Arsenoplatin-1 is a Dual Pharmacophore Anti-Cancer Agent

Abstract

Arsenoplatins are adducts of two chemically important anticancer drugs, cisplatin and arsenic trioxide, that have a Pt(II) bond to an As(III) hydroxide center. Screens of the NCI-60 human tumor cell lines reveal that arsenoplatin-1 (AP1), [Pt(μ-NHC(CH₃)O)₂ClAs(OH)₂], the first representative of this novel class of anti-cancer agents, displays a superior activity profile relative to the parent drugs As₂O₃ or cisplatin in majority of cancer cell lines tested. These activity profiles are important because the success of arsenic trioxide in blood cancers (such as APL) has not been seen in solid tumors due to the rapid clearance of arsenous acid from the body. To understand the biological chemistry of these compounds, we evaluated interactions of AP-1 with the two important classes of biomolecules – proteins and DNA. The first structural studies of AP-1 bound to model proteins reveal that platinum(II) binds the Nε of His in a manner that preserves the Pt-As bond. We find that AP-1 readily enters cells and binds to DNA with an intact Pt-As bond (Pt:As ratio of 1). At longer incubation times, however, the Pt:As ratio in DNA samples increases, suggesting that the Pt-As bond breaks and releases the As(OH)₂ moiety. We conclude that arsenoplatin-1 has the potential to deliver both Pt and As species to a variety of hematological and solid cancers.

Graphical Abstract

The serendipitous discovery of cisplatin more than 50 years ago¹ and its consequent approval in 1978 marked the beginning of the use of FDA-approved metal complexes as anti-cancer drugs. Cisplatin and its analogues continue to be widely used as single agents or especially in combination with other drugs. For instance, cisplatin is a component in more active clinical trials worldwide than any other anticancer agent². Unfortunately, intrinsic and acquired resistance due to mutations remain as major obstacles for the use of platinum drugs in treating cancer^3–5. Accordingly, many new types of platinum compounds have been designed to overcome platinum resistance: trans-Pt(II)-compounds⁶, polynuclear Pt(II)-compounds⁷, Pt(IV) prodrugs^8–9, Pt(IV)-prodrugs activated by visible light¹⁰, glucose-platinum conjugates,^11–12 as well as new drug delivery systems are being evaluated.^13–14 Cisplatin, carboplatin, oxaliplatin, and arsenic trioxide (As₂O₃)^15–17 are only non-radioactive metal/metalloid containing drugs approved by the FDA to treat cancer. These drugs are able to trigger apoptotic cell death; in the case of platinum compounds through direct platinum binding to nuclear DNA^18–19, and in the case of As₂O₃ most likely through the thiophilic As(III) binding to cysteine rich proteins and by its ability to displace zinc from zinc finger containing regulatory proteins.^20–22

The identification of a novel Pt-As bond in a nanoparticulate delivery system loaded with arsenic trioxide and cisplatin in our lab¹³ spurred the development of arsenoplatins (Chart 1).²³ These complexes can be synthesized directly from reaction of arsenic trioxide with cisplatin in a suitable solvent mixture. Arsenoplatins in their structure possess an arsenous acid moiety covalently linked to platinum(II) center with an unusual five coordinate As(III)-geometry. The presence of the coordinated As(III) distinguishes the reactivity of AP-1 from cisplatin reactivity. The substitution of Cl⁻ with small ligands such as SCN⁻ in an aqueous solution is rapid at room temperature due to a very strong trans effect of the arsenic atom.²³ We show here that substitution of Cl⁻ in AP-1 by DMSO does not occur in neat DMSO (Figure S1 and Table S1).

Chart 1.

AP-1 is stable in phosphate buffer saline, saline, DMSO, and methanol solutions for over 48 hours (Figures S1 and S2). ¹⁹⁵Pt NMR studies of AP-1 dissolved in DMSO²³ or methanol reveal that the Pt-Cl bond remains intact in both solvents (Figure S3 and Table S1). Unlike cisplatin, which is prone to solvolysis in DMSO solution at longer times²⁴ AP-1, oxaliplatin, and carboplatin are stable in neat DMSO²⁵.

Preliminary in vitro cytotoxicity evaluation suggested that arsenoplatins may have a distinct mode of action in comparison to cisplatin and As₂O₃, as well as the ability to overcome platinum resistance mechanisms.²³ To investigate the breadth of anti-cancer activity of this new class of anti-cancer agents, we assessed the cytotoxicity of a single 10 μM dose of AP-1, cisplatin, and As₂O₃ in the NCI-60 panel of human tumor cell lines.²⁷ For the NCI-60 screen, the compounds were dissolved in DMSO immediately before the assay. Stability studies of AP-1 in DMSO and PBS show no changes over 48 hours (see SI and Figures S1 and S2). The full activity profiles of AP-1, cisplatin, and As₂O₃ across 59 well characterized human cancer cell lines are presented in the NCI-60 form (i.e. one-dose mean graphs, Figures S4–S6). These graphs are a “fingerprint” of the activity of the investigated drugs. According to the NCI-60 screen, AP-1 showed superior activity to arsenic trioxide in all nine of the tumor types tested (Figure 1). When compared to cisplatin, AP1 showed greater activity in breast, leukemia, colon, and CNS cancer cell lines, and was comparable to cisplatin in ovarian and renal cancer cell lines.

Figure 1.

Summary of the NCI-60 human tumor cell lines screen. AP-1 shows superior activity compared to arsenic trioxide in all 9 indications tested; it also shows higher potency than cisplatin in breast, leukemia, colon, and CNS. Numbers in parentheses represent number of cell lines tested for each indication (Figures S4–S6).

Since arsenoplatin-1 was more potent than either cisplatin or arsenic trioxide in a majority of cancer cell lines evaluated, we tested whether there was a cooperative effect between platinum and arsenic pharmacophores. Synergy between arsenic trioxide and cisplatin has been reported in lung²⁸, oral squamous cell carcinoma²⁹, and ovarian cancer cell lines.³⁰ It has also been shown that arsenic trioxide increases apoptosis in OSSC cells²⁹ and enhances cellular and DNA bound platinum accumulation in ovarian cancer³¹. We first used the method of Chou³² to test for synergy between arsenic trioxide and cisplatin in a very aggressive triple negative breast MDA-MB-231 cancer cell line that is resistant to cisplatin.³³ Several cisplatin and arsenic trioxide ratios were examined (Figure 2 and Table S2). The results indicate significant synergy between two drugs at all ratios, with the cisplatin to arsenic trioxide ratio of 1:1 showing the optimal effects.

Figure 2.

Drug combination studies using the different arsenic trioxide: cisplatin ratios in the triple negative breast MDA-MB231 cancer cell line. The combination index (CI) values significantly less than 1 are found in all cases confirming strong synergy between the two drugs. The best results are obtained for arsenic trioxide to cisplatin ratio of 1:1.

Since arsenoplatin-1 in its structure possesses the Pt-As(OH)₂ moiety with the platinum to arsenic ration of 1:1, we asked if the Pt-As unit would remain intact upon DNA binding within the cell. MDA-MB-231 cancer cells were treated with 100 μM AP-1 or cisplatin for 4h and 8h. Nuclear DNA was isolated and the amount of platinum and arsenic in the DNA-adducts was measured using ICP-MS (Figure 3). After 8h exposure, we find similar Pt-DNA ratio for cisplatin and AP-1, even though AP-1 is significantly more cytotoxic (IC₅₀ = 9.5 ± 0.3 μM) compared to cisplatin (IC₅₀ = 22 ± 0.5 μM).²³ Thus, the greater cytotoxicity of AP-1 in this cancer cell line is not explained by the amount of Pt-bound to DNA. Intriguingly, the Pt-As ratio is close to 1 (0.97) after 4h; however, after 8h incubation we find significantly fewer moles of arsenic than platinum present in AP-1-DNA adducts (p < 0.0001 based on one-way-ANOVA with Bonferroni multiple comparisons). This suggests that the Pt-As bond in AP-1-DNA adducts is intact at early stage but dissociates at later times. This raises the possibility that As(OH)₂ moiety is released from AP-1-DNA adduct and could contribute to the overall cytotoxicity of AP-1. Support for this idea can be seen in higher Pearson correlation coefficient (PCC = .78) between AP-1 and As₂O₃ compared to AP-1 and cisplatin (.67), Figures S7 and S8. Arsenous acid has been shown to inhibit zinc finger containing regulatory proteins through direct arsenic binding to cysteine residues;³⁴ however, a detailed profile of biological activity is necessary to establish the mechanism of action of arsenoplatin-1. DFT calculations on AP-1-DNA and cisplatin-DNA bindings suggest that the hydrolysis of the Pt-Cl bond in AP-1 requires less energy than hydrolysis of cisplatin due to the trans effect of arsenic moiety (27.9 kcal/mol vs. 31.8 kcal/mol), and led Marino et al.³⁵ to propose that N7 of guanine is a kinetically preferred target site for AP-1 binding to the DNA (22.4 kJ/mol vs. 31.9 kcal/mol for the G and A platination processes).

Figure 3.

MDA-MB-231, triple negative breast cancer cells, were incubated with AP-1 or cisplatin for 4h and 8h and the extent of adduct DNA formation was determined by ICP-MS, where the amount of platinum and arsenic were in pmolM/μg DNA (M = Pt or As). Results shown are average of at least five independent experiments. Analysis of one-way-ANOVA using Bonferroni multiple comparison has shown that the difference in the amount of platinum and arsenic in DNA adducts in 8h experiment is highly significant (p ≤ 0.0001).

We next examined the interactions of arsenoplatin-1 with another important class of biomolecules – proteins, following an experimental protocol based on the integrated use of mass spectrometry and crystallography.^36–37 It is well known that platinum-protein adducts are crucial for pharmacokinetics, biodistribution, resistance, and toxicity and thus play a critical role in the mechanism of platinum-based drugs.³⁸ We examined AP-1 reactions with two small model proteins, hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A), for which crystal structures with cisplatin were known.³⁹ ESI-MS measurements on AP-1-adducts of HEWL (Figure 4) and RNase A (Figure S9) offered clear evidence for the formation of adducts where one or more [AP-1]⁺ fragments are associated with these proteins; these adducts were confirmed by crystallographic results (Figures 5 and 6, and Figure S10 and Tables S3 and S4 in SI).

Figure 4.

Deconvoluted ESI-MS spectrum of lysozyme-AP-1 (Cl) complex.

Figure 5.

Binding site of the AP-1 in the AP-1- HEWL adduct. (PDB code 5NJ1).

Figure 6.

Binding sites of the AP-1 moieties in the two molecules of RNase A in the asymmetric unit (PDB code 5NJ7).

In both crystals, the overall structures of HEWL and RNase A are well conserved. The best AP-1-HEWL crystals diffract X-rays at 1.85 Å resolution. In the AP-1-HEWL complex, the Pt(II)-center coordinates the Nε2 atom of the His15 side chain of HEWL (Figure 5 and Figure S10) and arsenoplatin-1 (-Cl) complex has occupancy equal to .60. Partial occupancy of metal containing fragments is frequently observed in the crystal structures of the metal-based drugs/protein adducts.³⁸ Cisplatin shows the same coordination mode in cisplatin-HEWL adduct.³⁹

The best AP-1 soaked RNase A crystals diffract X-rays at a 2.15 Å resolution (Figure 6). The asymmetric unit of these crystals contains two RNase A molecules, labeled as A and B. In both RNase A molecules, two AP-1 moieties with intact Pt-As bond and occupancy between 0.60 and 0.75 are present. The Pt(II) centers coordinate N-atoms of the solvent exposed His105 and His119 side chains on the surface of the protein. Since His119 residues are crucial for the catalytic activity of the RNase A, the binding of AP-1 inhibits the enzyme function, as indicated by a catalytic activity assay (Figure S11). In contrast to HEWL adducts, cisplatin and AP-1 form different adducts with RNase A. In the crystal structure of the cisplatin-RNase A adduct coordination of Met29 residue takes place, and the Pt-S bond is observed in both protein molecules present in the asymmetric unit.⁴⁰ We find no evidence of interactions of AP-1 binding to the methionine sulfur atoms in either protein under these conditions. In contrast, cisplatin and carboplatin both interact with Met29 of RNase A.⁴⁰ These results, coupled with negligible solvolysis of AP-1 in DMSO (Figure S1 and Table S1) support the idea that AP-1 may be less susceptible to inactivation by sulfur compounds than Pt-containing drugs currently in clinical use.⁴¹

These protein-AP-1 crystallographic studies provide key insights into AP-1 reactivity: a) the adduct formation process involves loss of chloride followed by platinum ligation to surface exposed histidines; b) the Pt-As bond in the protein adduct remains intact; c) AP-1 does not always follow the same coordination mode as cisplatin in the protein adducts.

Based on the results of this study, arsenoplatin-1 exhibits antiproliferative effects in hematological and solid tumors that are distinct and superior in a majority of the NCI-60 human tumor cell lines relative to cisplatin or arsenic trioxide when tested as single agents. The protein-AP-1 crystallographic studies reveal that adduct formation involves loss of chloride followed by platinum ligation to surface exposed histidines and that the PtAs bond in the protein adducts remains intact. Analytical studies of AP-1/DNA adducts in the triple negative breast MDA-MB-231 cancer cell line are consistent with a model in which the gradual release of As(OH)₂ moiety in the cellular milieu contributes to the increased toxicity of arsenoplatin-1 compared to cisplatin. Intriguingly, arsenoplatin compounds, AP-1 and AP-2²³, also satisfy the Lipinski rule of five used to predict not only the permeability through the cell membrane, but also the overall drug-likeness (Table S5).⁴² Taken together, these data suggest that arsenoplatin-1 acts as a unique and dual pharmacophore anti-cancer agent which has the potential to simultaneously deliver platinum and arsenic species to a variety of hematological and solid cancers.