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An Innovative DNA-Targeted Metallo-Prodrug Strategy combining Histone Deacetylase Inhibition with Oxidative Stress Tadhg J.P. McGivern, Creina Slator, Andrew Kellett, and Celine J. Marmion Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00652 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 9, 2018
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Molecular Pharmaceutics
An Innovative DNA-Targeted Metallo-Prodrug Strategy combining Histone Deacetylase Inhibition with Oxidative Stress Tadhg J. P. McGiverna,b, Creina Slatorb, Andrew Kellettb* & Celine J. Marmiona* a
Centre for Synthesis and Chemical Biology, Department of Chemistry, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland. b School of Chemical Sciences and National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. ABSTRACT: Cancer remains a global health challenge. There is an urgent need to develop innovative therapeutics that can overcome the shortcomings of existing cancer therapies. DNA enzymes involved in nucleic acid compaction and organization are an attractive cancer drug target for therapeutic exploitation. In this work a family of Cu(II) prodrugs containing suberoylanilide hydroxamic acid (SAHA), a well-established histone deacetylase inhibitor (HDACi) and clinically approved cancer drug, and phenanthrene ligands as DNA intercalative components, have been rationally developed. The complexes, of general formula [Cu(SAHA1H)(N,N6-phenanthrene)]+, exhibit excellent DNA recognition with binding affinity of lead agents in the order of ~107 M(bp)-1. Biophysical studies involving nucleic acid polymers indicate intercalative binding at both adenine-thymine (A-T) and guaninecytosine (G-C) rich sequences but thermodynamically stable interactions are favoured in G-C tracts. The complexes mediate DNA damage by producing reactive oxygen species (ROS) with spin trapping experiments showing superoxide, the hydroxyl radical, and hydrogen peroxide play critical roles in strand scission. The agents were found to have promising anti-proliferative effects against a panel of epithelial cancers and, in two representative cell lines possessing mutated p53 (SK-OV-3 and DU145), enhanced cytotoxicity was observed. Significantly, mechanistic experiments with the most promising candidates revealed HDAC inhibition activity was achieved over a shorter timeframe as compared to clinical standards with DNA damage-response markers identifying upregulation of both DNA synthesis and nucleotide excision repair (NER) pathways. Finally, confocal imaging and gene expression analysis show this metallodrug class to exert cytotoxic activity predominantly through an apoptotic pathway.
Keywords: Histone Deacetylases (HDAC), Suberoylanilide Hydroxamic Acid (SAHA), DNA, Metallodrug, Cu(II), Reactive Oxygen Species (ROS), Nuclease agent. Introduction: In the last 50 years, our understanding of DNA molecular processes including nucleolar organization has aided the development of new, targeted therapeutics. Nucleolar organization is mediated by chromatin. The fundamental repeating unit of chromatin is the nucleosome, consisting of core histone proteins, around which DNA is coiled ca. 1.65 times1. Central to regulating chromatin structure are enzymes including histone acetyltansferases (HATs) and histone deacetylases (HDACs). These enzymes are responsible for the acetylation or deacetylation of core histone lysine residues, respectively, which protrude from the N-terminal tail of core histone proteins. The acetylated state is associated with a relaxed chromatin conformation and this results in gene transcription activation. Deacetylation, in contrast, leads to a condensed chromatin structure resulting in attenuated gene expression2. In the late 1990s the link between HDAC inhibition and a reduction in cancer cell progression was identified.3,4 A range of structurally diverse HDAC inhibitors (HDACi) have since emerged as possible therapeutic agents5,6. Suberoylanilide hydroxamic acid (SAHA; Vorinostat®) was the first compound of this class to enter the clinic, gaining FDA approval in 2006 for the treatment of advanced primary cutaneous T-cell lymphoma7. It is also in advanced stages of clinical trials both as a monotherapy and as a combination therapy for the treat-
ment of leukemia, glioblastoma and ovarian cancer to name but a few7,8. While the precise mechanism of action of SAHA has still to be fully elucidated, crystallographic studies reveal SAHA can bind directly to the Zn(II) ion in the HDAC8 active site through O,O’-hydroxamate chelation9. This binding interaction leads to inhibition of HDAC function resulting in the accumulation of acetylated histones which in turn can trigger a series of cellular events within cancerous cells including induction of apoptosis, autophagy, ROS production and cell cycle arrest10. SAHA is also considered as an ‘epigenetic regulator’ affecting the expression profile of a number of genes such as p21WAF1, TBP-2, gelsolin, metallothionein 1L, histone H2B, cyclin D1, ErbB2, thymidylate synthase and importin b11. SAHA is also well tolerated by patients12,13 and, as such, is of significant therapeutic interest given its targeting effects on transformed cells14–16. Previous work by Marmion et al. demonstrated that dual HDAC inhibition and DNA platination could be achieved by covalent modification of clinically used HDAC inhibitors including SAHA17–20. But since these conjugates require the introduction of a dicarboxylate coordinating group specific to platinum(II), we reasoned the hydroxamate moiety—which has known affinity for first row transition metals21—was an ideal site for introducing a DNA damaging copper(II) agent. Our interest in using Cu(II) stems from recent observations on the clinical potential of the Casiopeinas® (specifically Cas-IIIia ([Cu(4,4’-dimethyl-2,2’bipyridine)(glycinato)(H2O)](NO3))22,23 and related copper(II)
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phenanthroline systems examined in collaboration with the National Cancer Institute24,25. Additionally, Cu(II) complexes are known to have excellent DNA targeting properties26,27 with the incorporation of designer metal-binding intercalating groups such as pyrazino[2,3-f][1,10]phenanthroline (DPQ) and dipyrido[3,2-a:2′,3′-c]phenazine (DPPZ)] demonstrating high potential utility as DNA-directing metallodrug compon ents28. We sought to combine, therefore, the DNA binding and nuclease activity of a Cu(II)-1,10-phenanthroline (Cu-Phen) core with a known HDAC inhibitor such as SAHA. A major driving force in our design strategy was the potential synergy gained through combining HDAC inhibition and oxidative
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DNA damage; upon entry into the reducing environment of the tumor cell we hypothesized the Cu(II) ion would be reduced to Cu(I) with concomitant release of the HDACi. Since HDAC inhibition should also render DNA in an ‘open’ chromatin structure (Figure 1A), the Cu-Phen core may also have greater access to its nucleic acid target. Additionally, a major limitation of SAHA is the susceptibility of the hydroxamate moiety to metabolic degradation through hydrolysis and glucuronidation29,30, which negatively impacts its half-life and pharmacokinetic profile31. Introducing Cu(II) at this site may essentially protect the hydroxamate functionality, thus enhancing its therapeutic potential.
A
B
Figure 1 Metallodrug design and mechanistic overview Panel A: HATs and HDACs acetylate or deacetylate lysine residues of histone proteins respectively regulating chromatin to a ‘wound’ or ‘unwound’ state. We hypothesized that inhibition of HDACs by the SAHA ligand would lead to an open chromatin structure allowing binding of the [Cu(N,N’-phenanthrene)]+ oxidant; Panel B: Chemical structures of the [Cu(SAHA-1H)(N,N’-phenanthrene)]2+ complexes synthesized in this study.
In this contribution, we describe the synthesis and biological characterization of a family of Cu(II) prodrugs as novel antiproliferative agents. These prodrugs constitute a Cu(II) core coordinating (i) a phenanthrene-based DNA intercalating moiety such as Phen, 1,10-phenanthroline-5,6-dione (Phendio), 2,2ʹ-bipyridine (Bipy), DPQ and DPPZ and (ii) the HDACi ligand, SAHA (Figure 1B). Using this structureactivity relationship approach, a family of complexes was developed and a range of mainstream and cutting edge molecular techniques were employed to elucidate the biological potential and mechanism of action of these novel agents. The DNA binding and nucleotide binding preference of the Cu(II) complexes was assessed using biophysical experiments such as fluorescence spectroscopy, viscosity, and thermal melting. DNA damage effects were then observed using electrophoresis, the Comet assay, quantification of double strand breaks by γH2AX foci, and by the uptake of an alkyne-tagged DNA building block - 5-ethynyl-2′deoxyuridine (EdU). Additionally, in vitro analyses revealed the Cu(II) complexes exert their DNA damaging effects predominantly through a nucleotide excision repair (NER) associated pathway whilst having a significant effect on DNA synthesis. Further gene expression and confocal imaging analysis then showed the Cu(II) complexes to apply cytotoxic effects predominantly through the apoptotic pathway. Experimental:
Complex synthesis: Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (Arklow, Ireland) as reagent grade and used without further purification. SAHA was synthesized through a two step coupling method reported previously32. The first step involved coupling suberic acid with aniline, the isolated suberanilic acid was then coupled to hydroxylamine to form SAHA. Phendio, DPQ and DPPZ were prepared by previously reported methods.28 Briefly, oxidation of 1,10-Phenanthroline (Phen) in acidic conditions formed the Phendio intermediate. Schiff-base condensation of Phendio with ethylenediamine or 1,2-phenylenediamine afforded DPPZ and DPQ respectively. Infrared (IR) spectra were recorded on a PerkinElmer Spectrum 100 spectrometer equipped with a universal ATR accessory. Electrospray ionization mass spectrometry (ESIMS) experiments were carried out on an Advion Expression Compact Mass Spectrometer, ESI-MS samples were prepared (10
1.99 ± 0.26
>10
8.82 ± 1.13
Cu-SAHA-Bipy
>10
2.45 ± 0.14
>10
2.29 ± 0.18
>10
>10
Cu-SAHAPhen
4.54 ± 0.20
1.14 ± 0.10
2.64 ± 0.14
1.94 ± 0.20
>10
4.82 ± 0.32
Cu-SAHADPQ
2.64 ± 0.17
1.60 ± 0.20
1.06 ± 0.24
0.95 ± 0.08
>10
>10
Cu-SAHADPPZ
1.31 ± 0.27
0.74 ± 0.20
3.43 ± 0.31
3.51 ± 0.20
2.68 ± 0.26
4.89 ± 0.19
Cu-SAHAPhendio
2.29 ± 0.16
