaction, acetylated α-tubulin,
immunoregulation, combination therapy
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ABSTRACT
Selective inhibition of histone deacetylase 6 (HDAC6) is being recognized as a
therapeutic approach for cancers. In this study, we designed a new HDAC6 inhibitor,
named Suprastat, using in silico simulations. X-ray crystallography and molecular
dynamics simulations provide strong evidence to support the notion that the
aminomethyl and hydroxyl groups in the capping group of Suprastat establish
significant hydrogen bond interactions, either direct or water-mediated, with residues
D460, N530, and S531, which play a vital role in regulating the deacetylase function of
the enzyme and which are absent in other isoforms. In vitro characterization of
Suprastat demonstrates subnanomolar HDAC6 inhibitory potency and a hundred- to a
thousand-fold HDAC6 selectivity over the other HDAC isoforms. In vivo studies reveal
that a combination of Suprastat and anti-PD1 immunotherapy enhances anti-tumor
immune response, mediated by a decrease of pro-tumoral M2 macrophages and
increased infiltration of anti-tumor CD8+ effector and memory T-cells.
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INTRODUCTION
Reversible acetylation of lysine side chains on the surface of enzymes and other
proteins is modulated by histone acetyltransferases (HATs) and histone deacetylases
(HDACs). Protein lysine acetylation or deacetylation serves as a key regulatory
pathway for various cellular processes, such as transcription, cell cycle, and cellular
metabolism.1-3 HDACs have been demonstrated to be effective targets for the treatment
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of cancer, neurological diseases, and immune disorders.4-6 Up to date, 11 zinc ion
2+
(
Zn )-dependent HDACs (Class I, II, and IV) and seven nicotinamide adenine
+
dinucleotide (NAD )-dependent sirtuins (SIRTs) (Class III) have been identified.
Unlike other members, HDAC6 in Class IIb is unique due to its ability to deacetylate a
number of non-histone proteins as its preferred substrates, such as α-tubulin and HSP-
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0.7, 8 HDAC6 inhibitors (HDAC6is), Ricolinostat (ACY-1215), Citarinostat (ACY-
41), and KA2507 are being evaluated in clinical trials for various types of cancers
9
through either monotherapy or a combination approach, although they are only
partially selective HDAC6is. Therefore, HDAC6is have emerged as a promising
approach for cancer therapy.
Immunomodulatory properties of HDAC6is have deemed them as therapeutic agents
for cancer immunotherapy. It has been reported that HDAC6 interacts with the
transcription factor STAT3, which is a primary regulator of immune responses in the
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tumor microenvironment, and that it regulates STAT3-mediated gene expression. In
antigen-presenting cells (APCs), such as macrophages and dendritic cells, selective
inhibition of HDAC6 leads to a decreased production of immunosuppressive cytokine
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IL-10, thereby retaining the proinflammatory state of APCs. In melanoma tumor cells,
HDAC6 inhibition leads to a decreased level of the immunosuppressive molecule PD-
L1 by affecting the recruitment and activation of STAT3.12 Furthermore, using the
syngeneic murine melanoma mouse model, we recently demonstrated that a
combination therapy comprised of selective HDAC6is and PD-1 antibody leads to
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significantly improved effects on tumor growth compared to a single therapy, thereby
underscoring the immunomodulatory capability of selective HDAC6is.
The structure of one HDAC6i typically contain: a) a zinc-binding group (ZBG)
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+
establishing essential coordination with the Zn ion in the active site; b) a linker region
filling the hydrophobic space between the catalytic site and the outer surface; c) a
capping group (cap) interacting with residues on the surface. The phenylhydroxamate-
based HDAC6is feature the critical ZBG and linker for selective and potent HDAC6
inhibition that originated from the invention of Tubastatin A (TubA, Figure 1A).9, 14-18
9
HDAC6is are largely comprised of arylhydroxamate-based analogs. However, the
recent development of oxadiazole-based selective HDAC6is by pharmaceutical
9
companies may provide a new opportunity to refine the druglike properties (e.g.,
metabolic stability, permeability, and mutagenicity)19-21 of HDAC6is. Crystallographic
studies of several ligands complexed with Danio rerio HDAC6 (zHDAC6) have
revealed that most selective HDAC6is containing phenylhydroxamate exhibit an
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22, 23
unusual monodentate Zn coordination geometry.
On the other hand, a typical
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canonical bidentate Zn coordination was observed in a number of zHDAC6 crystals
complexing with HDACis bearing alkylhydroxamate or capless arylhydroxamate.24-26
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In addition, the phenylhydroxamate moiety is sandwiched by F583 and F643 in the
hydrophobic tunnel, establishing a double π-stacking interaction.
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Figure 1. Crystal structures of zHDAC6 complexes with selective HDAC6is (A)
Tubastain A (yellow, PDB code 6THV) (B) RTS-V5 (green, PDB code 6CW8) (C)
ACY-1083 (pink, PDB code 5WGM) (D) HPB (blue, PDB code 5WGK) (E) SS-208
(cyan, PDB code 6R0K) (F) CBZ (purple, PDB code 6PZU). Selective amino acid
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residues in HDAC6 are indicated in stick representation with carbon atoms in grey,
oxygen atoms in red, nitrogen atoms in blue colors, whereas active-site zinc ion and
water molecules are indicated as silver and red spheres, respectively. IC values for
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HDAC1 and HDAC6, along with selectivity indices, are indicated for each HDAC6
inhibitor.
Among zinc-dependent HDACs, only HDAC6 contains a large open basin
approximately 14 Å wide. Thus, a lot of selective HDAC6is consist of a bulky and rigid
cap to occupy the broad rim of the pocket.22 Polycyclic aromatic rings are often
considered as useful caps in selective HDAC6is that can form robust hydrophobic
engagements with the L1 loop pocket defined by key residues H463, P464, F583, and
L712, which is considered as the selectivity-determining area.25-27 The crystal structure
of the enzyme in complex with TubA (PDB code 6THV) is shown in Figure 1A as an
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example. Recent findings provide additional examples, such as Resminostat (PDB
code 6PZR), whose cap engages with another pocket defined by the L2 loop.29
Moreover, the bifurcated cap of the dual HDAC-proteasome inhibitor RTS-V5 can
occupy both L1 and L2 pockets and interact with the nearby residues G640-N645
Figure 1B, PDB code 6CW8).30, 31
(
Besides hydrophobic and π-stacking interactions, HDAC6 crystals in complex with
selective HDAC6is also indicate that capping or linker groups form additional
hydrogen bonds with the residue S531 inside the pocket: a) The NH group on the linker
of pyrimidinylhydroxamate-based ACY-1083 shows a direct hydrogen-bonding
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interaction to the hydroxyl group of S531 (Figure 1C, PDB code 5WGM). b) The
hydroxyl group on the n-propyl tail chain of HPB forms a water-mediated hydrogen
23
bond with S531 (Figure 1D, PDB code 5WGK). c) The carbonyl group on the amide
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connecting unit of isoxazole-3-hydroxamate-based SS-208 interacts with S531 at a
distance of 3.3 Å (Figure 1E PDB code 6R0K).26 Moreover, hydrogen-bonding
interactions with other residues (e.g., D460 and F643) are also observed in zHDAC6
crystal complexes: a) Ricolinostat establishes water-mediated hydrogen-bonding
interactions with D460 and S531 through its aminopyrimidinyl core and amide linkage,
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respectively (PDB code 5WGL). b) zHDAC6 in the complex with a Cbz-protected
dipeptide (Leu-Ala) capped HDAC6i CBZ (Figure 1F, PDB code 6PZU) reveals that
the phenyl ring of the Cbz protecting group forms a carbon-hydrogen bond with D460.
In contrast, hydrogen-bond interactions mediated by water molecules are observed with
29
the imidazole ring of H614 and the amino group of F643. Noteworthy, S531 is critical
for substrate recognition and engages with the NH group of the N-acetyllysine moiety
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through a hydrogen-bond interaction. It was also found that a single mutation (S531A)
in sequence encoding zHDAC6-CD2 decreased the catalytic efficiency by 258-fold.24
Moreover, It was reported by the Matthias group that double mutations (W459A and
D460A) and other single mutations (N530A, N530D, or S531A) significantly reduced
α-tubulin deacetylation.22 It should be noted that N530 of zHDAC6 corresponds to
D567 in the human ortholog. Surprisingly, the N530D substitution in zHDAC6 led to
a substantial decrease in the deacetylation efficacy of α-tubulin. At the same time, it
has been reported by the Christianson group that the N530A substitution in zHDAC6
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and D567A substitution in hHDAC6 did not significantly influence HDAC6
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deacetylase activity using a fluorogenic substrate derived from histone H4 Apparently,
the residue might play an important role in the interaction interface with proteinaceous
substrates (e.g., tubulin), but not small peptides. The detailed understanding of the N530
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(D567) role in substrate recognition warrants further studies.
Overall, we hypothesized that establishing specific hydrogen-bonding interactions with
these key residues, which play crucial roles in regulating the deacetylation function of
HDAC6 and which are absent in other isoforms, might lead to enhanced potency and
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excellent HDAC6 isoform selectivity. Currently, a typical strategy in the discovery of
selective HDAC6is relies on modifications of aromatic capping groups to strengthen
hydrophobic interactions with residues of the L1-loop pocket.9, 16, 17 As an alternative
approach, we designed new analogs based on the Nexturastat A (NextA) scaffold by
incorporating polar groups to interact with key residues of the HDAC6 pocket.
Integrated structural and biological characterization demonstrates that one analog,
named Suprastat, represents a new generation of selective HDAC6i.
RESULTS AND DISCUSSION
Design and synthesis of Suprastat (6a). NextA (Figure 2A, left) comprises a classical
phenylhydroxamate zinc-binding core linked to a urea-based cap featuring a phenyl
ring and an n-butyl side chain, which exhibits good affinity and selectivity for
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HDAC6. The HDAC6/NextA crystal (PDB code 5G0I, Figure 2B) reveals that there
is no direct enzyme-inhibitor hydrogen bonding interaction between the cap and the
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pocket. Instead, the steric complementarity between the bulky cap and the 3D contour
of the pocket drives important hydrophobic interactions. NextA exhibited minimal
antiproliferative activity against a variety of human cancer cell lines and induced G1
cell-cycle arrest without eliciting apoptosis.26, 34 Moreover, it was found that NextA
down-regulated the level of the immunosuppressive molecule PD-L1 (CD274) in
melanoma cells and additionally impaired melanoma tumor growth in
immunocompetent mice mediated by increased tumor-specific immunogenic signals.13
Recent findings further demonstrated that a combination treatment employing NextA
along with anti-PD1 immune blockade resulted in enhanced T-cell infiltration coupled
with a decrease of pro-tumor M2 macrophages in the tumor microenvironment, leading
to significant tumor growth inhibition and a higher survival rate compared to
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monotherapy. Given the well-understood molecular basis for its selective inhibition
of HDAC6 as well as its efficacy in established murine melanoma models, we selected
NextA as a parent scaffold for designing the next generation of HDAC6is.
We reasoned that the derivatization of the NextA cap with polar groups could further
increase potency and selectivity via potential interactions with the residues at the rim
region of the HDAC6 catalytic core. Molecular docking studies using the
HDAC6/NextA complex as a template were performed for a series of newly designed
NextA derivatives. We examined their capacity to extend the interaction interface of
the inhibitor cap with the HDAC6 rim residues. The most promising derivative, named
Suprastat, contains an aminomethyl group at the para position of the phenyl ring and a
hydroxyl group at the end of the n-butyl chain attached to the proximal urea nitrogen
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(Figure 2A, right). Based on the in silico predictions, the positively charged
aminomethyl and hydroxybutyl branches of Suprastat interact with the carboxyl group
of D460 and the main chain amino group of F643, respectively (Figure 2B).
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Figure 2. In silico design of Suprastat by NextA derivatization. (A) Structures of
phenylhydroxamate-based Nexturastat A (left) and Suprastat (right). (B) Molecular
docking of Suprastat (orange) based on the HDAC6/Nexturastat A complex (PDB code
5
G0I; silver) with an indication of the additional interactions created by the
aminomethyl and hydroxybutyl polar groups with residues D460 and F643 at the rim
of the catalytic pocket of HDAC6.
Moreover, we performed relative binding free energy calculations35 on the
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HDAC6/NextA and HDAC6/Suprastat complexes, respectively. The results indicate
that the HDAC6/Suprastat complex shows a much lower free energy of binding (ΔG0
-1
0
-1
=
-67.25 kcal mol ) compared to HDAC6/NextA (ΔG = -38.85 kcal mol ), suggesting
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an improved affinity of Suprastat for the HDAC6 catalytic pocket. The enthalpic (ΔH0)
0
0
and entropic (ΔS ) contributes to both ΔG values, determined by the Gibbs free energy
equation (Figure 2A). The results suggest that the improved affinity of Suprastat is
accompanied by a more substantial entropic gain (-TΔ S0).
Scheme 1. Synthetic route to 6a-b and 6d-fa
a
BocHN
O
BocHN
N
O
NH2
H
1
2
c
R1
b
R1
H
N
R2
O
a: R1 = COOMe
b: R1 = CN
c: R1 = Br
4a: R1 = COOMe, R2 = OH;
3
3
3
4b: R1 = COOMe, R = H;
2
4c: R1 = CN, R2 = OH;
d: R1 = Br, R2 = OH;
4
R2
H
R2
d-g
R1
R1
H
N
N
N
N
BocHN
O
H2N
O
6
6
a: R1 = CONHOH, R2 = OH;
5a: R1 = COOMe, R2 = OH;
b: R = CONHOH, R2 = H;
1
5
b: R1 = COOMe, R2 = H;
6d: R = COOH, R2 = OH;
1
5
5
c: R1 = CN, R2 = OH;
6
6
e: R1 = CONH2, R2 = OH;
f: R1 = B(OH)2, R2 = OH;
d: R1 = Br, R2 = OH;
a
Reagents and conditions. (a) phenyl chloroformate, K CO , acetone, rt, 2 h; (b) i) 4-
2
3
amino-1-butanol for 4a, 4c, and 4d and n-butylamine for 4b, EtOH, reflux, 2 h; ii)
NaBH , MeOH, 0°C-rt, 2 h; (c) 2, TEA, THF, reflux, 2 h; (d) for 6a and 6b: i) aq.
4
NH OH (50%), NaOH, THF/MeOH, 0°C, 15 min; ii) TFA, THF, rt, 0.5 h; (e) for 6d:
2
i) 1N NaOH, THF/MeOH, rt, overnight; ii) TFA, THF, rt, 0.5 h; (f) for 6e: i) H O
2
2
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(30%), K CO , DMSO, rt, 5 h; ii) TFA, THF, rt, 0.5 h; (g) for 6f: i) B pin , KOAc,
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3
2
2
Pd(dppf)Cl , DMF, 80°C, overnight; ii) NaIO , NH OAc, acetone-H O, rt, overnight;
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iii) TFA, THF, rt, 0.5 h.
0
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The synthetic route to Suprastat was initiated by the preparation of a carbamate
intermediate 2 from phenyl chloroformate and aniline 1 under K CO /acetone
2
3
conditions. On the other hand, methyl 4-formylbenzoate 3a underwent a rapid reductive
amination with 4-amino-1-butanol to provide intermediate 4a. Subsequently, the
combination reaction between 2 and 4a under TEA/THF conditions afforded the key
urea precursor 5a, which was further converted to the final hydroxamate product 6a
(Suprastat) using aqueous hydroxylamine under basic conditions followed by
TFA/THF to remove the Boc group. For evaluation together with Suprastat in the
following biological experiments, analog 6b bearing an aminomethyl group and the
original n-butyl chain attached to the proximal urea nitrogen was also prepared from 3a
and n-butylamine using the same synthetic route to Suprastat. The synthesis of 6c
(Table 1), which contains a hydroxylbutyl side chain but no aromatic substituent, has
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been reported in our prior work. Moreover, non-hydroxamate analogs 6d-f containing
the same cap with Supratstat were prepared and evaluated to explore if additional
hydrogen binding interactions would be able to retain activity without a hydroxamate
ZBG, inspired by ketone/amide-based Class I HDACis.37-39 The carboxylic acid analog
6d was directly afforded from the urea ester 5a through hydrolysis under basic
condition and Boc deprotection. To synthesize the amide analog 6e, 4-
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formylbenzonitrile 3b underwent the two-step reductive amination followed by the
reaction with carbamate 2 to generate the intermediate urea 5c. The nitrile group in 5c
was further converted to an amide group by treating with aqueous hydrogen peroxide
solution under basic condition, and the final product 6e was afforded through Boc
deprotection as described above. The synthetic route to the boronic acid analog 6f
initiated with the reductive amination of 4-bromobenzaldehyde 3c followed by urea
formation using the same procedures as above to give the intermediate urea 5d. The
precursor 5d underwent coupling reaction with bis(pinacolato)diboron under
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KOAc/Pd(dppf)Cl conditions. In the end, the desired boronic acid product 6f was
2
obtained through pinacol deprotection and Boc deprotection under NaIO /NH OAc and
4
4
TFA/THF, respectively.
In vitro HDAC potency assessment. To evaluate the influence of each additional
functional group on the potency and isoform selectivity, we tested the potency of 6a-c
along with NextA against human HDACs 1-9 and 11 under optimized conditions in
vitro.26 It should be mentioned that our highly pure (>98%) HDAC10 preparations
heterologously expressed in HEK293T cells did not show any appreciable activity
using either acetylspermidine40 or RHKK(Ac)AMC (typically used by Reaction
Biology Corp, Malvern, PA) as the substrate for HDAC10 profiling. Therefore, the IC50
values of HDAC10 were not included. Results in Table 1 suggest that 6a and 6c are
more potent and selective HDAC6is (IC50 = 0.4 vs. 0.5 nM) with at least 290-fold
selectivity over Class I isoforms HDAC1-3 and 8 and a thousand-fold selectivity over
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Class IIa isoforms HDAC4, 5, 7, and 9. Moreover, 6a-c did not show activity against
the Class IV isoform HDAC11 up to 50 µM. It shall be noted that experimental IC50
values in the range of 0.2–0.4 nM for HDAC6 are at the limit of our assay that uses
approximately 0.6 nM concentration of the full-length enzyme (as determined by
absorbance measurements at 280 nm). IC values for Suprastat and 6c are thus at the
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limit of our assay, and these two inhibitors can theoretically have an even higher
potency than reported here. Overall, the inhibition data suggest that the hydroxylbutyl
chain contributes more prominently towards the increase in HDAC6 affinity and
selectivity compared to the aminomethyl group. Additionally, the incorporation of the
polar aminomethyl and hydroxylbutyl groups into the inhibitors 6a-c increases the
4
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number of heavy atoms but decreases their clogP (calculated by SwissADME ) relative
to NextA (Table 2), which leads to significantly elevated lipophilic ligand efficiencies
(
LipE),42 especially 6a ((LipE = 7.57 (6a) vs. 6.19 (NextA)), although the ligand
4
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efficiencies (LE) of 6a and 6b are slightly lower. Taken together, compared to NextA
and related analogs, Suprastat (6a) bearing both hydroxybutyl and aminomethyl
moieties showed improved potency against HDAC6 and excellent selectivity over the
other HDAC isoforms. At the same time, it also exhibits the highest LipE value due to
its significantly decreased clogP. On the other hand, the inhibitory potency of the non-
hydroxamate-based analogs 6d-f comprised of carboxylic acid, amide, or boronic acid
as alterative ZBGs against HDAC6 was severely compromised (Supplementary Table
S1), which would indicate that the additional hydrogen bonding interactions between
the cap and HDAC6 pocket are not strong enough to maintain nanomolar potency when
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the critical hydroxamate-Zn coordination is absent.
Table 1. In vitro HDAC profiles of 6a-c and NextA a
R2
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
OH
N
H
H
N
N
O
R1
Compound
R1
Suprastat (6a)
CH NH
OH
6b
6c b
H
NextA
2
2
CH
2
NH
2
H
H
R2
H
OH
Isoform
HDAC6
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC7
HDAC8
HDAC9
HDAC11
IC50, nM
SI c
1
IC50, nM
SI
1
IC50, nM
SI
1
IC50, nM
SI
1
0.4 ± 0.0
117 ± 10
176 ± 21
352 ± 2
0.9 ± 0.7
80 ± 45
0.5 ± 0.4
148 ± 9
1.6 ± 0.4
151 ± 20
293
440
880
89
296
536
1,160
94
281 ± 28
312
492
268 ± 16
581 ± 18
276 ± 96
173
887
443 ± 108
23,100 ± 226
10,100 ± 162
9,000 ± 1,110
614 ± 58
1,420 ± 145
8,250 ± 1,350 20,600
25,700 14,000 ± 1,380 27,900 14,800 ± 1,700 9,250
3,420 ± 373
1,470 ± 56
498 ± 58
8,550
3,680
1,250
11,200
10,000
682
9,130 ± 119
1,930 ± 70
478 ± 97
18,300
3,870
956
6,620 ± 2,660
2,430 ± 300
988 ± 264
4,140
1,520
618
6,270 ± 177
>50,000
15,700 17,300 ± 4,990 19,200 29,700 ± 4,190
>50,000 >50,000
59,50
-
2,000 ± 770
1,250
-
-
10,600 ± 2,200 6,630
a
IC50 values are the mean of two experiments ± SEM calculated by non-linear
b
regression analysis from experimental v /v values for each HDAC isoform. 6c was
i
0
c
originally published as compound 7b in Ref 36. SI: HDAC6 selectivity index over
other HDAC isoforms.
In vitro ADME profiling of 6a-c. As a part of the initial ADME profiling, we
determined the stability of studied compounds in PBS, simulated gastric fluid (SGF),
human plasma, and rat liver microsomes, as well as protein binding in human plasma
(
Table 2). Overall, the stability of Suprastat (6a) is excellent, ranging from >24 h in
PBS to a half-life (t ) of 173 min in rat liver microsomes. In line with predicted
1
/2
physicochemical characteristics, plasma binding of Suprastat is much lower (2% of
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plasma protein-bound fraction), which is beneficial for elevating free drug
concentration in vivo, compared to the more hydrophobic parent compound NextA
(89%). In comparison, plasma-bound fractions of singly modified analogs 6b and 6c
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7
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both are approximately 50%.
Table 2. Ligand efficiency and in vitro ADME profiling of 6a-c and NextA
Compound
clog Po/w a
LE b
LipEc
PBS stability (t1/2, h) d
SGF stability (t1/2, h) d
Human plasma stability (t1/2, h) d
Rat liver microsomes (t1/2, min) e
Human plasma binding (%) f
Suprastat (6a)
1.23
6b
6c
NextA
2.61
2.21
0.47
6.59
>24
1.82
0.50
6.98
>24
0.47
0.49
7.57
6.19
>24
>24
>5
>5
>5
>5
>5
>5
>5
>5
177 ± 6
1.9 ± 3.5
173 ± 71
47 ± 2.0
198 ± 32
48 ± 0.9
423 ± 104
89 ± 0.7
a
41
b
clog Po/w values were calculated by SwissADME (http://www.swissadme.ch/). LE:
c
ligand efficiency = 1.4 × pIC /number of heavy atoms. LipE: lipophilic ligand
5
0
d
efficiency = pIC − clog P . Data were obtained from two independent experiments
5
0
o/w
run in triplicates. e Data are presented as the mean ± standard error from two
f
independent experiments run in triplicates. Data are presented as the mean ± standard
error from two independent experiments run in duplicates.
In vitro characterization of 6a-c in melanoma cells. To assess the potency and
isoform selectivity of 6a-c in cells, we performed in vitro analysis using the WM164
human melanoma cell line. WM164 cells are mutant for BRAF V600E, a mutation that
4
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is frequently seen in melanoma patients. WM164 cells were treated with 6a-c and
NextA with a concentration range from 0.1 to 10 µM, respectively. Their abilities to
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increase the levels of acetylated α-tubulin (Ac-α-tubulin) were determined by
immunoblot analysis and compared side-by-side. Figures 3A-D show an increase in
Ac-α-tubulin with increasing concentrations of HDAC6is but with varying magnitudes
(Figure 3E). However, the increase in Ac-α-tubulin was also associated with a slight
increase in the levels of acetylated histone H3 (Ac-H3), albeit at higher concentrations
(Figure 3F). Treatment with Suprastat led to the highest Ac-α-tubulin levels ranging
from 0.1 to 10 µM and the most significant elevation at 10 µM compared to other
HDAC6is. At higher concentrations, we did observe a slight increase in the levels of
Ac-H3 with Suprastat, but it was much lower than with other HDAC6is. These data
thus demonstrate that Suprastat, containing both aminomethyl and hydroxylbutyl
groups, exhibits better HDAC6 potency and selectivity when tested in live cells. Based
on the concentration range for the α-tubulin/histone acetylation experiments, additional
cytotoxicity assays were performed in SM1 murine melanoma cells. The results shown
in the Supplementary Figure S1 reveal that NextA and 6b begin to induce cytotoxicity
at a concentration of 10 µM, while Suprastat and 6c were not cytotoxic upon to 25 µM.
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Figure 3. Suprastat is a highly selective HDAC6 inhibitor. WM164 human melanoma
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cells treated overnight with increasing concentrations ranging from 0.1 μM to 10 μM
for HDAC6 inhibitors Suprastat (6a, A), 6b (B), 6c (C), and NextA (D). Immunoblot
analysis was performed for Ac-α-tubulin and Ac-H3. Total α-tubulin and total H3 are
loading controls. (E) and (F) are densitometric analyses of the Ac-α-tubulin and Ac-H3
bands, respectively. The immunoblots were repeated at least twice to confirm accuracy,
and the best representation is shown.
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Functional characterization of Suprastat. Immune cells, such as macrophages, are a
major cellular component of the tumor microenvironment. Tumor-associated
macrophages are often tumor-promoting by secreting anti-inflammatory cytokines such
as TGFβ and IL-10. We previously established that HDAC6 forms a complex with
STAT3, and either pharmacological inhibition or shRNA mediated knockdown of
HDAC6 decreased STAT3 recruitment at the IL10 promoter region in antigen-
1
1
presenting cells. In line with these experiments, as shown in Figure 4A, the treatment
of mouse bone marrow-derived macrophages with 5 µM Suprastat resulted in decreased
expression of IL10 gene compared to vehicle-treated macrophages as determined by
mRNA quantification. IL10 gene expression is normalized to β-actin (ACTB) as the
reference gene. A dose-dependent increase in the levels of Ac-α-tubulin was observed
in RAW 264.7 macrophages (Supplementary Figure S2) when they were treated with
increasing concentrations (1, 5, and 10 µM) of Suprastat, indicating that Suprastat
affects macrophages and melanoma cells in a similar fashion. Furthermore, as shown
in Figure 4B, immunoblot analysis of lysates obtained from WM164 melanoma cells
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pre-treated with either Suprastat or NextA followed by exposure to IL-6 cytokine (30
ng/mL) for 20 min resulted in decreased Y705 phosphorylation of STAT3 compared to
IL-6 alone. This result indicates that Suprastat, similar to NextA, mediates
immunomodulatory effects by affecting HDAC6 interaction with the STAT3
transcription factor.
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Figure 4. Suprastat modulates immune pathways through interaction with STAT3. (A)
IL10 gene expression was determined by quantitative PCR in bone marrow-derived
macrophages exposed to interferon-gamma (20 ng/mL) and LPS (100 ng/mL). (B)
WM164 murine melanoma cells were pre-treated with 5 µM of Suprastat or NextA
followed by treatment with IL-6 (30 ng/mL) for 20 min.
In vivo combination study with immunotherapy. Immunotherapy is emerging as a
primary treatment modality for solid tumors; however, patients will often develop
resistance, and currently, there is a need for combination therapies to increase the
4
5
effectiveness of immunotherapy while overcoming resistance. Using the syngeneic
SM1 murine melanoma model, we previously demonstrated that pre-treatment with
NextA significantly decreased tumor size in C57BL/6 mice. These mice have an active
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immune system, which enables us to test immune checkpoint inhibitors such as anti-
PD1 therapy. The combination of NextA and anti-PD1 treatment resulted in substantial
control of tumor growth compared to single-arm therapies, suggesting that HDAC6
inhibition plays a significant role in enhancing anti-tumor immunity.13 Following a
similar approach, C57BL/6 mice harboring SM1 melanoma tumors were administered
25 mg/kg of Suprastat intraperitoneally (IP) before starting the anti-PD1 immune
checkpoint blockade therapy (15 mg/kg, IP). The pre-treatment modality was
performed to pre-condition the tumor microenvironment (TME) in a manner conducive
to eliciting an anti-tumor immune response. As shown in Figure 5A, compared to the
control (PBS) group, single-arm therapies with Suprastat similarly reduced the tumor
burden as indicated by the tumor volume. However, we did not see a significant
difference between Suprastat and anti-PD1 therapy. On the contrary, a combination of
Suprastat and anti-PD1 therapy showed a substantial decrease in tumor burden
compared to control and single therapy groups, suggesting that pre-treatment with
Suprastat enhances the anti-tumor immune response resulting from the anti-PD1
therapy. Figure 5B shows the tumor growth of each mouse in the respective treatment
groups. It was noted that the Suprastat and anti-PD1 therapy combined group exhibited
enhanced inhibitory effects on the tumor growth before Day 14 compared to other
groups. On the other hand, the combination of NextA and anti-PD1 therapy started to
exhibit distinct antitumor effects after Day 20 relative to single therapy groups in our
previous studies,13 suggesting that Suprastat is capable of promoting the
immunotherapy at an earlier stage.
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Figure 5. The combination of Suprastat with anti-PD1 immunotherapy significantly
decreases tumor burden in the SM1 murine melanoma model. (A) Cumulative tumor
growth rates in C57BL/6 mice (n = 10) after treatment with Suprastat or anti-PD1
therapy or a combination of both. Tumor growth rates of treatment groups were
compared to control groups treated with PBS. (B) Tumor growth rates of individual
mice in respective treatment groups.
Immunomodulatory properties of Suprastat. To understand the immunomodulatory
properties of Suprastat, we performed a comprehensive immune cell phenotyping by
flow cytometry. The number of F4/80+ CD80+ H2+ anti-tumor M1 macrophages as a
percentage of CD45+ cells did not significantly change in all of the treatment groups
(Figure 6A). However, Suprastat significantly decreased F4/80+ CD206+ pro-tumor
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M2 macrophages (Figure 6B), thus shifting the balance towards an anti-tumor immune
response as indicated by the considerably higher M1/M2 ratio (Figure 6C) in the
Suprastat and combination groups relative to control and anti-PD1 groups. Interestingly,
the enhanced M1/M2 ratio in the combination group was not observed in our prior work
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with NextA. Analysis of lymphoid cells shows a significant increase in CD8+ effector
T-cells and effector memory (EM) cells in all of the treated groups compared to the
control group (Figure 6D) in which the measured fold-increases were more significant
in all the groups than prior studies performed with NextA. However, we only observed
an increase in the percentage of CD8+ central memory (CM) cells with the Suprastat
treated group, suggesting that it can enhance the effector memory function of CD8 T-
cells for prolonged anti-tumor immune responses. Analysis of CD4+ T-cells did not
show any significant changes in central memory (CM) or effector memory (EM)
functions (Figure 6E). We also did not see a substantial change in immunosuppressive
T-regs (Figure 6F). Further analysis of natural killer (NK) cells demonstrated an
increase in the anti-PD1 group and combination group but did have a positive trend in
the Suprastat group (Figure 6G), which was not observed in previous combination
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studies using NextA. NK T-cells were significantly decreased in all treatment groups
compared to the control group (Figure 6H), which may be speculated to a significant
increase in CD8+ effector T-cells. Overall, the immune cell phenotyping indicates that
Suprastat has improved immunomodulatory properties by decreasing pro-tumoral M2
macrophages and increasing the infiltration of anti-tumor CD8+ effector T-cells and
memory cells relative to parent compound NextA. These factors are, in turn, likely
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responsible for the enhanced immunomodulatory effects observed in vivo and the
improved anti-tumor immune response in combination with anti-PD1 therapy.
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Figure 6. Suprastat has immunomodulatory properties in the tumor microenvironment.
Macrophages (A-C), T-cells (D-F), and NK (G and H) cells were stained with cell
surface markers and analyzed by multi-color flow cytometry.
X-ray crystallography. In vitro and in vivo characterization demonstrates that
Suprastat is a highly selective HDAC6 inhibitor with significant antitumor effects
through its immunomodulatory properties. To corroborate our in-silico models
experimentally, we solved the crystal structure of the zHDAC6/Suprastat complex
(PDB code 6TCY) to the resolution limit of 1.60 Å. The complex crystallized in the
P12 1 monoclinic space group with two monomers in the asymmetric unit. There are
1
no major conformational differences between the two monomers, as documented by the
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RMSD of 0.12 Å for 314 corresponding Cα atoms. Interestingly though, the inhibitor
can be modeled in two different conformations for each monomer in the asymmetric
unit (Figure 7A and 7B). In the first and more populated inhibitor conformation,
monodentate coordination is observed for the zinc atom with the hydroxamate N–O−
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group with a Zn --O distance of 2.24 Å. It is interesting to note that typical Zn --O
interatomic distances for monodentate HDAC6 complexes reported previously are
shorter, falling into the range of 1.8 – 2.0 Å.23-25, 46 At the same time, however, the Zn2+-
−
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O interatomic distance in the NextA complex is 2.2 Å that is virtually identical to
the distance reported here. Clearly, the capping group of NextA (as well as its Suprastat
derivative) might impose steric constraints on inhibitor positioning that leads to the
somewhat atypical Zn2+ coordination. In the less populated conformation, the
hydroxamate moiety coordinates with the active-site zinc ion in a bidentate fashion with
−
interatomic distances of 2.25 and 2.34 Å for the hydroxamate N–O and C=O groups,
respectively.
The phenyl rings of the linker fully overlap between the two conformers and are located
in the internal tunnel delineated by the side chains of F583, F643, and H614 (Figure
7
C). As for the capping group, the phenyl ring packs against a hydrophobic patch
defined by the side chains of H463, P464, F583, and L712 of the L1-loop pocket (Figure
7D). As predicted by docking simulations, the positively charged aminomethyl group
(pKa = 9.46, calculated by MarvinSketch Version 20.11) forms a salt bridge as well as
a water-mediated hydrogen bond (3.0 Å) with the carboxylate group of D460 (Figure
7
E). On the other hand, contrary to predictions, the hydroxybutyl group extends away
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from the protein, and its distal hydroxyl group thus does not engage any residue of the
HDAC6 rim directly but forms a solvent-mediated hydrogen bond with the side chain
of N530 (Figure 7E). A 1.99 Å-resolution crystal structure of the zHDAC6/NextA
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complex was reported by the Matthias group in 2017 (PDB code 5G0I). Superposition
of the two complexes reveals virtually identical positions of the capping groups as well
as surrounding HDAC6 residues, suggesting that our derivatization strategy did not
elicit any (additional) conformational strain on the binding pose of the inhibitor (Figure
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F).
Figure 7. Crystal structure of the zHDAC6/Suprastat complex. (A) Both monodentate
2+
(
left) and bidentate (right) coordination of the active-site Zn ion is observed in the
complex. (B) The omitted Fo-Fc difference electron density map for Suprastat is shown
green mesh) for the more prevalent monodentate binding mode (contoured at the 2σ
(
level). (C, D) Non-polar interactions between the phenylhydroxamate function of
Suprastat and residues delineating the internal tunnel (C); and the capping group and
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the hydrophobic patch of the L1 loop (red surface representation; D). (E) Newly
introduced polar/ionic contacts between the aminomethyl and hydroxylbutyl groups of
Suprastat and D460 and N530, respectively (black dashed lines). (F) Superposition of
binding modes of NextA (yellow, PDB code 5G0I) and Suprastat (cyan) documenting
minimal structural differences between the two structures. Both structures were
superposed on corresponding 320 Cα atoms with an rmsd of 0.192 Å. Inhibitors are
shown in stick representation, carbon atoms are colored cyan (Suprastat) or yellow
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(NextA), oxygens are red, and nitrogens are blue. The catalytic zinc ion and water
molecules are depicted as purple and red spheres, respectively.
Molecular dynamics simulations. X-ray crystallography is invaluable for the
structure-assisted design of inhibitors yet, and crystal structures typically offer only a
static snapshot of a single inhibitor binding pose disregarding the flexibility of
inhibitors’ functional moieties and protein residues. Moreover, the protein/inhibitor
interaction pattern can also be influenced by crystallographic contacts. In solution, an
inhibitor can interact only either with water molecules or the target protein. In crystals
(due to crystal packing), functional groups (that stick outside HDAC6 pocket) can also
interact “non-specifically” with neighboring molecules from the crystal. Consequently,
the interaction pattern can be influenced by such contact and differ from the solution –
thus, one does simulations to model contacts in solution. To explore the flexibility and
ensemble of interactions between the Suprastat capping group and HDAC6, we carried
out molecular dynamics (MD) simulations using the GROMACS software (2019). MD
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simulation results (Figures 8A-C) indicate the ability of the hydroxybutyl chain to
engage in two hydrogen-bond interactions with N530 (Figure 8A) and S531 (Figure
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C) at the rim of the catalytic pocket. At the same time, Figure 8B shows the transition
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state between each interaction. The direct frequency of contact (Figure 8D) determined
for amino acids within the vicinity of the hydroxybutyl chain and the aminomethyl
group indicates that the interactions with N530 and S531 repeatedly occurred during
the entire simulation length (100 ns) (Supplementary Figures S3 and Video S1). On the
contrary, direct contact between the ligand and D460 was not observed. There is an
indirect interaction between the aminomethyl group and D460 mediated by two water
molecules. Unlike both bidentate and monodentate hydroxamate-Zn2+ coordination
modes were observed in the crystal complex, Suprastat mainly exhibited bidentate
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coordination mode with Zn during the entire MD simulation process (Figures 2A-C
and Supplementary Video S1). Overall, these findings suggest that the hydroxybutyl
chain can directly engage in hydrogen bonding interactions with the side chains of N530
and S531, which were not observed in the zHDAC6/Suprastat crystal complex. It
should be noted that in the case of the monoclinic crystal form reported here, the
capping group of inhibitor comes to a short distance from a symmetry-related molecule,
and both added polar functional groups engage with a symmetry-related HDAC6
molecule via solvent-mediated interactions. The observed crystallographic contacts
may thus be responsible for differences in interaction patterns observed for the
zHDAC6/Suprastat crystal complex as compared to MD simulations. Given the results
from crystallographic studies and MD simulation, we conclude that Suprastat has a high
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ability to interact with the residues D460, N530, and S531 that play crucial roles in
regulating the tubulin deacetylation function of HDAC6.22
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Figure 8. Representative poses of Suprastat bound to zHDAC6-CD2 catalytic pocket
after Molecular Dynamics (MD) simulations. (A) The flexible hydroxylbutyl chain
engages in H-bonding interaction with N530. (B) Transition state between each stable
conformer. (C) H-bonding interaction between Suprastat and S531. (A-C) The
aminomethyl group establishes a consistent water-mediated interaction with D460
during the MD simulation process. (D) Direct contact frequency between the
hydroxylbutyl chain and the aminomethyl group of Suprastat and the amino acids at the
rim of zHDAC6-CD2 along with the MD simulation. Carbon atoms are depicted as
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grey. Nitrogen atoms are depicted as blue. Oxygen atoms are depicted as red. The Zn2+
ion is represented as a grey sphere. Water molecules are represented as red spheres. The
protein is depicted in grey. Hydrogen-bonding interactions are depicted as green-
dashed lines. Distances are reported in angstroms (Å). Images were generated in the
PyMOL 2.1 software.
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CONCLUSIONS
HDAC6 is unique among the histone deacetylases in possessing two functional
catalytic domains and a C-terminal ubiquitin-binding domain.47, 48 HDAC6 is
predominantly cytosolic due to the presence of a nuclear export signal, and it thereby
regulates the acetylation status of numerous cytosolic proteins. Among the many effects
of HDAC6, it appears to possess immune-modulatory properties through regulation of
the expression of immunosuppressive molecules such as IL-10 and PD-L1, thus leading
to enhanced anti-tumor activity when combined with anti-PD1 immune checkpoint
blockade therapy. For these reasons, there is an increasing interest in the identification
of potent and highly selective HDAC6 inhibitors. Suprastat was rationally designed
based on the crystal structure of Nexturastat A in complex with zHDAC6, wherein it
was observed that the introduction of key functional groups would likely enhance
HDAC6 potency and selectivity over other HDAC isoforms by establishing new
hydrogen-bonding interactions with key residues in the HDAC6 pocket in addition to
providing hydrophobic interactions between the cap and the rim region. In comparison
with Nexturastat A and related analogs, Suprastat shows improved HDAC6 activity,
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