Anticancer ruthenium complexes with HDAC isoform selectivity

Article abstract of DOI:10.3390/molecules25102383

The histone deacetylase (HDAC) enzymes have emerged as an important class of molecular targets in cancer therapy, with five inhibitors in clinical use. Recently, it has been shown that a lack of selectivity between the 11 Zn-dependent HDAC isoforms may lead to unwanted side-effects. In this paper, we show that piano stool Ru complexes can act as HDAC inhibitors, and variation in the capping arene leads to differences in HDAC isoform selectivity.

Full text of DOI:10.3390/molecules25102383

molecules
Article
Anticancer Ruthenium Complexes with HDAC
Isoform Selectivity
Jasmine M. Cross 1, Tim R. Blower 2 , Alexander D. H. Kingdon 1 , Robert Pal 1,
David M. Picton 2 and James W. Walton 1,
*
1
Department of Chemistry, Durham University, Lower Mountjoy, South Road, Durham DH1 3LE, UK;
jmcross92@gmail.com (J.M.C.); aleckingdon@gmail.com (A.D.H.K.); robert.pal@durham.ac.uk (R.P.)
Department of Biosciences, Durham University, Stockton Road, Durham DH1 3LE, UK;
timothy.blower@durham.ac.uk (T.R.B.); david.m.picton@durham.ac.uk (D.M.P.)
Correspondence: james.walton@durham.ac.uk
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Kogularamanan Suntharalingam
Received: 20 April 2020; Accepted: 16 May 2020; Published: 21 May 2020
Abstract: The histone deacetylase (HDAC) enzymes have emerged as an important class of molecular
targets in cancer therapy, with five inhibitors in clinical use. Recently, it has been shown that a lack of
selectivity between the 11 Zn-dependent HDAC isoforms may lead to unwanted side-effects. In this
paper, we show that piano stool Ru complexes can act as HDAC inhibitors, and variation in the
capping arene leads to differences in HDAC isoform selectivity.
Keywords: histone deacetylase inhibitors; ruthenium in medicine; selective enzyme inhibition
1. Introduction
The histone deacetylase (HDAC) enzymes control the extent of acetylation of
within the histone core. Hypoacetylation leads to a charged histone core and a condensed chromatin
structure, resulting in the suppression of gene expression [ ]. HDACs are involved in the deacetylation
of p53, a transcription factor involved in tumour suppression, which leads to its degradation and
hence allows cancer cell progression [ ]. HDACs are also associated with other functions, including
angiogenesis, DNA damage repair and cell cycle control [ ]. Five HDAC inhibitors have been approved
for clinical use [ ]. The archetypal inhibitor is suberanilohydroxamic acid (SAHA, Figure 1A) [ ].
ε-lysine residues
1–3
4
5
6
7,8
It incorporates a hydrophobic chain (red) that terminates with a hydroxamic acid (blue), which binds
to a Zn(II) ion located at the bottom of a hydrophobic channel in the enzyme active site. A phenyl head
group (green) sits in the cavity entrance of the active site (Figure 1B) [
clinically approved drugs, is a pan-inhibitor, acting on all 11 known Zn-dependent HDAC isoforms.
However, it has recently been shown that pan-inhibition may lead to genotoxicity [10 11], and that
targeting specific HDAC isoforms could be a better approach to target cancer progression [12 14].
Hence, the ability to inhibit isoforms selectively is at the forefront of research in this area, with several
isoform-selective HDAC inhibitors in clinical trials [ ]. As shown in Figure 1B, key differences in HDAC
9]. SAHA, along with each of the
,
–
5
isoforms are present in the cavity entrance region, where the capping phenyl group of SAHA binds.
As such, variation in the inhibitor head group has the potential to lead to isoform-selective inhibitors.
Transition metal complexes have emerged as promising candidates for selective enzyme
inhibition [15,
16]; they have more complex structural geometry than simple sp2/sp3-centred organic
molecules, and their coordinated ligands can exchange with biological targets. Meggers has led the way
in this field, with a series of PIM-1 kinase inhibitors, in which an organic heterocycle in staurosporine
is replaced by a Ru complex, leading to an increase in selectivity towards PIM-1, a proto-oncogene that
is implicated in multiple human cancers [17]. Other examples of metal-based inhibitors of carbonic
anhydrase [18,19] and glutathione-S-transferase [20] have also been demonstrated.
Molecules 2020, 25, 2383; doi:10.3390/molecules25102383
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Figure 1. (A) HDAC pan inhibitor SAHA and Ru complexes 8a–f, described in this work. (B) Docking
models of SAHA with isoforms HDAC1 (upper) and HDAC6 (lower), showing the differences in active
site cavity entrances.
Metal-based HDAC inhibitors have been reported, in which either the hydroxamic acid group
acts as a ligand to the metal [21] or the phenyl capping group of SAHA is replaced by a metal
complex (e.g., ferrocene [22–24], square planar Pt(II) [25,26], octahedral Ru(II) [27], Re(I) [28] and
Ir(III) [29]). Examples of isoform-selectivity for metal-based HDAC inhibitors have appeared for
ferrocene [22,24,30] and Ir complexes [31].
We recently showed that replacing the phenyl head group in SAHA for Ru piano stool complexes
gives viable HDAC inhibitors [32]. We hypothesised that increasing the size of the capping arene
group of the Ru complex could lead to improved selectivity towards HDAC6, which is seen to have a
wider active site cavity entrance. In the work herein, we show that variation in the η
6-coordinated
arene of Ru piano stool complexes (Figure 1A) leads to modulation of isoform selectivity between
HDAC1 and HDAC6, and we use computational docking experiments to rationalise these differences.
We also show that this family of Ru complexes have potential as anticancer agents in vitro.
2. Results and Discussion
2.1. Synthesis and Characterisation
As previously described, complex
the dimer [(p-cymene)RuCl2]2 (Scheme 1A) [32]. To introduce structural variation in the capping
6-coordinated arene, aryl precursors benzylamine and 4-methylbenzylamine were reacted via Birch
reduction to give 1,4-cyclohexadienes and , respectively (Scheme 1B). Compounds and were
coupled with acyl chlorides to give amides 6a , which were reacted with RuCl3 xH2O to give the
corresponding Ru metal dimers 7a , which were
. Complexation with L1 afforded complexes 8a
1
was synthesised through the reaction of ligand L1 with
η
4
5
4
5
–
f
·
–
f
–f
purified by preparative reverse phase high performance liquid chromatography (HPLC). Formation
and purity of the complexes were confirmed using 1H NMR spectroscopy, mass spectrometry, analytical
HPLC and elemental analysis. The resulting complexes fall into two sets: 8a
capping group, and 8d , with a
6-tolyl capping group. Within each set, the amide group in the
capping ligand incorporates a methyl, t-butyl or phenyl group at position R2. The aqueous stability of
complex
was monitored by 1H-NMR. After 1 h in D2O, the Ru-Cl bond remained fully intact, and
after 96 h only around 10% hydrolysis was observed (Figure S2).
–c, with a η
6-phenyl
–
f
η
1

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Scheme 1. (A) Synthesis of complex 1 and (B) synthesis of complexes 8a–f.
2.2. Enzyme Inhibition Assays
To begin to understand whether variation in the Ru capping ligand gives rise to HDAC isoform
selectivity, we carried out fluorimetric assays of complexes 8a
, SAHA and L1, against HDAC1 (Class I)
–
f
and HDAC6 (Class IIb) isoforms at two inhibitor concentrations. These isoforms were chosen as they
provide contrasting binding topographies, and HDAC6 in particular is closely linked with cancer
metastasis (Figure 2).
Figure 2. HDAC1 and HDAC6 inhibition assays of Ru complexes
1 and 8a–f, and control compounds,
measured using commercially available assay kits. All assays carried out in triplicate. See Supplementary
Materials for details.
All new complexes 8a
HDAC6 activity to less than 25% at 1
showed very similar activity at a given concentration. Against HDAC6, SAHA and L1 showed high
inhibitory potency. Within the series of Ru complexes, those with the
6-tolyl capping group (8d
show improved inhibitory activity compared to complexes with the –c).
6-phenyl capping group (8a
–
f
demonstrated HDAC-inhibitory activity, reducing both HDAC1 and
µ
M inhibitor. Against HDAC1, SAHA, L1 and all complexes
η
–f)
η
The more bulky and more hydrophobic tolyl group likely provides more favourable interaction with
the catalytic channel rim of HDAC6, which is known to be wider and shallower than other HDAC
isoforms [33]. In a similar way, the selectivity of the known HDAC6-selective inhibitor tubastatin A
originates from its ability to interact with the wide HDAC6 opening [34]. Of the new Ru complexes,
8f, which incorporates a phenyl group at the R2 amide position, shows the highest activity against
HDAC6, potentially due to aromatic
aromatic amino acids found at the cavity entrance of HDAC6 (e.g., Phe199, Phe200, Figure S21).
For the candidates with the leading HDAC-inhibitory profiles, complexes and 8f, inhibitory
concentration (IC50) values were measured against HDAC1 and HDAC6 (Table 1). Selectivity factors
π
-stacking interactions between the R2 phenyl group and the
1

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were calculated as the ratio of the IC50 values for HDAC1 and HDAC6. Data for L1 and SAHA were
also collected for comparison. Under the assay conditions, SAHA was more active against HDAC6,
compared to HDAC1, with a selectivity factor of 2.5. Complexes 8f and L1 showed slightly reduced
activity compared to SAHA, with selectivity close to that of the control compound. Of all compounds
tested, complex
compared to the ligand L1 alone. For complex
but HDAC1 activity was reduced, leading to the observed selectivity. HDAC1 has a narrower entrance
to the catalytic channel, and the bulky p-cymene capping arene in complex may restrict access to the
1
displayed the largest selectivity factor of 7.5, a three-fold improvement in selectivity
1
, HDAC6 inhibition was very close to that of 8f and L1
,
1
channel. This result demonstrates how addition of the Ru metal complex can impart HDAC isoform
selectivity compared to the purely organic ligand.
Table 1. IC50 values measured for selected compounds against HDAC1 and HDAC6. Selectivity factor
defined as ratio of the two IC50 values. All assays carried out in triplicate. See Supplementary Materials
for details.
Compound
HDAC1 IC50 (nM)
HDAC6 IC50 (nM)
Selectivity Factor
1
8f
240 ± 30
80 ± 10
105 ± 15
30 ± 4
32 ± 1
35 ± 6
41 ± 2
12 ± 1
7.5
2.3
2.6
2.5
L1
SAHA
2.3. In Vitro Assays
To further investigate the biological activities of the complexes, studies were carried out into
the in vitro cytotoxicity, the pathway of cell uptake, and cell uptake quantification. Cell viability
assays were carried out in triplicate, using the MCF7 human breast adenocarcinoma cell line as a
representative cancer cell line, and effective concentration (EC50) values were calculated for complexes
1 and 8a–f, alongside control compounds L1 and SAHA (Table 2).
Table 2. In vitro EC50 cell anticancer activity and cellular uptake measured against the MCF7 human
breast adenocarcinoma cell line (96 h, using ChemoMetec Via1-Cassette assay). All assays carried out
in triplicate. See Supplementary Materials for details.
Compound
EC50 (µM)
Cellular Uptake (%)
Compound
EC50 (µM)
Cellular Uptake (%)
SAHA
1
8a
8b
8c
1.5 ± 0.2
1.5 ± 0.4
32 ± 2
-
8.0
0.5
18.9
13.8
8d
8e
8f
1.2 ± 0.3
5.1 ± 1.5
2.1 ± 0.2
3.0 ± 0.6
13.1
6.2
11.2
-
1.4 ± 0.2
1.7 ± 0.3
L1
The majority of the Ru complexes showed cytotoxicity equalling that of the clinically used inhibitor,
SAHA. Variation of the capping arene in the Ru complexes does not appear to give any definitive trend
in cytotoxicity. However, it can be noted that the EC50 value for L1 is double that of SAHA, whereas
the presence of the metal-arene in complexes
EC50 values closer to the SAHA control. Furthermore, complex
upon cell line. Against MCF7 cells, complex 1 and SAHA have EC50 values of approximately 1.5 µM.
Against H460 non-small lung carcinoma cells, complex
and SAHA returned EC50 values of 21 ± 6 µM
and 1.5 0.4 M, respectively [32]. The origin of the selectivity of complex is unclear, but the varying
1,
8b
,
8c and 8d slightly improves activity and provides
1
shows variable behaviour depending
1
±
µ
1
behaviour compared to SAHA could imply an alternative mechanism of cell uptake and/or mode of
action. Indeed, Ru complexes are known to exert antiproliferative activity via several different modes
of action [35–37].
Inductively coupled plasma mass spectrometry (ICP-MS) was employed to quantify intracellular
Ru and discern any relationship between toxicity and cellular accumulation (Table 2).

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The data show a clear trend between cellular uptake and toxicity, with the most potent Ru
complexes (8b 8c and 8d) showing the highest cellular uptake (18.9%, 13.8% and 13.1%, respectively).
,
Conversely, complex 8a with the lowest toxicity showed only 0.5% cellular uptake. It can be concluded
that variation in toxicity between the complexes likely arises from the variation in cell uptake, although
the reason for this variation is not clear.
◦
Finally, through a cell assay with complex
1
at 4 C [38
,39], it was determined that an active
transport mechanism of cell uptake is responsible for Ru complex ingress, as cells treated with
1
remained viable at this temperature, suggesting no Ru uptake (see Supplementary Materials for details).
2.4. Computational Docking Study
In an attempt to rationalise variation in HDAC-inhibitory activity, docking studies were carried
out for SAHA, L1
, 1, 8c and 8f against HDAC1 (hHDAC1, 4bkx) and HDAC6-CD2 (hHDAC6-CD2,
5edu, see Supplementary Materials for details). Comparative analysis of the outputs was conducted,
based on: (a) the docking ChemScore rankings (a prediction of total free energy change upon inhibitor
binding to protein); (b) the number of docking poses (out of the 10 generated) showing zinc-hydroxamic
acid coordination, and (c) the zinc-coordination mode (monodentate vs. bidentate). Previously solved
crystal structures typically show hydroxamic acid-based HDAC inhibitors with bidentate coordination
of the hydroxamic acid to zinc [33,40], with a few examples showing monodentate coordination with a
secondary bridging water molecule coordination [41].
From the 10 docking predictions made between SAHA and HDAC1, only 1/10 predicted
zinc-hydroxamic acid binding (monodentate binding, Figure 3A,B), while docking between SAHA
and HDAC6 showed 9/10 poses with Zn-hydroxamic acid binding, comprised of 2 monodentate and
7 bidentate (Figure 3C,D). ChemScore values of 17.1 and 23.7 were calculated against HDAC1 and
HDAC6, respectively. This follows the experiment results, which showed a preference of SAHA to
HDAC6 over HDAC1, validating the docking parameters.
Figure 3. Computational docking studies showing (
) active site cavity entrance surface view of SAHA with HDAC1; (
SAHA with HDAC6; ( ) active site cavity entrance surface view of SAHA with HDAC6; (
cavity entrance surface view of L1 with HDAC1; ( ) active site cavity entrance surface view of L1 with
HDAC6; ( (teal) overlapped with HDAC6; ( ) active site
) Zn(II) coordination view of L1 (gold) and
cavity entrance surface view of L1 and 1 overlapped with HDAC6.
A
) Zn(II) coordination view of SAHA with HDAC1;
) Zn(II) coordination view of
) active site
(
B
C
D
E
F
G
1
H
Ligand L1 also appeared to favour HDAC6 over HDAC1, with all 10 docking positions binding
to the zinc in the former, compared to 8/10 for the latter. The ChemScore was also 25.9 vs. 23.7 for
HDAC6 vs. HDAC1. The surface view of the docking shows that in HDAC1, the phenanthroline
moiety extends out of the cavity entrance (Figure 3E), whereas for HDAC6 the phenanthroline moiety
fits more comfortably in the wider cavity entrance (Figure 3F). Similarly, complex
ChemScore against HDAC6, relative to HDAC1. Overlapping the highest scoring docking for L1 and
shows very similar binding, with the Ru complex benefiting from additional interaction between
1 returned a higher
1

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the p-cymene capping arene and a hydrophobic cleft on the surface of HDAC6 (Figure 3G,H and
Figure S22).
Experimental data suggests that the η6-tolyl capped complexes show increased HDAC6 activity,
compared to the η
6-phenyl capped complexes. In docking studies (Figures S18–S20), the ChemScore
was higher for 8f than 8c (19.1 vs. 13.6) against HDAC6, which reflects the increase in binding affinity
for 8f. While docking studies are used with caution, this preliminary study does suggest that the wider
cavity entrance of HDAC6 is able to accommodation the larger L1 and Ru complexes better than the
narrower HDAC1 cavity entrance. This is reflected in the IC50 inhibition assay experimental data,
where HDAC6 inhibition is more efficient.
3. Conclusions
In conclusion, six novel Ru piano stool complexes have been synthesised as inhibitors of the
HDAC enzymes. The novel complexes show excellent in vitro anticancer activity, which correlates
with the extent of cell-uptake. All complexes are able to inhibit HDAC1 and HDAC6, and variation in
inhibitory potency arises from variation in the capping arene of the piano stool complex. Varying the
capping
η
6-arene from phenyl-derived (8a
–
c
) to tolyl-derived (8d
–
f
) leads to an increase in HDAC6
shows
inhibition. Comparing the HDAC activity and selectivity of the ligand L1 and the Ru complex
1
that, while addition of the transition metal complex does not increase overall potency, it does have a
beneficial effect on selectivity between the two isoforms. Preliminary docking studies provide some
evidence for this variation and will be used in future work as a guide for designing inhibitors with
increased potency and selectivity. Overall, this proof of concept study highlights the potential for
organometallic complexes to address the challenge of isoform-selective HDAC inhibitors.
Supplementary Materials: The following are available online: synthetic procedures; hydrolysis studies; details on
HPLC purification and analysis; enzyme inhibition assay; cell viability assays; cell uptake assays; computational
docking studies; NMR spectra.
Author Contributions: Project conception, design and management by J.W.W. Synthesis and characterisation of
compounds by J.M.C. Enzyme inhibition studies by J.M.C., T.R.B. and D.M.P. Cytotoxicity studies by J.M.C. and
R.P. Computational docking study by A.D.H.K. Manuscript written and edited by all authors. All authors have
read and agreed to the published version of the manuscript.
Funding: This work was partially funded through the Durham University Biophysical Sciences Institute.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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