Discovery of the First-in-Class Dual Histone Deacetylase-Proteasome Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.8b01487

Dual- or multitarget drugs have emerged as a promising alternative to combination therapies. Proteasome inhibitors (PIs) possess synergistic activity with histone deacetylase (HDAC) inhibitors due to the simultaneous blockage of the ubiquitin degradation and aggresome pathways. Here, we present the design, synthesis, binding modes, and anticancer properties of RTS-V5 as the first-in-class dual HDAC-proteasome ligand. The inhibition of both targets was confirmed by biochemical and cellular assays as well as X-ray crystal structures of the 20S proteasome and HDAC6 complexed with RTS-V5. Cytotoxicity assays with leukemia and multiple myeloma cell lines as well as therapy refractory primary patient-derived leukemia cells demonstrated that RTS-V5 possesses potent and selective anticancer activity. Our results will thus guide the structure-based optimization of dual HDAC-proteasome inhibitors for the treatment of hematological malignancies.

Full text of DOI:10.1021/acs.jmedchem.8b01487

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Article
Discovery of the first-in-class dual histone deacetylase-proteasome inhibitor
Sanil Bhatia, Viktoria Krieger, Michael Groll, Jeremy Osko, Nina Reßing, Heinz Ahlert,
Arndt Borkhardt, Thomas Kurz, David W. Christianson, Julia Hauer, and Finn K. Hansen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01487 • Publication Date (Web): 26 Oct 2018
Downloaded from http://pubs.acs.org on October 27, 2018
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Discovery of the first-in-class dual histone
deacetylase-proteasome inhibitor
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Sanil Bhatia,‡,1 Viktoria Krieger,‡,2 Michael Groll, Jeremy D. Osko, Nina Reßing,
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,§,1
Heinz Ahlert, Arndt Borkhardt, Thomas Kurz, David W. Christianson, Julia Hauer*
and Finn K. Hansen*,§,5
1Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical
Faculty, Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf,
Germany
2Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University
Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany
3Center for Integrated Protein Science at the Department Chemie, Lehrstuhl für
Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching,
Germany
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4Roy and Diana Vagelos Laboratories, Department of Chemistry, University of
Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, United States
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5Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Medical Faculty, Leipzig
University, Brüderstr. 34, 04103 Leipzig, Germany
ABSTRACT
Dual- or multi-target drugs have emerged as a promising alternative to combination
therapies. Proteasome inhibitors (PIs) possess synergistic activity with histone
deacetylase (HDAC) inhibitors due to the simultaneous blockage of the ubiquitin-
degradation and aggresome pathways. Here, we present the design, synthesis, binding
modes and anticancer properties of RTS-V5 as the first-in-class dual HDAC-proteasome
ligand. The inhibition of both targets was confirmed by biochemical and cellular assays
as well as X-ray crystal structures of the 20S proteasome and HDAC6 complexed with
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RTS-V5. Cytotoxicity assays with leukemia and multiple myeloma cell lines as well as
therapy-refractory primary patient-derived leukemia cells demonstrated that RTS-V5
possesses potent and selective anticancer activity. Our results will thus guide the
structure-based optimization of dual HDAC-proteasome inhibitors for the treatment of
hematological malignancies.
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INTRODUCTION
The approach “one drug multiple targets” or “multi-target drugs” is gaining major
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consideration in drug discovery and has been termed polypharmacology. Despite the
highly significant therapeutic relevance of combination therapies, potential advantages of
a targeted therapy based on a single drug acting through two or more independent modes
of action include (a) a more predictable pharmacokinetic profile, (b) increased patient
compliance, and (c) the simultaneous presence of the molecule in tissues where the
active principles are intended to work.1
cap
linker
O
S
zinc-binding group (ZBG)
N
N
N
NH
N
H
O
O
N
N
N
OH
N
N
O
H
O
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OH
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CUDC-907
Cl
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H N
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OH
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H
O
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tinostamustine
4SC-202
Figure 1. HDACi-based multi target drugs in clinical trials.
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Histone deacetylases (HDACs) are clinically validated cancer targets and four inhibitors
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thereof (HDACi) have been approved by the FDA for cancer therapy. HDACi are
characterized by a cap–linker–zinc-binding group pharmacophore model (Figure 1).3
Fortunately, the HDACi pharmacophore tolerates a variety of cap groups which allows
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scope for hybridization approaches. Consequently, the incorporation of a second
pharmacophore in the cap region has been used to engineer several HDACi-based multi-
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target drugs. Notably, the dual kinase-HDAC inhibitors CUDC-101 and CUDC-907, the
nitrogen mustard-HDACi hybrid tinostamustine, as well as the dual LSD1-HDAC inhibitor
4SC-202, are currently being investigated in clinical trials (Figure 1).4-5 In regards to
combination therapy, the best investigated synergism of HDACi has been identified with
proteasome inhibitors (PIs) leading to dual proteasome and aggresome blockage and
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apoptosis-induction due to the accumulation of misfolded proteins. However, to the best
of our knowledge, no dual HDAC-proteasome inhibitor has been reported so far.
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Herein, we present the design, synthesis, biological evaluation, and binding modes of
RTS-V5 as the first-in-class dual HDAC-proteasome inhibitor.
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RESULTS
Design and synthesis of RTS-V5. PIs can be divided into covalent and non-covalent
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binders. We decided to focus on non-covalent scaffolds to suppress several drawbacks
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such as excessive reactivity, lack of specificity, and/or stability. Moreover, highly reactive
electrophilic warheads might cause chemical incompatibilities with the typical HDACi zinc-
binding groups (ZBGs) such as hydroxamic acids, aminoanilides or thioles. The first non-
covalent acting PI was identified in the crystal structure of the yeast proteasome in
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complex with the natural product TMC-95A. In the following years, binding modes of
TMC-95A derivatives10 as well as non-covalent linear peptide mimetics have been
reported.11 In particular, a promising PI turned out to be compound ML16 (Figure 2)
obtained from an elaborate study published by Blackburn and colleagues.11a The high
affinity of ML16 and several analogs is primarily achieved by a P3-neopentyl-Asn residue
(Figure 2). The comparison of currently available crystal structures of the proteasome in
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complex with peptidic ligands12 revealed that this bulky residue indeed represents a
superb side chain to occupy the entire S3 specificity pocket of the chymotrypsin-like site
of the 20S core particle. We, therefore, decided to use ML16 as a starting point for the
design of dual HDAC-proteasome inhibitors. The S4 binding site does not resemble a
pocket-like structure and a careful inspection of a series of X-ray structures of ML16 and
its analogs indicated that the P4 residue is solvent exposed.7, 11a As a result, we aimed at
the design of a HDAC-proteasome hybrid inhibitor by incorporating the HDACi part at the
P4 position (Figure 2). The most obvious synergy between PIs and HDACi is derived from
the inhibition of HDAC6.6, 13 Thus, we chose an N-hydroxybenzamide scaffold as HDACi
part as this moiety provides HDAC6 selectivity.14
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S3
Hybridization
S3
NH
S1
Cl
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HN
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H
N
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O
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S1
O
O
O
H
S4
N
N
N
N
H
H
ML16
non-covalent PI
O
O
NH
HO
RTS-V5
HDACi-PI hybrid
S4
O
N
H
O
cap
linker
NH
zinc-binding group (ZBG)
HO
I
selective HDAC6 inhibitor
Figure 2. Design of RTS-V5 as the first-in-class dual HDAC-proteasome inhibitor.
Compound I (Figure 2) is a representative example of a selective HDAC6 inhibitor
based on an N-hydroxybenzamide group. Furthermore, the solvent-exposed 4-picolyl
group in the P2 position was replaced by a methyl group in order to reduce the molecular
weight of the hybrid compound. Our hybridization strategy thus yielded the prototype
HDAC-proteasome hybrid inhibitor RTS-V5 (Figure 2).
RTS-V5 was synthesized as outlined in Scheme 1. The readily available building blocks
1 and 2 were combined by HATU-mediated coupling to generate dipeptide 3. Next, the
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deprotection of 3, followed by introduction of 4-((benzyloxy)carbamoyl)benzoic acid via
another amide coupling reaction afforded the protected hydroxamic acid 5. Finally,
catalytic hydrogenolysis of 5 provided the target compound RTS-V5.
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Scheme 1. Synthesis of RTS-V5.a
NH
a)
NH
b)
NH
H
N
O
+
O
O
H N
2
O
O
H
H
O
Boc
OH
Boc
N
N
N
N
N
H N
2
N
H
H
H
H
O
O
O
1
2
3
4
NH
NH
c)
O
O
d)
O
O
O
O
H
H
N
N
N
N
N
N
H
H
H
H
H
H
N
O
N
O
BnO
HO
O
O
5
RTS-V5
a
Reagents and conditions: a) HATU, DIPEA, DMF, rt, 16 h. b) TFA, CH Cl , rt, 4 h. c) 4-
2
2
((Benzyloxy)carbamoyl)benzoic acid, HATU, DIPEA, DMF, rt, 24 h. d) Pd/C, H , rt, 4 h.
2
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RTS-V5 inhibits histone deacetylase and proteasomal activity. RTS-V5 was evaluated
for its ability to inhibit both histone deacetylase and proteasomal activity. First, we tested
the compound in a biochemical assay for activity against recombinant HDAC6. The
screening demonstrated potent submicromolar activity with an IC50 value of 0.27 µM
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(Figure 3a). To assess the selectivity of RTS-V5 for HDAC6, it was further tested for
activity against all class I isoforms (HDACs 1, 2, 3, and 8, Figure 3a). Our analysis
revealed that RTS-V5 has low activity against HDACs 1, 2, and 3. However, HDAC8 was
blocked at submicromolar concentrations as well (HDAC8 IC : 0.53 µM) which can be
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Figure 3. Functional specificity of RTS-V5 against HDAC6 and the proteasome. a)
Inhibitory activities of compounds RTS-V5 against HDAC isoforms 1, 2, 3, 6, and 8. b),
HL-60, SEM, and SUP-B15r cells were exposed to bortezomib, vorinostat, ricolinostat,
and RTS-V5 at the indicated concentration for 24 h, after which lysates were
immunoblotted with anti-acetyl-α-tubulin, anti-acetyl-Histone H3, poly (ADP-ribose)
polymerase (PARP), and anti-glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH)
antibodies. c) HL-60, SEM, and SUP-B15r cells were treated for 2 h with bortezomib,
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vorinostat, ricolinostat and RTS-V5 at concentrations ranging from 4 nM-25 µM. The
proteasomal activity was measured after 2 h using the cell-based Proteasome-Glo
Chymotrypsin-Like assay by taking Suc-LLVY-aminoluciferin (Succinyl-leucine-leucine-
valine-tyrosine-aminoluciferin) as a substrate. The compounds were printed on a 384-
well plate using a randomization feature (n = 3).
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In the following, we aimed to evaluate the inhibition of RTS-V5 against HDAC6 in a
cellular environment. Therefore, we treated the acute myeloid leukemia (AML) cell line
HL-60 as well as the B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines
SEM and SUP-B15r (Tyrosine Kinase Inhibitor (TKI) - resistant)16 with RTS-V5, the
preferential HDAC6 inhibitor ricolinostat and the FDA-approved proteasome inhibitor
bortezomib for 24 h. Next, the cell lysates were immunoblotted with anti-acetyl-α-tubulin
and acetyl-Histone H3 antibodies (Figure 3b). Compared to bortezomib, the treatment
with RTS-V5 enhanced the expression of acetyl-α tubulin and acetyl-histone H3 in
accordance to ricolinostat. Furthermore, RTS-V5 upregulated the expression of cleaved
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PARP, a marker of apoptosis, corresponding to ricolinostat and bortezomib (Figure 3b).
The inhibition of proteasome activity by RTS-V5 was evaluated using a cell-based
chymotrypsin-like Glo assay (Promega) by taking bortezomib as a positive control and
ricolinostat (HDAC6i) or vorinostat (pan-HDACi) as a negative marker (Figure 3c). In all
selected leukemic cell lines (HL-60, SEM and SUP-B15r), RTS-V5 blocked the
chymotrypsin-like proteasome activity while vorinostat and ricolinostat were unable to
inhibit the protease. Furthermore, it was shown that RTS-V5 acts specifically on the
chymotrypsin-like activity, i.e. RTS-V5 was unable to inhibit the trypsin- and caspase-like
proteasome activities (Figure S1a, S1b, Supporting Information). Thus, these results
demonstrate that RTS-V5 is the first-in-class dual HDAC-proteasome inhibitor.
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Co-crystal structures of RTS-V5 in complex with HDAC6 and the 20S proteasome.
Encouraged by the functional specificity of RTS-V5 against HDAC6 and the proteasome,
we set out to elucidate its binding modes in the vastly differing targets. First, the crystal
structure of catalytic domain 2 (CD2) of Danio rerio (zebrafish) HDAC6 complexed with
RTS-V5 was determined at 1.90 Å resolution (Rfree = 0.190, PDB ID: 6CW8, Table S1,
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Supporting Information). The crystal structures of zebrafish and human CD2 enzymes are
identical,17a so zebrafish HDAC6 CD2 (henceforth simply "HDAC6") serves as a more
readily studied surrogate of the human enzyme. The crystal structure of the enzyme-
inhibitor complex depicts no major conformational changes between the inhibitor-bound
and unliganded states of the enzyme, and the root-mean-square (rms) deviation is 0.14
Å for 287 Cα atoms (unliganded HDAC6, PDB accession code 5EEM). Notably, there are
two independent and essentially identical monomers in this crystal form (rms deviation =
0.15 Å for 299 Cα atoms). Electron density for RTS-V5 is generally well defined in both
monomers (monomer A, Figure 4; monomer B, Figure S2, Supporting Information).
Enzyme-inhibitor interactions are quite similar in both monomers, except for alternative
interactions resulting from individual conformations of the benzyl-L-alanyl moiety in
monomers A and B, respectively (Figure S3, Supporting Information).
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Figure 4. Stereoview of the Polder omit map of RTS-V5 bound to monomer A of HDAC6
(contoured at 3.0 σ) (PDB ID 6CW8). Atoms are color-coded as follows: C = orange (RTS-
2+
V5) or light blue (protein), N = blue, O = red, Zn = gray sphere, solvent = red spheres.
Metal coordination and hydrogen bond interactions are indicated by solid and dashed
black lines, respectively. The Zn2+ coordination geometry is pentacoordinate square
pyramidal.
In both monomers, the hydroxamate moiety of RTS-V5 coordinates to the active site
2+
Zn ion in monodentate fashion, in a similar manner to that observed in complexes with
other bulky phenylhydroxamate inhibitors such as HPOB, HPB, and ACY-1083.17 This
binding mode is characterized by the coordination of the ionized hydroxamate hydroxyl
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group to Zn (average Zn ---O separation = 2.0 Å), while the hydroxamate carbonyl
2+
group accepts a hydrogen bond from a Zn -bound water molecule (average O---O
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separation = 2.8 Å). The Zn -bound N-O group also accepts a hydrogen bond from Y745
(
average O---O separation = 2.6 Å).
Beyond the Zn2+ coordination polyhedron, intermolecular interactions observed for
RTS-V5 in both monomers contribute to inhibitor affinity and selectivity. The aromatic ring
of the phenylhydroxamate is sandwiched between two fully conserved residues, F583
and F643. The para-substituted amide NH group forms a hydrogen bond with S531 on
the L2 loop (average N---O separation = 3.0 Å). Notably, S531 is unique to HDAC6 and
plays an important role in substrate binding.17a Thus, hydrogen bonds with S531
presumably contribute to HDAC6 inhibitor selectivity.
In monomer A, the carbonyl group of the neopentylamide moiety accepts a hydrogen
bond from H463 in the L1 loop with an O---N separation of 2.9 Å; in monomer B, the O--
-N separation is 3.3 Å, which is slightly too long for, but perhaps within experimental error
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of, a hydrogen bond. Interestingly, H463 is unique to vertebrate HDAC6 isozymes, so this
interaction may confer additional selectivity toward HDAC6.
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Figure 5. Yeast 20S proteasome in complex with RTS-V5. a) Cartoon representation of
the yeast 20S proteasome core particle (yCP) in complex with RTS-V5 (PDB ID: 6H39).
The decarboxylated ligand is presented as a sphere model, which is located at the
intersection of the β –β ′-rings. The molecule solely binds to the nonprimed substrate
binding channel of the chymotrypsin-like active site, which is composed of subunits β5
(
gold) and β6 (cyan), respectively. b) The 2F -F electron density map of the non-
O
C
covalent inhibitor is illustrated as blue mesh and contoured to 1σ. Hydrogen bonds
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forming the antiparallel β-sheet between ligand and protein main chain residues are
indicated by black dashed lines. RTS-V5 intensely interacts with the S1 and S3 sites,
whereas the P2-Ala side chain is solvent-exposed. Amino acid numbering is according to
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Löwe et al. and Groll et al. . c) Structural superposition of RTS-V5 with the covalently
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acting aldehyde inhibitor MG132 (PDB ID: 4NNN) depicts a uniform arrangement. The
hemiacetal bond is highlighted in gold.
Next, the crystal structure of the yeast 20S proteasome core particle (yCP) in complex
with RTS-V5 was determined to 2.5 Å resolution (Rfree = 0.217, PDB ID: 6H39, Table S2,
Supporting Information). Intriguingly, the 2F -F electron density map displays the entire
O
C
inhibitor molecule only bound to the chymotrypsin-like active site by adopting an
antiparallel β-sheet structure (Figure 5a, b). Importantly, the HDACi hydroxamic acid ZBG
of RTS-V5 is solvent exposed and thus, not in contact with protein residues. In agreement
with our predictions, the complex structure depicts that the ligand acts non-covalently on
the proteasome. Compared to standard inhibitors bound to the CP, such as the tripeptide
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aldehyde MG132, our study revealed that RTS-V5 is solely stabilized by Van der Waals
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interactions with its P1 benzene ring to Val31, Ala49 and predominantly Met45 of subunit
β5, while its P3-neopentyl-Asn-moiety forms elaborate interactions with yβ5-Ala49 as well
as Asp114, Val116 and Ser118 of yβ6 (Figure 5b, c). Notably, the electron density map
uncovered the presence of an N-morpholino-ethane-sulfonic acid molecule (MES) in
proximity to the inhibitor, which is derived from the crystallization buffer. Hereby, the
sulfonate moiety of MES interacts with β5Gly47NH and hence, occupies the oxyanion
hole, an area normally populated by the active residue of ligands such as i) the oxygen
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anion of the scissile peptide bond in its tetrahedral intermediate, or ii) functional groups
of covalently bound inhibitors.22 Taken together, the crystallographic insights at the
molecular resolution confirmed our structure-activity relationships demonstrating that the
non-covalent proteasome inhibitor RTS-V5 fulfils elaborate interactions with the distinct
specificity pockets of the chymotrypsin-like substrate binding channel, hereby generating
target specificity.
Specific cytotoxic activity of RTS-V5 against cancerous cells. In order to investigate the
anticancer properties of our dual HDAC-proteasome inhibitor, RTS-V5 was screened for
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cytotoxicity against a panel of leukemia and multiple myeloma cell lines using ricolinostat
as a positive control (Table 1). Hereby, RTS-V5 showed comparable or higher cytotoxicity
than ricolinostat with IC50 values in the single-digit micromolar to submicromolar
concentration range. The highest activity of RTS-V5 was observed against the BCP-ALL
cell line SEM (IC : 0.89 µM). Our dual inhibitor was also active against TKI-resistant
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SUP-B15r and KCL-22r cells with IC values of 1.83 and 2.58 µM, respectively (Table
50
1). Due to its encouraging activity against chemosensitive and chemoresistant BCP-ALL
cell lines, RTS-V5 was further tested for activity against primary BCP-ALL cells derived
from four therapy-refractory patients (Patient 1 and 2 from initial diagnosis and Patient 3
and 4 from the relapse cohort) revealing IC values ranging from 1.51 to 5.23 µM (Table
50
1).
Next, we evaluated the cell viability in peripheral blood derived mononuclear cells
(PBMCs) from healthy individuals. Strikingly, RTS-V5 showed only marginal toxicity
against PBMCs (IC50 >25 µM, Figure S4, Supporting Information). In contrast, the
reference compounds ricolinostat, vorinostat and bortezomib caused significant
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cytotoxicity against PBMCs with IC50 values in the single-digit micromolar (ricolinostat,
vorinostat) or even submicromolar concentration range (bortezomib) (Figure S4,
Supporting Information). These data emphasize that RTS-V5 possesses promising
anticancer properties against several leukemic and multiple myeloma cell lines as well as
patient-derived BCP-ALL cells. Intriguingly, RTS-V5 acts in an encouraging therapeutic
window.
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Table 1. Cytotoxicity of RTS-V5 and ricolinostat against selected leukemia and multiple
myeloma cell lines as well as patient-derived BCP-ALL cells.
Cell line
Characteristic
RTS-V5
Ricolinostat
IC50 [µM]
IC50 [µM]
HL60
AMLa
1.55 ± 0.02
0.89 ± 0.01
1.77 ± 0.02
3.14 ± 0.03
1.83 ± 0.03
2.58 ± 0.04
1.75 ± 0.32
2.04 ± 0.37
2.06 ± 0.16
1.84 ± 0.07
5.23 ± 0.13
1.51 ± 0.05
2.36 ± 0.07
1.61 ± 0.02
1.92 ± 0.07
3.75 ± 0.09
3.54 ± 0.02
3.38 ± 0.03
1.97 ± 0.12
3.52 ± 0.38
0.29 ± 0.01
0.58 ± 0.04
4.45 ± 0.14
0.54 ± 0.01
SEM
BCP-ALLb
BCP-ALLb
CMLc
BCP-ALLb,d
CMLc,d
SUP-B15
KCL-22
SUP-B15r
KCL-22r
RPMI-8226
U266
MMe
MMe
Patient 1
Patient 2
Patient 3
Patient 4
BCP-ALLb
BCP-ALLb
BCP-ALLb
BCP-ALLb
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Acute myeloid leukemia. B-cell precursor acute lymphoblastic leukemia. Chronic
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myeloid leukemia. Imatinib resistant. Multiple myeloma.
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Based on these promising results, the biological properties of RTS-V5 were analyzed
in more detail using the BCP-ALL cell line SEM. RTS-V5 significantly inhibited the
proliferation of SEM cells at its IC or 2x IC concentrations, comparable to ricolinostat
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(
Figure S5, Supporting Information). RTS-V5 induced apoptosis in SEM cells as
illustrated by Annexin V and PI staining with ~ 5-fold increase in the apoptotic
+
+
(Annexin PI ) cells upon 48 h treatment (Figure 6a). Comparable results were observed
in a caspase 3/7 enzyme-dependent apoptosis assay with an induction of ~ 6-fold of
apoptotic cells at its IC50 concentration (Figure 6b). SEM cells were dose-dependently
arrested in S phase and a reduction in G2/M phase was observed after exposure to RTS-
V5 for 48 h (Figure 6c). The exposure of RTS-V5 induced early differentiation of SEM
cells in the liquid medium marked by the expression of (CD14 and CD11b) myeloid
markers (Figure S6, Supporting Information) and moreover, 48 h exposure of RTS-V5 to
SEM cells significantly reduced their colony forming capacity (Figure 6d). In addition,
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exposure of RTS-V5 to SEM cells induces the heat shock response or HSR (marked by
the overexpression of Grp94, HSP70, HSP40, and HSP27 proteins), unfolded protein
response or UPR (marked by the overexpression of BIP, ATF4, ATF6, and pJNK proteins)
in response to combat the proteotoxic stress and autophagy (marked by the
overexpression of LC3B and p62 proteins) (Figure 6e).
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Figure 6. RTS-V5 as a potent inhibitor in a leukemic cell line. a) SEM cells were treated
with RTS-V5 and ricolinostat at IC or at 2x IC concentration for 48 h. Subsequently
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dual staining was performed with annexin V/PI and measured by FACS. Viable cells (-ve
for annexin V/PI) were analyzed if they are necrotic (+ve for PI) and are either in an early
(+ve for annexin V) or in a late (+ve for both annexin/PI) apoptotic stage. The bar graph
is depicting the percentage of living, apoptotic and necrotic cells after 48 h exposure to
RTS-V5 or ricolinostat. b) SEM cells were treated with RTS-V5 for 48 h followed by
determining the enzymatic activity of caspase 3/7 by applying a Glo assay (absorbance
at 405 nm) to record the induction of apoptosis. c) SEM cells were treated with RTS-V5
for 48 h, and after propidium iodide staining cell cycle analysis was carried out by FACS.
d) SEM cells were seeded in a semisolid methylcellulose-based medium after 48 h
treatment with RTS-V5 or controls. Next, the impact of RTS-V5 on the differentiation
ability of leukemic cells was evaluated. The bar-graphs depict the colonies counted after
14 days. e) SEM cells were treated with bortezomib, ricolinostat, and RTS-V5 at the
indicated concentration for 18 h, after which the expression of proteins involved in the
HSR, UPR and autophagy were analyzed by western blot analyses. The achieved values
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depicted in the Figure 5 are plotted as a bar graph. Columns depict the mean of 3
independent experiments (n = 3). Significance analyses of normally distributed data with
variance similar between groups used paired, two-tailed student’s t-test. * p < 0.05, ** p
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Aggresomes are inclusion bodies produced in response to inhibition of the ubiquitin-
proteasome machinery. HDAC6 together with the motor protein dynein is required to
recruit cytotoxic, ubiquitylated proteins to aggresomes. The effect of RTS-V5 on the
aggresome accumulation was studied using fluorescence microscopy and FACS upon
staining with an aggresome dye (Figure 7a, b). The well-known proteasome inhibitors
MG132 and bortezomib were used as positive controls whereas ricolinostat served as
negative control. RTS-V5 significantly blocked aggresome accumulation at its inhibitory
concentration as opposed to bortezomib and MG132, but in accordance with ricolinostat.
These results led us to conclude that RTS-V5 induces apoptosis and blocks
proliferation, cell cycle, colony formation, and aggresome accumulation in the SEM cell
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Journal of Medicinal Chemistry
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line. Furthermore, the exposure of RTS-V5 leads to the activation of HSR and UPR.
Hence, our findings together with the crystallographic and biochemical data demonstrate
that RTS-V5 eradicates cancer cells by dual blockage of the aggresome-proteasome
pathway.
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a)
b)
Figure 7. Effect of RTS-V5 on the aggresome accumulation. a) SEM cells were treated
with the respective compounds at their indicated concentration for 18 h. In the following,
the Enzo proteostat aggresome detection kit was used to stain the cells along with their
DNA. Pictures were taken with a 63X objective using fluorescence microscopy. Scale =
10 µm. b) Treated SEM cells were analyzed by FACS upon staining with aggresome dye
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to determine the aggresome propensity factor according to the relative mean
fluorescence intensity (MFI).
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DISCUSSION AND CONCLUSIONS
The ‘one-disease-one-drug’ paradigm has dominated drug development strategies for
23
decades. However, the so-called magic bullets, molecules that exhibit high selectivity
and potency for one target, are often not effective to treat multifactorial diseases such as
4a
cancer or neurological disorders. Consequently, combination therapy is a cornerstone
of cancer therapy: the combination of anti-cancer drugs enhances efficacy compared to
the mono-therapy approach because it modulates key pathways in an additive or even
synergistic manner.24 Bortezomib is often given in combination with the pan-HDACi
panobinostat. The combination of HDAC6i and proteasome inhibitors leads to increased
α-tubulin acetylation as well as to accumulation of misfolded proteins.25 Misfolded
proteins accumulate because both clearance routes, the proteasome and the aggresome
26
pathway, are blocked; in turn, this leads to apoptosis of the cell. Thus, the simultaneous
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Journal of Medicinal Chemistry
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inhibition of both pathways could be of high clinical importance to combat hematological
malignancies.
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A phase I/II trial conducted for patients with relapsed or refractory multiple myeloma
showed that therapy with ricolinostat as a single agent resulted in neither significant
toxicity nor clinical responses.27 However, combination therapy with the proteasome
inhibitor bortezomib and dexamethasone achieved a response rate of 37%.27 Similar
results were reported in a study using a combination therapy including the proteasome
inhibitor MG132 and vorinostat, which induced synergistic cytotoxicity in leukemia cells
by downregulating BCR-ABL1 expression and by inducing intracellular ROS levels.28
In recent years, multi-target drugs have emerged as a powerful alternative to
combination chemotherapy. Although several HDACi-based multi-target drugs have been
described before, it is surprising that no dual HDAC-proteasome inhibitors have been
reported to date. In this work, we have designed and synthesized RTS-V5 as a first-in-
class dual HDAC-proteasome inhibitor. We have shown that this compound inhibits both
HDAC6 and the chymotrypsin-like proteasome activity. RTS-V5 induces apoptosis, HSR,
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UPR and autophagy in the SEM cell line. Furthermore, it blocks cell cycle, colony
formation, and aggresome accumulation. It is an encouraging finding that RTS-V5
displayed potent anticancer activity against a panel of chemosensitive, chemoresistant
leukemic and multiple myeloma cell lines, as well as against therapy-refractory primary
patient-derived leukemia cells without imposing toxicity against PBMC cells from healthy
volunteers. In future studies the efficacy and toxicity of RTS-V5 or improved analogues
will be investigated in in vivo models in comparison to a combination treatment with a
HDAC6 inhibitor and a proteasome inhibitor (e.g. ricolinostat combined with bortezomib)
in order to further evaluate the therapeutic potential of this promising new class of multi-
target ligands.
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To the best of our knowledge, this is also the first report of a dual target binder with
accompanying co-crystal structures of complexes with both protein targets. The X-ray
structures confirmed several important features that might lead to fewer side effects. The
non-covalent and selective inhibition of chymotrypsin-like proteasome activity may
explain the selective toxicity profile of RTS-V5 compared to covalent proteasome
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