A novel anti-cancer bifunctional platinum drug candidate with dual DNA binding and histone deacetylase inhibitory activity

Article abstract of DOI:10.1039/b916715c

The successful design and synthesis of a novel multifunctional platinum drug candidate with DNA binding, histone deacetylase inhibitory activity and enhanced selectivity for cancer cells are described. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b916715c

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A novel anti-cancer bifunctional platinum drug candidate with dual
DNA binding and histone deacetylase inhibitory activityw
Darren Griffith,a Maria P. Morganb and Celine J. Marmion*a
Received (in Cambridge, UK) 14th August 2009, Accepted 14th September 2009
First published as an Advance Article on the web 12th October 2009
DOI: 10.1039/b916715c
The successful design and synthesis of a novel multifunctional
platinum drug candidate with DNA binding, histone deacetylase
inhibitory activity and enhanced selectivity for cancer cells are
described.
A range of structurally diverse HDAC inhibitors (HDACi’s)
have been shown to cause cell cycle arrest, differentiation
and/or apoptosis of tumour cells7,8 and several of these are
now undergoing clinical trials.9 Suberoylanilide hydroxamic
acid (SAHA, vorinostat), Fig. 1, is the first FDA-approved
pan-HDACi to enter the clinic10 as a treatment for cutaneous
T-cell lymphoma. Crystal structures of human HDAC’s with
SAHA bound11–13 show the hydroxamic acid coordinated to
the active-site zinc (Zn) ion. Several HDACi’s have thus been
designed based on these data in which their structural
motif consists of a metal binding group, a linker domain
(that mimics the Ca functional group of lysine) that occupies
the enzyme’s narrow channel and a cap group which interacts
with residues on the enzyme surface.
Nearly 50% of all cancer therapies are platinum (Pt)-based,1
yet surprisingly to date only three Pt drugs have been
approved for world-wide clinical use, namely cisplatin,
carboplatin and oxaliplatin.1,2 The cytotoxicity of Pt drugs
is attributed to their ability to bind DNA nucleobases and
induce apoptosis.2 Despite their enormous success, their
widespread application and efficacy are hindered by toxic side
effects, their limited activity against many human cancers and
their susceptibility to acquired drug resistance.2 The need to
overcome these drawbacks has stimulated the search for new
molecular targets in addition to DNA which may present
unique opportunities for therapeutic exploitation. The recent
correlation between the inhibition of enzymes that regulate
chromatin structure/function and tumour growth suppression
has, for example, validated chromatin control as a promising
new molecular target in contemporary medical oncology.
Chromatin is a complex structure that plays a key role as an
epigenetic regulator of gene expression in eukaryotic cells. The
fundamental repeating unit of chromatin is the nucleosome
consisting of core histones around which DNA coils.3 Some
histone residues protrude the nucleosome and are subject to
numerous enzyme-catalysed post-translational modifications.4,5
Histone acetyltransferases (HAT’s) and histone deacetylases
(HDAC’s) are two such enzymes that work in harmony to
acetylate and deacetylate core histone lysine residues, respectively.
Histone acetylation leads to an open chromatin structure that
upregulates transcription whereas deacetylation leads to a
condensed structure and transcriptional repression.6 Inhibition
of one or other of these enzyme classes can therefore
dramatically affect chromatin structure and thus function.
HAT’s and HDAC’s have therefore emerged as novel
molecular targets for which inhibitors are sought that could
reprogram transcription and inhibit tumour cell growth and
progression.
We devised a strategy to derivatise SAHA in such a way so
as to facilitate its binding to Pt while not compromising its
HDAC inhibitory activity. We anticipated that the novel
bifunctional Pt–HDACi conjugate should bind DNA in much
the same way as classical Pt drugs but because of its additional
functionality it might be active against a broader spectrum of
human cancer cells and/or cells that have acquired resistance
to Pt-based regimes. An important property of some HDACi’s
such as SAHA is their selectivity for tumour cells over normal
cells.6,14 Because Pt drugs react indiscriminately in the body
giving rise to many of their drawbacks, we speculated that the
presence of the inhibitor, with its known affinity for tumour
cells, might also confer selectivity to the drug candidate
thereby reducing the non-selective toxicity of classical Pt drugs
as well as potentially offering an advantage over concurrent
administration of classical Pt drugs with HDACi’s.15 Despite
the prevalence of Pt-based therapeutics, there have been no Pt
or indeed metal drug candidates reported to date with dual
DNA binding and HDAC inhibitory properties. Herein we
describe the synthesis, characterisation and pharmacological
evaluation of a novel Pt drug candidate with such dual
functionality.
We derivatised SAHA by adding a malonic acid substituent
to its cap group or phenyl ring to give malSAHA (2),
Scheme 1. We anticipated that Pt would bind malSAHA
in an O,O0-bidentate mode in much the same way as
cyclobutane-1,1-dicarboxylate binds to Pt in carboplatin2
a Centre for Synthesis and Chemical Biology, Department of
Pharmaceutical & Medicinal Chemistry, Royal College of Surgeons
in Ireland (RCSI), 123 St. Stephen’s Green, Dublin 2, Ireland.
E-mail: cmarmion@rcsi.ie; Fax: +353 1 4022168;
Tel: +353 1 4022161
b Department of Molecular & Cellular Therapeutics, Royal College of
Surgeons in Ireland (RCSI), 123 St. Stephen’s Green, Dublin 2,
Ireland
w Electronic supplementary information (ESI) available: Experimental
procedures and pharmacological evaluation. See DOI: 10.1039/b916715c
Fig. 1 Structure of suberoylanilide hydroxamic acid (1).
Chem. Commun., 2009, 6735–6737 | 6735
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Scheme 1 Synthesis of malSAHA (2) and its Pt complex cis-[PtII(NH3)2(malSAHAHꢁ2)] (3).
but leaving the linker domain unchanged and the hydroxamic
acid moiety free to bind the HDAC active-site Zn2+ ion.
Although hydroxamic acids are known to be strong metal ion
chelators,16,17 we previously reported that they have a low
affinity for Pt.18–20 We were therefore confident that the
Pt would bind selectively to the malonato substituent.
We also believed it important that the two functional entities
of the Pt–HDACi conjugate, upon reaching the nucleus,
would separate to allow each to work independently of
the other. Carboplatin has been shown to undergo hydrolysis
via a classical ring opening process followed by hydration and
consequent displacement of cyclobutane-1,1-dicarboxylate, to
increasing amounts of cis-[PtII(NH3)2(malSAHAHꢁ2)] have
bound to negatively supercoiled (SC), closed circular
pUC19 plasmid DNA. The rate of migration of the SC band
decreases until it co-migrates with the open circular (OC)
relaxed band as seen in lane 9. This DNA mobility shift
confirms binding of the Pt complex 3 to nucleotides causing
unwinding of the DNA. Similar effects are shown for cisplatin,
Fig. 2, lanes 14–16.25
The ability of malSAHA and cis-[Pt(NH3)2(malSAHAHꢁ2)]
as well as known HDAC inhibitors (trichostatin A and
SAHA) to inhibit HDAC1 was investigated in triplicate using
Cayman’s HDAC1 Inhibitor Screening Assay Kit, Table 1.
This is a cell-free assay which utilises an acetylated lysine
substrate incubated with human recombinant HDAC1. The
IC50 value for malSAHA (142 ꢂ 29 nM) compares favourably
with that found for SAHA (83 ꢂ 23 nM) which suggests that
give DNA binding adducts such as [Pt(NH3)2(H2O)2]2+ 21,22
.
We envisaged that our Pt–malSAHA conjugate should act in
much the same way as carboplatin releasing the malSAHA
derivative thus free to inhibit HDAC’s and the resulting Pt
moiety free to bind DNA nucleobases.
The new ligand malSAHA (2) was synthesised according
to Scheme 1.w The synthetic protocol for generating
Pt–dicarboxylato complexes is well established.23 Reaction of
the disodium salt of malSAHA with cis-[PtII(NH3)2(H2O)2]2+
,
generated from the reaction of iodoplatin and silver nitrate,
gave cis-[PtII(NH3)2(malSAHAHꢁ2)] (3), Scheme 1, in good
yield (66%) and excellent purity.w cis-[Pt(NH3)2(malHꢁ2)]
(where mal is malonic acid) was also synthesised as a reference
standard for biological tests.w
A well established electrophoretic technique was used to
investigate the effect of cis-[PtII(NH3)2(malSAHAHꢁ2)] on
DNA supercoiling, where changes in DNA mobility are taken
as evidence of a direct metal–DNA interaction leading to
DNA unwinding.24 Fig. 2 shows an agarose gel in which
Fig. 2 Unwinding of closed circular supercoiled pUC19 plasmid
DNA by 3. The top bands correspond to the open circular (OC) form
of plasmid DNA and the bottom bands to closed, negatively
supercoiled (SC) plasmid DNA. pUC19 DNA (30 mM) was incubated
for 72 hours with 0, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM of 3
(lanes 2–13) and 0, 30 and 40 mM cisplatin (lanes 14–16). DNA ladder
(lanes 1 and 17).
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Table 1 IC50 values (nM) against HDAC1 at 200 mM
have developed cis-[PtII(NH3)2(malSAHAHꢁ2)], the first Pt
complex of its kind with dual DNA binding and HDAC
inhibitory activity. The structure of this lead compound
will be optimised to further enhance its efficacy and safety
profile with a view to generating a new class of cancer
chemotherapeutics beyond those currently in use.
IC50/nM
Trichostatin A
SAHA
MalSAHA
8 ꢂ 0.3
83.5 ꢂ 23.1
142 ꢂ 29.8
1143 ꢂ 83.2
cis-[PtII(NH3)2(malSAHAHꢁ2)]
This material is based upon works supported by the Science
Foundation Ireland under Grant No. [07/RFP/CHEF570].
We also gratefully acknowledge the Programme for Research
in Third Level Institutions (PRTLI), administered by the HEA
for funding. We also thank colleagues in EU COST D39 for
fruitful discussions.
the presence of the malonate substituent in malSAHA
does not adversely affect its HDAC1 inhibitory activity.
cis-[PtII(NH3)2(malSAHAHꢁ2)] exhibits HDAC1 inhibitory
activity at low micromolar concentrations, despite the
fact that we would not expect the complex to significantly
hydrolyse in this cell-free assay.
Finally, the cytotoxicities of cis-[PtII(NH3)2(malSAHAHꢁ2)],
malSAHA, SAHA, cis-[Pt(NH3)2(malHꢁ2)] and cisplatin were
studied by means of a colorimetric cell proliferation microculture
assay (MTS assay) against cisplatin-sensitive and cisplatin-
resistant ovarian cancer cell lines A2780P and A2780cisR,
respectively, and the non-tumorigenic, normal human dermal
fibroblast cells, NHDF, Table 2. cis-[PtII(NH3)2(malSAHAHꢁ2)],
while having a similar cytotoxicity (IC50 9 ꢂ 3 mM) as
compared to cisplatin (IC50 2.9 ꢂ 0.1 mM) against A2780P,
differs greatly to cisplatin in that it is significantly less toxic
to NHDF’s (IC50 83 ꢂ 7.6 mM versus IC50 10 ꢂ 1.8 mM).
Whilst we did not observe any synergistic effect or improved
efficacy against the cisplatin-sensitive or cisplatin-resistant cell
lines, nevertheless we did observe marked selectivity for
tumour cells relative to the non-tumorigenic, normal cells.
cis-[PtII(NH3)2(malSAHAHꢁ2)] offers a distinct advantage
therefore over treatments involving cisplatin alone as the
non-toxic malSAHA may act as a Trojan horse delivering
the DNA binding agent PtII and the HDACi, malSAHA, to their
target sites, thereby reducing non-specific effects. We also observed
that cis-[PtII(NH3)2(malHꢁ2)] is nearly twofold less cytotoxic
compared to cis-[PtII(NH3)2(malSAHAHꢁ2)]. The presence of
the HDACi, malSAHA in cis-[PtII(NH3)2(malSAHAHꢁ2)], is
thus enhancing its cytotoxicity.
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A2780P
A2780cisR NHDF
Cisplatin
SAHA
MalSAHA
2.9 ꢂ 0.1
3.5 ꢂ 0.1
28.5 ꢂ 1.5 10 ꢂ 1.8
3.5 ꢂ 0.1
4.5 ꢂ 0.3
205 ꢂ 44.2 258 ꢂ 15.9 335 ꢂ 3.1
cis-[PtII(NH3)2(malSAHAHꢁ2)] 9 ꢂ 3.1
70 ꢂ 3.5
81 ꢂ 5.8
83 ꢂ 7.6
48 ꢂ 2.2
cis-[Pt(NH3)2(malHꢁ2)]
16 ꢂ 4.3
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Chem. Commun., 2009, 6735–6737 | 6737