Synthesis, chemical characterization, PARP inhibition, DNA binding and cellular uptake of novel ruthenium(II)-arene complexes bearing benzamide derivatives in human breast cancer cells
Graphical abstract
Designing the single molecule that modulates multiple and specific targets, is paradigmin antitumor drug discovery. Here, we describe the synthesis, characterization, and in vitro antitumor activity of ruthenium(II)-arene complexes conjugated with 3-aminobenzamide derivatives, affecting poly(ADP-ribose) polymerase 1 enzymatic activity in vitro and interacting with DNA in human breast cancer cells.
Introduction
Rapid increase in the number of cancer deaths worldwide makes drug development research highly important. Nowadays, it is well known that novel antitumor drugs developed to replace cisplatin need to outweigh severe adverse effects and accompanying resistance induced by cisplatin. [[1], [2], [3]]. Ruthenium-based compounds are one of the most promising metal-based anticancer drug candidates [4]. The synthetic chemistry of ruthenium complexes is very well established and gives access to a large variety of compounds [5]. Among all synthesized ruthenium complexes so far, two ruthenium(III) complexes, NAMI-A (imidazolium-trans-[tetrachloro-(dimethylsulfoxide)imidazole ruthenium(III)]), which acts on metastases of solid tumors [6], and KP1339 (sodium-trans-[tetrachlorobis(1H-indazole)ruthenium(III)]), which is effective against platinum-resistant tumors [7], completed phase II clinical trials [[8], [9], [10], [11], [12], [13]]. Organometallic ruthenium(II)-arene compounds with completely different metallodrug scaffold, in which three of the coordination sites are occupied by η6-coordinated arene that stabilizes Ru+2 oxidation state, are being under intensive development [14]. RAPTA-C ([Ru(η6-p-cymene)(pta)Cl2], pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane), RAED-C ([Ru(η6-p-cymene)(en)Cl][PF6], en = ethylenediamine), and RAPTA-T ([Ru(η6-toluene)(pta)Cl2]), showed to be effective in reducing the number and weight of lung metastases [[15], [16], [17], [18], [19]]. To date, a myriad of structurally different ruthenium(II)-arene complexes have been prepared, with some of them exhibiting notable anticancer activities in vitro [[20], [21], [22], [23], [24], [25], [26]]. Ruthenium(II) have similar ligand exchange kinetics to platinum(II), and the octahedral geometry of ruthenium complexes offers unique possibilities for binding to nucleic acids [27,28].
The numerous still ongoing studies of mechanisms of action of cisplatin and ruthenium-based drugs emphasize the existence of more variable cellular targets of these agents beyond nuclear DNA [[29], [30], [31], [32]]. The particular interest is in investigating enzyme inhibition by metal complexes [33]. In that aspect, Kilpin and Dyson proposed the classification of metal-based enzyme inhibitors to several groups [34], depending on whether the activity of metal complexes relies mostly on the chemical properties of metal centre, bioactive ligand, or was influenced by the combination of both.
Particularly interesting family of enzymes present in eukaryotes are poly(ADP-ribose) polymerases (PARPs), which are involved in various cellular processes, including DNA repair, chromatin remodeling, transcriptional regulation, and cell death [[35], [36], [37]]. PARP-1 is one of the most abundant chromatin-bound protein, accounting for >90% of total PARP activity [38]. It is considered to be a key player in DNA base excision repair (BER) and repair of single-strand DNA breaks (SSBs) in response to ionizing radiation, oxidative stress, or DNA-binding agents [39,40]. Structurally, PARP-1 is comprised of three distinct functional domains: (1) N-terminal DNA-binding domain containing three zinc fingers important for the PARP-1 binding to DNA breaks, (2) central automodification domain serving as an acceptor of adenosine diphosphate ribose (ADP-ribose) moieties, and (3) C-terminal catalytic domain forming active site of PARP-1 (“PARP signature”) that transfers ADP-ribose subunits from nicotinamide adenine dinucleotide (NAD+) to protein acceptors (Fig. 1) [41,42].
PARP-1 utilizes beta nicotinamide adenine dinucleotide (β-NAD+) as a substrate to covalently add poly(ADP-ribose) (PAR) chains onto itself and other nuclear acceptor proteins, in a process termed PARylation [43]. The role of PARP-1 in DNA damage response and cell death regulation prompted the development of potent small molecules named PARP inhibitors (PARPi) [39]. Generally, PARPi are designated as competitive inhibitors, since they impair the catalytic activity of PARP-1 by interacting with the NAD+ binding site [44]. Nicotinamide, benzamide, and 3-aminobenzamide (3-AB) were identified as the first generation of “classical” PARPi [45]. Despite the limited potency and specificity to be used in clinics, these compounds are significant for research purposes [46]. Structure-activity relationship analysis showed that potent PARPi should have an electron-rich aromatic or polyaromatic heterocyclic system, bearing a pharmacophore with cis-configurated carboxamide, imide, or a carbamoyl group [47]. To date, four PARPi of the third generation (olaparib, rucaparib, niraparib, and talazoparib) were approved by the United States Food and Drug Administration (U.S. FDA) (Fig. 2) [42,[48], [49], [50]], as single-agent therapy, for targeting breast cancer susceptibility genes (BRCA)-mutated breast, ovarian, prostate, and pancreatic cancers [51]. PARPi are the first clinically approved drugs designed to exploit synthetic lethality concept in tumors harboring mutations in BRCA1 or BRCA2 genes, responsible for the repair of double-strand DNA breaks (DSBs) by homologous recombination (HR) [52,53]. Synthetic lethality is a genetic concept based on the idea that defect in either one of two genes has little effect on the cell or organism, but a combination of defects in both genes results in cell death [54]. The cells with impaired HR pathway are dependent on alternative ways for DNA repair and survival, and thereby PARPi became promising therapy for BRCA-mutated cancers.
Although recent reports suggested that the application of PARPi could be extended to “BRCAness” tumors with defects in HR repair genes other than BRCA1 and BRCA2 [[55], [56], [57]], the acquired resistance caused by arising secondary mutations in HR repair genes that restore the function of HR repair pathway limits the clinical efficacy of PARPi as single agents [58]. Therefore, combination therapy is a reasonable approach to improve the utility of these inhibitors to serve as chemopotentiators of commonly used cytotoxic chemotherapeutics, such as cisplatin [[59], [60], [61], [62]]. Unfortunately, multicomponent drug cocktails can display adverse effects caused by complex pharmacokinetics and unpredictable drug-drug interactions. Hence, multi-targeting single drugs offer some advantages of pharmacokinetic simplicity and improved outcomes [35].
Growing tendency in the drug design area is to connect metals with pharmacophoric moieties of bioactive ligand, to achieve different spectra of biological activity and improve the properties of both constituents [29,32,63,64]. Reports up to date showed that different ruthenium(II)-arene complexes entered tumor cells efficiently and bound DNA [24,65]. Therefore, they may present appropriate scaffold to bring bioactive ligands with PARP-1 inhibitory potential more closely to their targets in the cell. Additionally, the study performed by Mendes et al has shown that compounds based on platinum, ruthenium, or gold may display high PARP-1 inhibitory activity, supporting the model whereby displacement of zinc from the zinc finger motif of PARP-1 by metal ions led to decreased PARP-1 activity [66].
In the present study, novel ruthenium(II)-arene complexes carrying 3-AB derivatives as ligands, were synthesized and examined for the inhibitory potential against the catalytic activity of PARP-1. Further, their growth inhibitory effects were investigated in a panel of human breast cancer cell lines, which were either BRCA1-mutant, triple-negative, or hormone-responsive. To reveal the mechanism of action and intracellular targets, we analyzed cell cycle progression, binding to DNA, cellular uptake, and distribution across cellular compartments of treated cells. The influence of the particular structural changes in the molecules on the different aspects of anticancer potential is discussed.
Section snippets
Material and methods
RuCl3·3H2O was purchased from Johnson Matthey (London, UK). A 2-amino-4-methylbenzamide (L1) and 3-amino-N-methylbenzamide (L2) were purchased from Sigma Aldrich. [Ru(η6-p-cymene)Cl2]2 was prepared according to a published procedure [67]. [Ru(η6-toluene)Cl2]2 was prepared according to a published procedure [68]. Solvents were obtained from different companies and used without further purification. Infrared spectra were recorded on Nicolet 6700 FTIR spectrometer, using the ATR technique. 1H and
Cell culture
MDA-MB-231 and MCF-7 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich Co), while HCC1937, MDA-MB-453, MDA-MB-361, and BEAS-2B cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 nutrient medium (Sigma-Aldrich Co). All media were supplemented with 10% fetal calf serum (FCS) (pH 7.2) (Sigma-Aldrich Co), 2 mM l-glutamine, 25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, Capricorn), and Penicillin-Streptomycin solution
Synthesis and characterization
Four new ruthenium(II)-arene complexes (C1-C4) were synthesized in the reaction of 3-aminobenzamide analogues, 2-amino-4-methylbenzamide (L1) and 3-amino-N-methylbenzamide (L2), to get potentially good PARP inhibitors (Scheme 1). Starting Ru(II) complexes were Ru(II) dimmers with toluene or p-cymene moiety. Synthesis of Ru(II)-p-cymene-based complexes with L1 and L2 has to be done in an oxygen-free atmosphere, as it was previously reported for similar complexes [65]. All syntheses were
Conclusions
In the present study, four new ruthenium(II)-arene complexes, C1-C4, carrying derivatives of 3-aminobenzamide, with anticipated multifunctional potential, were synthesized and tested for their anticancer activity in vitro. Complexes exhibited efficiency in inhibiting PARP-1 activity, at micromolar concentrations, in the following order: C2 > C4 > 3-AB>C1 > C3. Complexes bearing η6-p-cymene moiety (C2 and C4), demonstrated better PARP-1 inhibition than their starting complex C6 and the benchmark
Abbreviations
NAMI-A Imidazolium-trans-[tetrachloro(dimethylsulfoxide)imidazoleruthenium(III)]
KP1339 Sodium-trans-[tetrachlorobis(1H-indazole)ruthenium(III)]
RAPTA-C Ruthenium(II)-dichloro(1,3,5-triaza-7-phosphaadamantane)(η6-p-cymene)
RAED-C Ruthenium(II)chloro(η6-p-cymene)ethylenediamine-hexafluorophosphate
RAPTA-T Ruthenium(II)-dichloro(1,3,5-triaza-7-phosphaadamantane)(η6-toluene)
PARP Poly(ADP-ribose) polymerase
ADP Adenosine diphosphate
BER Base excision repair
SSBs Single-strand breaks
β-NAD+ Beta nicotinamide adenine
Acknowledgements
This work was financed by Ministry of Education, Science and Technological Development of the Republic of Serbia, contract numbers: 451-03-68/2020-14/200168 and 451-03-68/2020-14/200043.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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2021, Coordination Chemistry ReviewsCitation Excerpt :Notably, both complexes showed improved bioactivity over their free ligands which highlights the potential for synergistic targeting properties through transcription inhibition and DNA binding. Complexes N-30a and N-30b containing a 3-aminobenzamide-derived PARP inhibitor (N-ah) (Fig. 20) were assessed for inhibition of cell growth against five human breast cancer cell lines (Hcc1937, MDA-MB-231, MDA-MB-453, MDA-MB-361, MCF-7) as well as non-cancerous bronchial epithelium BEAS-2B cells [173]. Both complexes showed moderate anticancer activity across all cell lines without selectivity for cancer cells (Table S1).