Developing an anticancer copper(II) multi-target pro-drug based on

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Developing an anticancer copper(II) multi-target pro-drug based on the His146 residue in the IB sub-domain of modified human serum albumin Jun Wang, Yi Gou, Zhenlei Zhang, Ping Yu, Jinxu Qi, Qipin Qin, Hongbin Sun, Xiaoyang Wu, Hong Liang, and Feng Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00045 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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Molecular Pharmaceutics

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Developing an anticancer copper(II) multi-target pro-drug based on the His146 residue in the IB sub-domain of modified human serum albumin Jun Wang1§, Yi Gou1,2§, Zhenlei Zhang1§, Ping Yu1, Jinxu Qi1, Qipin Qin1, Hongbin Sun1, Xiaoyang Wu3, Hong Liang1*, Feng Yang1* 1

State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal

Resources, Guangxi Normal University, Guilin, Guangxi, China. 2

School of Pharmacy, Nantong University, Nantong, Jiangsu, China.

3

Ben May Department for Cancer Research, University of Chicago, Chicago, IL, USA.

*

Corresponding author:

Hong Liang, [email protected] Feng Yang, [email protected] Phone/Fax: 86-773-584-8836 Address: 15 Yucai Road, Guilin, Guangxi, China. Zip code: 541003 §

J Wang, Y Gou and ZL Zhang pay the same contribution for the article.

KEYWORDS: albumin; metal complex; pro-drug; drug carrier; target therapy.

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Abstract Designing a multi-target anticancer drug with improved delivery and therapeutic efficiency in vivo presents a great challenge. Thus, we proposed to design an anticancer multi-target metal pro-drug derived from thiosemicarbazone based on the His146 residue in the IB sub-domain of palmitic acid (PA) modified human serum albumin (HSA-PA). The structure-activity

relationship

of

six

6-methyl-2-formylpyridine-4N-substituted

Cu(II)

compounds

thiosemicarbazones

with were

investigated, and then the multi-target capability of 4b was confirmed in cancer cell DNA and proteins. The structure of the HSA-PA-4b complex (HSA-PA-4b) revealed that 4b is bound to the IB sub-domain of modified HSA, and that His146 replaces the nitrate ligand in 4b, coordinating with Cu2+, whereas PA is complexed with the IIA sub-domain by its carboxyl forming hydrogen bonds with Lys199 and His242. In vivo data showed that 4b and the HSA-PA-4b complex inhibit lung tumor growth, and the targeting ability and therapeutic efficacy of the PA-modified HSA complex was stronger than 4b alone.

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1 Introduction Designing and synthesizing metal compounds that are derived from organic ligands with anticancer activities has been the focus of many studies. This is a promising strategy for the development of novel anticancer agents since metal compound may have stronger biological and chemical activities than organic compound alone.1, 2 Metal anticancer agents with individual anticancer mechanism not only produce severe side effects and drug resistance, but also are not sufficient for achieving tumour recession since cancers are very complicated and multigenic disease.3 Currently, multi-target anticancer drugs are promising for overcoming the shortcoming of anticancer drug acting against single target.4 In the last few decades, a lot Cu compounds derived from thiosemicarbazones

lignads

were

synthesized,

and

many

Cu

thiosemicarbazone compounds have considerable anticancer activity against murine and human cultured cells.5-20 Indeed, the studies of Cu thiosemicarbazone compounds are probably one of the most heavily areas of medicinal inorganic chemistry.21-24 Interestingly, they also kill cancer cells possibly using multiple different mechanisms.25-27 This suggests the possibility that a multi-target anticancer Cu agent could be developed, which destroys cancer cells by several different means, thus avoiding the resistance of cancer cells to drugs that treat cancer via a single mechanism.28 Meanwhile, increasing the in vivo delivery efficiency and target ability as well as decreasing the side effects of metal agents should also be addressed.29 To achieve these goals, Yang et al. proposed developing metal pro-drugs that are based on special N-donor residue(s) in the human serum albumin (HSA) IIA subdomain.30-33 This strategy, however, does not work well when metal agents bind weakly to the HSA IIA subdomain. The reasons 3


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for this are the following: 1) most metal agents can be released from HSA during in vivo blood circulation32, and 2) fatty acids are the main endogenous ligands that bind to HSA. Therefore, when the binding affinity of the drug is weaker than that of fatty acids, the fatty acids will displace the drug and bind with the HSA IIA subdomain.34,35 These potential risks can lead to side effects in vivo; thus, developing a novel pro-drug strategy based on the other properties of HSA is necessary. Currently known structures of HSA complexes have revealed that metal agents bind to the IB subdomain via the His146 residue of HSA, replacing one ligand in the metal agent.36-38 Taking into consideration the above factors, to increase the target ability and therapeutic efficacy as well as delivery efficiency of metal agent in vivo, we proposed to design metal pro-drug based on the His146 residue of modified HSA: 1) modify HSA through palmitic acid (PA)-saturated binding to HSA, thus blocking the HSA IIA sub-domain, and 2) design a metal compound with a leaving group. The His146 residue of HSA will then replace the leaving group and coordinate to a metal ion, forming a stable HSA complex upon the metal compound bound to the IB sub-domain of PA-modified HSA (HSA-PA complex). Subsequently, the coordination ability of His146 to the metal compound dramatically decreases because the N-donor residue is protonated in the acidic environment of the cancer cell lysosomal compartment, which allows release of the metal compound from HSA (Figure 1). Interestingly, West et al. synthesized a series of Cu(II) compounds derived from 6-methyl-2-formylpyridine-4N-substituted thiosemicarbazones, and characterized them mainly by elemental analyses.9,39 In addition, Ali et al.

also

synthesized

several

Cu(II)

6-methyl-2-formylpyridine-4N-substituted 4

compounds

derived

thiosemicarbazones,


from and


characterized them by X-ray diffraction.12,13,40 Obviously, their studies provided a possibility for developing potential anticancer metal compounds with 6-methyl-2-formylpyridine-4N-substituted thiosemicarbazones. Thus, to further study the anticancer function and mechanism of Cu(II) compounds with

6-methyl-2-formylpyridine-4N-substituted thiosemicarbazone, and

investigate their structure-activity relationship, we re-synthesized a series of Cu

compounds

with


thiosemicarbazones (1b-6b) reported by West and Ali groups, and characterized them by X-ray crystallography (Figure 2).39,40 In addition, to confirm our hypothesis the above-mentioned by us, we used 4b and lung cancer (A549) cells to conduct the following studies: First, the multi-target capability of these compounds was confirmed in A549 cells. Second, the development of a Cu pro-drug based on the His146 residue in the IB subdomain of the HSA-PA was shown to be feasible. Third, the side effects, in vivo targeting ability, and therapeutic efficacy of the HSA-PA complex were compared with the Cu compound. Fourth, the release behavior of the Cu compound from HSA-PA and the possible mechanism of the HSA-PA complex’s penetration of cancer cells were investigated. 2 Experimental sections HSA and palmitic acid (PA) were purchased from Sigma-Aldrich. All other solvents and chemicals were of high purity, and were purchased from commercial sources. Distilled H2O was used in all reactions. Elemental analyses (C, N, H, and S) were performed on a Perkin-Elmer 2400 analyzer for all the samples. 2.1 Synthesis and characterization of 1a -6a ligands 5


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A 10 mL methanol solution of 6-methylpicolinaldehyde (10 mmol) was added to a 10 mL methanol solution of thiosemicarabazide (10 mmol). The acetic acid (1-2 drops) was then added to the mixture and the yellow solution was stirred at 60 °C for 6 h. Subsequently, the mixture was filtered, and the white or off-white precipitate was placed in a freezer. The characterization of compounds has been deposited into Supporting Information. 2.2 Synthesis and characterization of Cu(II) compounds The Cu(II) compounds 1b-6b were synthesized according to literature procedures.39,40 In brief, the relevant ligands (1 mmol) and the Cu(II) salts (1 mmol) were dissolved in an aqueous methanol solution (20 mL), respectively, and stirred for 2 h. Dark blue single crystals suitable for X-ray diffraction analysis were obtained after 5 days. The characterization of Cu compounds has been deposited into Supporting Information. 2.3 Binding characterization of 4b to HSA-PA We determined the binding characterization of metal pro-drug to HSA-PA by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS), fluorescence quench (Shown in Supporting Information) and X-ray crystallography. In addition, the binding efficiency of Cu(II) agent with HSA-PA and Cu agent-loading capacity of HSA-PA were determined by the [1] and [2] equations: Binding efficiency (%) = (Cu agent binding/Cu agenttotal) × 100% [1]; Cu agent-loading capacity (%) = (weight of Cu agentbinding/weight of HSA-PA-Cu agent complex) × 100% [2]. X-ray crystallography: Briefly, HSA (100 µL and 100 mg/mL) and

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palmitic acid (PA) (960 µL, 2.5 mM) were incubated for 2 h, and then 60 µL of the Cu compound (2.5 mM and 5 mM) were added to the mixture for 24 h. The mixture solution had a concentration of 100 g/L with a Millipore spin filter (10,000 Da cut-off). Crystallization was performed according to a previous study.41 The data of HSA complexes were collected at 100 K using a BL17U beam line of the

Shanghai Synchrotron Radiation Facility and then

processed with HKL2000.42,43 The HSA complex structure was determined by molecular replacement method using the PHASER program in PHENIX suites with an initial HSA-MYR (PDB:1bJ5) model. However, the ligand was stripped as an initial searching model. All ligands were built into the model with the LigandFit program in PHENIX suites and manually PA modified and adjusted in COOT.44,45 The figures that depict the structures were prepared by PyMOL software.46 Data collection details and unit cell parameters are listed in Table 1. 2.4 Determining the possible mechanism for uptake of the HSA-PA-4b by cells The possible mechanism of uptake of the HSA-PA-4b complex by cells was determined according to previous methods (shown in Supporting Information).30-33 2.5 Cu pro-drug release from the HSA or HSA-PA complex The releasing behavior of the Cu compound from the HSA or the HSA-PA complex was assessed according to a previously reported method.30-33 Briefly, 2 mL of the HSA-PA-4b suspension in a dialysis pocket

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were dispersed in a tube containing 50 mL of pH 7.4 and 4.7 buffers for 48 h, respectively. The amount of the Cu compound that was released from the HSA-PA complexes was examined with a graphite furnace atomic absorption spectrometer (GF-AAS). 2.6 In vitro anticancer activity The cytotoxicity assay (MTT) of ligands and Cu compounds to cells was performed according to a previous protocol (shown in Supporting Information).30-33 2.7 Determine of multi-target anticancer mechanism of 4b and the HSA-PA-4b complex Some potential anticancer mechanism of 4b and the HSA-PA-4b complex were designed and conducted according to a previous protocol (shown in Supporting Information).30-33 2.7.1 In vitro proteasome activity assay47 Purified 20S proteasome (17.5 ng) was incubated for 1 h at 37˚C in 100 µL of the assay buffer (pH 7.5, 50 mM Tris-HCl) with or without different concentrations of 4b and 10 µmol/L fluorogenic peptide substrate Suc-LLVY-AMC. After incubation, production of hydrolyzed AMC groups was measured using a multi-well plate Wallac Victor 3 multilabel counter with an excitation filter of 365 nm and an emission filter of 460 nm. 2.7.2 TRAP-silver staining assay48

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A telomerase extract was prepared from the A549 cells. A total of 1.0 × 107 A549 tumor cells that were untreated or treated with the complexes 4b (1 µM) and HSA-PA-4b (1 µM) were pelleted. The cells were washed with 5.0 mL of PBS. Then the cells were scraped and lysed for 30.0 min on ice. Finally, the lysate was centrifuged at 13,000 rpm for 0.5 h at 4.0 °C. The supernatant was collected and subsequently stored at −80 °C before use. A modified version of the TRAP assay was utilized. Afterward, PCR was performed in a reaction solution composed of reaction mix (45.0 mL) containing Tris-HCl (pH 8.0, 20.0 mM), deoxynucleotide triphosphates (50.0 mM), BSA (20.0 mg/mL), EGTA (1.0 mM), MgCl2 (1.5 mM), KCl (63.0

mM),

Tween-20

(0.005%),

5’-AATCCGTCGAGCAGAGTT-3’), 5’-G3[T2AG3]3-3’),

primer primer

primer

Cxext

TS

(18.0

pmol;

H21T

(3.5

pmol;

(22.5

pmol;

5’-GTGCCCTTACCCTTACCCTTACCCTAA-3’), primer NT (7.5 pmol; 5’-ATCGCTTCTCGGCCTTTT-3’), TSNT internal control (0.01 amol; 5’-ATTCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3’),

Taq

DNA polymerase (2.5 U), and telomerase (100 ng), with a final volume of 50.0 mL. Distilled water (5.0 mL) was added. Finally, PCR was carried out in an Eppendorf Mastercycler equipped with a hot lid. The reaction was incubated for 30.0 min at 30.0 °C, followed by 92.0 °C for 30 s; 52.0 °C for 30 s, and 72.0 °C 30.0 s for 30 cycles. Loading buffer (8.0 mL; 5 × TBE buffer, 0.2% xylene cyanol, and 0.2% bromophenol blue) was added to the reaction after amplification. An aliquot (15.0 mL) was loaded onto a nondenaturing acrylamide gel (16.0%; 19:1) in 1 × TBE buffer. It was resolved at 200.0 V for 1.0 h. Gels were fixed and subsequently stained with silver nitrate. Commonly, the integrated optical density (IOD) data of TRAP-silver staining assay were captured using Gel pro4.0, and the 9


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inhibitory rates of complexes 4b and the HSA-PA-4b on the telomerase could be described by the following: Inhibitory rates (%) = (IOD

control group

-IOD treatment group)/IOD control group × 100%. 2.7.3 Cell migration inhibition assay49 A scratching wound healing assay was implemented in the A549 cell line to study the effect of the complexes on cancer cell migration. In brief, A549 cells were seeded in six well plates and grown to 90% confluence. Then the monolayer of the cells was scratched using a sterile microtip. After being washed three times with ice-cold PBS, the new medium was used and incubated with 4b (0.6 µM) or the HSA-PA-4b complex (0.6 µM). The phase images of the cells were captured at different time points (0, 12 and 24 h) using a bright field microscope. 2.7.4 Molecular docking Docking studies were carried out using Autodock Vina.50 The crystal structures of the B–DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA), DNA Topoisomerase 1 (PDB ID: 1T8I) and Bcl-xL (PDB ID: 2YXJ) were downloaded from the protein data bank. The DNA and protein structures were modified to include polar hydrogen atoms. The coordinates of the metal complex were taken from its crystal structure. During docking studies, the DNA and protein structures were kept rigid. Rotation in the Cu complex was permitted about all single bonds. 2.7.5 Gene expression profiles were generated by Agilent Human Gene Expression Array

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To evaluate which genes were up- or down-regulated in response to the different treatments, we created a set with a log2 fold-change cutoff of ≥2 and realized a hierarchical clustering of these differentially expressed gene sets, using a row-normalized data-set that was prepared using the Feature Extraction software Pre-process Dataset.51 2.8 In vivo animal studies 2.8.1 Animal subject and tumor models Athymic nude mice and Kunming mice (4 weeks of age) were obtained from the Shanghai Laboratory Animal Center and used at 6 weeks of age. The Animal Management Rules (document NO. 55, 2001) of the Ministry of Health of the People’s Republic of China as well as the Animal Care Committee of the Institute approved all animal experiments. 2.8.2 Acute toxicity study The acute toxicity studies of 4b and the HSA-PA-4b complex were performed on healthy mice as described previously.52 In brief, 24 Kunming mice were placed into three groups of eight mice each. The mice were treated with 20 µmol Cu/kg 4b or the HSA-PA-4b complex. Blood samples from each mouse were obtained after three days with intravenous (i.v.) injection to prepare the serum samples. The serum biochemical parameters were aspartate aminotransferase (AST), creatinine kinase (CK), alanine aminotransferase (ALT), and blood urea nitrogen (BUN). 2.8.3 In vivo anti-tumor activity study

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A total of 4 × 106 A549 cells were injected subcutaneously in the right flank region of the nude mice. The 24 mice were randomly divided into 3 groups for the anti-tumor activity study when the tumor volume was about 100 mm3. The mice in the different treatment groups were intravenously (i.v.) injected every two days with normal saline (vehicle), free 4b or the HSA-PA-4b complex at a dose of 3.0 µmol Cu/kg body weight. The body weight of the mice and the size of the tumors were measured before every injection before the end of experiment. The tumor volume (V) was calculated with the following equation: V = 1/2 × width2 × length. Mice were sacrificed, and major organs and tumor tissues were excised for histopathological analysis after 18 days of treatment. 2.8.4 H&E staining Tumor samples and major organs were subjected to a routine histopathological examination using standard hematoxylin and eosin (H&E) staining.

The

tissue

specimens

were

collected

and

placed

in

paraformaldehyde (4%) for proper fixation. They were subsequently dehydrated, cleared and embedded in paraffin wax. The tissue sections were then stained with H&E and visualized under an Eclipse E800 Nikon (Nikon, Japan). 2.8.5 TUNEL assay At 18 days after the final injection, the mice were sacrificed and the tumor tissues were excised. They were then fixed with paraformaldehyde for 48 h and embedded in paraffin. Each tissue section was cut into 5-µm slices. Apoptosis in the neuromasts was assayed with a TUNEL assay and an in situ

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cell detection kit (A-23210; Molecular Probes, Eugene, OR), per the manufacturer’s protocol. Samples were then visualized under a fluorescence microscope (AxioCam MRc5; Carl Zeiss). 2.8.6 In vivo targeting ability study31-33 At the end of the in vivo experiment, the tumor tissues and major organs of the mice were homogenized. 0.5 g of the sample was then placed in a Teflon container and mineralized in a microwave device under pressure (system Milestone MSL 1200) in the presence of 7 mL of concentrated nitric acid and 1 mL of 30% H2O2. ICP-AES was used to determine the contents of Cu in the tumors and major organs. 2.9 Statistical analysis A Student’s t test was used to evaluate the significance of the differences that were measured. The representative results are expressed as the mean ± standard deviation (SD), and they were considered to be significant when p < 0.05. 3 Results 3.1 Synthesis and structure of Cu pro-drugs Since thiosemicarbazone itself is a promising anticancer agent, a thiosemicarbazone was designed to be the primary ligand (pharmacophore) in the Cu compound, and another ligand, without anticancer activity, was designed to be a leaving group (Figure 2).53-55 To optimize anticancer Cu(II) compounds

with


13


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thiosemicarbazone, and further study their structure-activity relationship, we synthesized the six Cu(II) compounds according to the previous method. 39, 40 Single crystals that were suitable for X-ray diffraction were obtained from the compounds (1b-6b). As shown in Figure 2, single-crystal X-ray diffraction revealed that all Cu(II) compounds were mononuclear, and each thiosemicarbazone ligand was coordinated in a tridentate fashion, forming two 5-membered bis-chelate rings around the Cu(II) center. All Cu−N/S bond lengths fall in the range of 1.927−2.290 Å, which compare well with those observed for similar Cu(II) compounds.12,13,40 In compounds 1b, 2b, and 4b-6b, the coordination geometry surrounding all of the Cu(II) centers formed a distorted square pyramid geometry (τ = 0.03 for 1b, 0.51 for 2b, 0.35 for 4b, and 0.16 for 5b and 0.18 for 6b,). In compound 3b, the Cu(II) metal center, which is coordinated by one Cl atom and two nitrogen atoms of 3a ligand, adopts a square-planar coordination geometry. 3.2 Structure-activity relationship of Cu compounds The Cu compounds showed higher cytotoxicity against many types of cancer cells compared with the ligands (1a-6a) and Cu2+ alone (Table 2). This may imply that the chelation of ligands to Cu2+ had a synergistic effect with respect to cytotoxicity. Six Cu compounds showed higher cytotoxicity against A549 cells (≤ 13.69 ± 0.97 µM) than cisplatin (17.36 ± 0.75 µM) (Table 2). Compound 1b had the lowest cytotoxic activity against A549 cells (13.69 ± 0.97 µM) of the six Cu compounds. The cytotoxic activities of 2b and 3b were enhanced (6.77 or 2.75 µM) compared with 1b, and an even higher cytotoxicity was observed (0.88−1.47 µM) for 4b-6b (Table 2).

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Therefore, we can keep the pharmacophore of the ligand attached to Cu intact and modify the lipophilic group(s) of the ligand to regulate the Cu agent’s anticancer activity. Importantly, the cytotoxicity of the HSA-PA-Cu (II) complexes was approximately 1.0-3.0-fold higher in A549 cells relative to the Cu II) compounds alone. However, the cytotoxicity of the complexes was not higher in the normal HL7702 cells (Table 2). The HSA-PA-4b complex showed higher cytotoxic activity in A549 cells compared with the other Cu compounds in vitro. The above complex was selected for further investigation. 3.3 Feasibility of the Cu pro-drug design based on the IB sub-domain of the HSA-PA The intrinsic fluorescence properties of HSA are attributable to the single Trp214 residue in IIA subdomain of the HSA.56 Compounds that bind to IIA subdomain would diminish the fluorescence emission of HSA.56 The fluorescence of HSA was gradually quenched upon increasing the concentration of Cu compound (Figure S1A), indicating that the Cu compound binds to the subdomain IIA of HSA.56 The binding constant of HSA for Cu compound was estimated from the Stern−Volmer equation (Figure S1B).56 The binding constant of Cu compound to HSA was estimated to be K = 6.38 ± 0.07 × 103 M−1. Owning to Cu compound weakly bound to HSA IIA subdomain, the HSA was modified by creating a palmitic acid (PA)-saturated-HSA complex in vitro to protect the metal compound from being replaced by fatty acids in vivo. The matrix-assisted laser desorption/ionization time-of-flight-mass

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spectrometry (MALDI-TOF-MS) spectrum showed an increase of ～280 Da for the HSA-PA complex, which was equivalent to a molecular weight of one Cu compound (Figure S1C). The structure of the HSA-PA-4b complex was resolved to provide solid evidence of the feasibility of developing a Cu(II) pro-drug based on the His146 residue of the HSA-PA IB sub-domain. The electron density maps of the HSA-PA complex indicate clearly that there was one Cu compound molecule at the IB sub-domain and one PA molecule at the IIA sub-domain (Figure 3A and B). The HSA-PA-4b complex structure indicated that 4b could not compete with PA to bind in the IIA sub-domain. The structure of the HSA-PA-4b complex was heart-shaped overall (Figure 3C). The 4b portion binds to the sub-site of the narrow and long hydrophobic pocket in the IB subdomain of HSA. It was in contact with several hydrophobic residues of HSA, away from PA1. Nitrate is replaced by His146 of HSA in 4b and forms a coordination bond with the Cu ion (Figure 4A and C). PA7 binds to a large hydrophobic pocket in the IIA subdomain of HSA. This was delimited by Trp214, Phe223, Leu219, His242, Leu238, Ile264, Arg257, Ser287, Leu260, Ala291, and Ile290. Its carboxyl group forms hydrogen bonds with His242 and Lys199 (Figure 4B and D). Furthermore, although the incubation ratio of 4b to HSA-PA ranges from 1:1 to 1:3, just one 4b bound to per HSA-PA, indicating the binding efficiency of 4b to HSA-PA can reach ～100%, and the 4b-loading capacity to HSA-PA is ～0.6%. 3.4 Potential mechanism for HSA-PA-4b complex absorption by cancer cells

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The uptake efficiency of 4b and HSA-PA-4b in the cancer cells was investigated by treating A549 cells with 4b (0.6 µM) and HSA-PA-4b (0.6 µM) for 6 and 12 h. The results showed that the Cu content in A549 cells treated with the HSA-PA-4b is higher than that of in cells treated by 4b (Figure 5A), suggesting A549 cells can absorb more HSA-PA-4b than 4b. In addition, A549 cells at 4°C internalized less Cu than A549 cells incubated with the HSA-PA-4b complex at 37°C, indicating that the uptake of the HSA-PA-4b

complex

was

energy-dependent.

Glucose

deprivation

significantly decreases the uptake of the HSA-PA-4b complex in cells. The uptake of HSA-PA-4b complex by cells was inhibited in the presence of sodium azide or sodium cyanide relative to the control (Figure 5B). These data suggested the HSA-PA-4b complex absorbed by the cells was reliant on metabolic energy.57 Phenylglyoxal distinctly inhibited the uptake of the HSA-PA-4b complex in cells. Also, methyl-β-cyclodextrin, which is a known endocytosis inhibitor, stopped the albumin-induced increase in transendothelial HSA permeability (Figure 5C).58 The results suggested that the HSA-PA-4b complex possibly penetrated the A549 cells by energy- and temperature-sensitive endocytosis. 3.5 Releasing behavior of 4b from the HSA-4b and HSA-PA-4b complexes We examined the releasing behavior of the Cu compound from the HSA-4b complex without PA. Approximately 50% of the loaded 4b was released from the HSA within 72 h in the pH 7.4 buffer (Figure 5D). This implied that the HSA-4b complex was unstable in the circulating blood in vivo, and the majority of the Cu compound was released from the HSA before delivery into the cancer cells. The reason is that the IIA subdomain is

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available in unmodified HSA, and the Cu complex bound in the IIA subdomain is easily released at a pH of 7.4. The releasing behavior of the Cu compound from the HSA-PA inside cancer cells needs to be explained. Thus, we simulated the environment inside the cancer cells and the amount of Cu compound released from the HSA-PA was measured in pH 7.4 and pH 4.7 buffers. Approximately 5% of the loaded 4b was released from the HSA-PA within 72 h in the pH 7.4 buffer. Furthermore, about 7% of the loaded 4b was released from the HSA-PA in the Dulbecco's Modified Eagle's medium (DMEM) with 10% FBS during 0−72 h. About 80% of the 4b was released from the HSA-PA in the pH 4.7 buffer during the same time period (Figure 5D). 3.6 Confirmation of the multi-target anticancer Cu pro-drug The primary intracellular targets of many drugs used to treat cancer are cancer cell DNA or proteins.59, 60 Herein, we investigated the interaction of Cu compound with calf-thymus (CT)-DNA and supercoiled pBR322 plasmid DNA by multiple techniques. As shown in Figure 6A, upon the addition of CT-DNA, the absorption band of the 4b observed at 395 nm exhibited a hypochromism of about 5.1% with 1 nm red shift in the band position. Competitive binding studies with ethidium bromide (EB)-bound DNA were performed to further elucidate the binding nature of the Cu compound. On addition of the 4b to the EB-DNA system, the emission was quenched by about 56% (Figure 6B), and the observed binding constant (Kapp) value of the 4b is 2.2 × 106 M−1. The viscosity of CT-DNA increased on increasing the concentration of the 4b (Figure 6C). Docking studies further revealed that the Cu compound fit

18



snugly into the curved contour of the targeted DNA (Figure 6D). These results suggested the Cu compound involve in intercalative DNA interaction with moderate binding affinity.61-63 Particularly, to evaluate the DNA cleavage properties of the Cu compound, the pBR322 DNA as a substrate was incubated with the 4b, and the cleavage reactions on pBR322 DNA were investigated by agarose gel electrophoresis. The 4b is capable of converting supercoiled pBR322 DNA into Form II (open circular form) in a dose-dependent fashion (Figure 6E). The cleavage efficiency of the 4b kept unaffected in the presence of hydroxyl radical scavengers DMSO and MeOH (Lane 3 and Lane 4) and singlet oxygen scavenger NaN3 (Lane 5) (Figure 6F), which means that HO• and/or 1O2 were not involved in the DNA cleavage.64-69 When a DNA mimic phosphates bis(2,4-dinitrophenyl) phosphate (BDNPP) was added (Lane 6), the reaction of DNA cleavage was partially inhibited, suggesting that the hydrolysis pathway for the DNA cleavage process is possible.70 Notably, when hydrogen peroxide scavenger KI (Lane 7) was added, the DNA cleavage was significantly inhibited, implying that H2O2 was the reactive species in the cleavage process.64,71,72 Furthermore, EDTA (Lane 8) efficiently inhibited DNA cleavage, implying that the complexation of 4a ligand with Cu2+ is crucial to the cleavage. Taken together, these results implied the compound bound to DNA by moderate intercalative binding mode, and efficiently cleaved pBR322 DNA probably via an oxidative mechanism with the involvement of H2O2. Topoisomerase I (Topo I), as isomerase enzyme, plays an essential role in the topology of DNA.73 The Cu compound showed significant inhibition at 15 µM (Figure 7A). The docking model further revealed that the Cu compound was well-fitted into the site of DNA cleavage of the Topo I enzyme (Figure 7B). In addition, the Cu compound not only inhibited 20S 19


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proteasome activity (Figure 7C) and telomerase activity (Figure 7D) but also inhibited A549 cell cycle progression at the G0/G1 phase and promoted cell apoptosis (Figures S2 and S3) by regulating the activity of proteins related to the cell cycle and apoptotic progression of the cancer cell (Figure 8A-C). Interestingly, the Cu compound not only inhibited the transfer of cancer cells (Figure S4) but also the activity of metastasis-associated in colon cancer-1 (MACC1) protein related to tumor transfer (Figure 8D).74 Furthermore, gene array results showed that the Cu compound regulated the gene expression of cancer-related proteins (Figure 8E). Together, these results demonstrated that the Cu compound kills cancer cells by targeting DNA and proteins in cancer cells at the same time. 3.7 Evaluating the therapeutic efficiency of 4b and the HSA-PA-4b complex in vivo A lung cancer A549 xenograft mouse model was established to further evaluate whether the HSA-PA complex enhanced the therapeutic efficiency of the Cu compound in vivo. 3.7.1 Acute toxicity of 4b and the HSA-PA-4b complex in vivo The acute toxicities of 4b and the HSA-PA-4b complex were analyzed in the kidneys, hearts, and livers of normal mice by measuring levels of blood urea nitrogen (BUN), creatine kinase (CK), alkaline phosphatase (ALT), and aspartate aminotransferase (AST) 3 days after intravenous (i.v.) injection of drugs (Table S1). Compound 4b caused significant nephrotoxicity, which was indicated by a higher BUN value in the treated mice (23.6 ± 1.8 mmol/L) compared with

20



the control mice (8.6 ± 1.0 mmol/L). CK levels in mice treated with the HSA-PA-4b complex were similar to the levels in the control mice. This indicated there was low cardiotoxicity. In contrast, nephrotoxicity was significantly lower in the mice that were treated with the HSA-PA-4b complex, which indicated there was lower nephrotoxicity. The mice that were treated with 4b had significantly elevated levels of AST and ALT. However, the AST and ALT levels in mice treated with the HSA-PA-4b complex were similar to those in control mice, which indicated a lower level of liver damage in the mice treated with the HSA-PA-4b complex compared with those treated with 4b. 3.7.2 Anti-tumor activities of 4b and the HSA-PA-4b complex in vivo The tumor volumes in mice treated with 4b and NaCl were larger than the tumor volumes in mice that were treated with the HSA-PA-4b complex (Figure 9A). This suggested that the HSA-PA-4b complex was better at inhibiting tumor growth than 4b. The tumor weights in mice treated with 4b were higher than in mice treated with the HSA-PA-4b complex and compared with the NaCl group (Figure 9B). To further evaluate the anti-tumor effects of 4b and the HSA-PA-4b complex in mice, the tumor tissues were excised with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The TUNEL stained tissue sections indicated clear differences in tumor tissue morphology between the control groups and treated groups. The tumor cells of mice in the vehicle-treated group were normal, and showed no clear apoptosis (Figure 9C). However, different degrees of tumor cell apoptosis were observed in the 4b and the HSA-PA-4b treated groups (Figures 9C and

21


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S5). The HSA-PA-4b complex was particularly more effective at promoting cell apoptosis relative to 4b. 3.7.3 Comparing the side effects and targeting ability of 4b and the HSA-PA-4b complex Mice that were treated intravenously with the HSA-PA-4b complex showed no significant weight loss compared with the control mice (p > 0.05). There was, however, an approximate 9% reduction in body weight in the mice that were treated with 4b (Figure 10A). In addition, 4b caused damage to the liver (vacuolation and hepatic cord loss), heart (vacuolation), and kidneys (focal abnormalities) in mice. The damage to these tissues was less in mice treated with the HSA-PA-4b complex (Figure 10B). Together, the results demonstrated that the side effects induced by the HSA-PA-4b complex were lower compared with the side effects induced by 4b. Inductively coupled plasma atomic emission spectrometry (ICP-AES) indicated that the increase in Cu content in the tumors of mice that were treated with the HSA-PA-4b complex was approximately 1.3- higher than in the mice that were treated with 4b alone. The HSA-PA, however, was helpful for decreasing 4b accumulation in the kidney and liver (Figure 10C). In conclusion, the HSA-PA enabled the Cu compound to selectively accumulate in the lung tumor in vivo. 4 Discussion Anticancer functions and mechanisms may be diversified by combining metal ions and ligands with anticancer activities to form metal compounds.25 Indeed, many studies have confirmed that, compared with a ligand alone, the anticancer activity of metal compounds are optimized, and anticancer 22



mechanisms are diversified, especially in regard to regulating the activity of proteins related to cancer.75 Thus, a promising strategy is to rationally design a multi-target metal agent that can work on different intracellular targets in cancer cells at the same time. Indeed, the results of the current study showed that Cu compounds derived from thiosemicarbazones could kill cancer cells by targeting DNA and proteins in cancer (Figures 6-8). Telomerase, in particular, is a promising target for novel anticancer drugs (Figure 7D).76 Developing drugs that inhibit tumor migration are especially important and challenging since tumor migration is the main cause of death due to cancer. The MACC1 protein plays an important role in tumor metastasis. The Cu compound can inhibit the activity of the MACC1 protein and prevent cell migration (Figure 8D). Therefore, the Cu compound derived from thiosemicarbazone shows promise for development as a multi-target anticancer metal lead drug. Excitingly, many studies revealed that 64Cu2+ can be used as a cost-effective probe for positron emission tomography (PET) imaging of various types of cancers.77-80 It suggested that we can design multi-function

64

Cu2+ compound derived from thiosemicarbazide that

simultaneously diagnose and treat cancer. HSA is the most abundant protein in plasma, which has been promising drug carrier because HSA is a non-toxic, non-antigenic, biocompatible, and biodegradable endogenous protein.81 There are two commonly applied strategies for design HSA to deliver metal agent, namely, forming albumin-drug conjugate by chemical linker and encapsulating metal agent into albumin nanoparticles.29,81-83 However, the above-mentioned two methods can introduce exogenous chemicals or change albumin’s conformation. To overcome drawbacks associated with HSA-based delivery strategies for metal agent, we developed a novel approach of HSA carrier for 23


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metal agent by the specific residue(s) in HSA IIA subdomain coordinated to the metal centre, forming tightly complex of Cu compound and HSA.30-33 Unfortunately, our results revealed that the majority of 4b can escape from the HSA carrier in the blood circulation because of their weak bonds to the IIA subdomain of HSA (Figure 5D). Therefore, developing a novel strategy to render drugs tightly bound to HSA is necessary in order to avoid the release of the Cu agent from HSA prior to its delivery to the target site. We therefore proposed to develop a metal pro-drug based on the N-donor of the HSA-PA IB sub-domain by taking into account the nature of metal compounds and the cancer cells. The results of the current study support the feasibility of this idea. The HSA-PA-4b complex structure revealed that the His146 in the IB subdomain replaced the nitrate of the Cu compound and coordinated with Cu2+ (Figures 3 and 4). Afterwards, about 5% of the Cu compounds were released from the HSA-PA at pH 7.4. Approximately 80% of the Cu compounds were released from the HSA-PA in an acidic environment (pH 4.7) (Figure 5D). These release profiles indicated that the HSA-PA complex was stable in the circulating blood in vivo, and the Cu compound was released after accumulation in the lysosomes of cancer cells. Importantly, the HSA-PA complex has better therapeutic efficiency than the metal agent alone in vivo (Figures 9 and 10). The tumor inhibition rate (TIR) of the HSA-PA-4b complex-treated mice (almost 65.3%) was approximately 1.6 times higher than in the 4b-treated mice. The HSA-PA-4b complex not only caused less weight loss and was well tolerated but damage to the liver, heart, and kidney was less relative to 4b alone (Figure 10A, B). Furthermore, the HSA-PA enabled the Cu compound to selectively accumulate in the tumor in vivo (Figure 10C). The HSA-PA was helpful by improving the therapeutic efficiency of the Cu agent. This was due to 24



enhanced permeability the retention effect (EPR), and endocytosis of the HSA-PA in tumors.84 5 Conclusions Due to the rational design of the metal ion and ligand, the Cu pro-drug derived from thiosemicarbazone kills cancer cells mainly by targeting the DNA and proteins in cancer. Also, the delivery efficiency of the Cu pro-drug was improved when the leaving group of Cu pro-drug was replaced with His146 and coordinated with Cu2+ to form the HSA-PA complex. The HSA-PA complex showed better tolerance compared with the Cu compound alone and a higher drug accumulation in the tumor, a stronger capacity for inhibiting tumor growth, and lower toxicity in other tissues. These results may be used as a guide to rationally design an HSA carrier to deliver multi-target metal agents for targeted cancer therapy. ACKNOWLEDGMENTS This work received financial support from the Natural Science Foundation of China (21431001, 31460232), the Natural Science Foundation of Guangxi (2017GXNSFEA198002, AD17129007), National Basic Research Program of China (IRT-16R15), Ministry of Education of China (CMEMR2017-A03), Guangxi “Bagui” scholar program to HB Sun, and High-level innovation team and distinguished scholar program of Guangxi universities to F Yang.

ASSOCIATED CONTENT Supporting Information

25


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The Supporting Information is free of charge on the ACS Publications website. Characterization of 1a-6a ligands and 1b-6b compounds, determination of binding affinity of HSA for Cu compound, in vitro anticancer activity, DNA-binding and cleavage experiments, topoisomerase I inhibition assay, in vitro proteasome activity assay, western blot analysis, determining the possible mechanism of uptake of HSA-PA-4b by cells, serological and organ weights analysis, crystal data for Cu compounds, selected bond lengths (Å) and angles (deg) for Cu compounds, cell cycle distribution and apoptosis analysis, cell migration inhibition assay, apoptosis in tumors, 4b and HSA-PA-4b uptake by A549 cells, the change of mitochondrial membrane potential, intracellular ROS measurements,

1

H and

13

C NMR spectra of

1a-6a.

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(61) Barut, B.; Sofuoğlu, A.; Biyiklioglu, Z.; Özel, A. The water soluble peripherally

tetra-substituted

zinc(ii),

manganese(iii)

and

copper(ii)

phthalocyanines as new potential anticancer agents. Dalton Trans. 2016, 45, 14301-14310. (62) Li, M. J.; Lan, T. Y.; Cao, X. H.; Yang, H. H.; Shi, Y.; Yi C.; Chen, G. N. Synthesis, characterization, DNA binding, cleavage activity and cytotoxicity of copper(II) complexes. Dalton Trans. 2014, 43, 2789-2798. (63) Gou, Y.; Li, J.; Fan, B.; Xu, B.; Zhou, M.; Yang, F. Structure and biological properties of mixed-ligand Cu(II) Schiff base complexes as potential anticancer agents. Eur. J. Med. Chem. 2017, 134, 207-217. (64) Chen M.; Tang X. Y.; Yang S. P.; Li H. H.; Zhao H. Q.; Jiang Z. H.; Chen

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Five

water-soluble

zwitterionic

copper(II)-carboxylate polymers: role of dipyridyl coligands in enhancing the DNA-binding, cleaving and anticancer activities. Dalton Trans. 2015, 44, 13369-13377. (65) Arjmand, F.; Yousuf, I.; Hadda, T.; Toupet, L. Synthesis, crystal structure and antiproliferative activity of Cu(II) nalidixic acid-DACH conjugate: comparative in vitro DNA/RNA binding profile, cleavage activity and molecular docking studies. Eur. J. Med. Chem. 2014, 81, 76-88. (66) Manikandamathavan, V. M.; Rajapandian, V.; Freddy, A. J.; Weyhermüller, T.; Subramanian, V.; Nair, B. U. Effect of coordinated ligands on antiproliferative activity and DNA cleavage property of three mononuclear Cu(II)-terpyridine complexes. Eur. J. Med. Chem. 2012, 57, 449-458. (67) Tabassum, S.; Zaki, M.; Ahmad, M.; Afzal, M.; Srivastav, S.; Srikrishna, S.; Arjmand, F. Synthesis and crystal structure determination of copper(II)-complex: In vitro DNA and HSA binding, pBR322 plasmid 35


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cleavage, cell imaging and cytotoxic studies. Eur. J. Med. Chem. 2014, 83, 141-154. (68) Liang, J. W.; Wang, Y.; Du, K. J.; Li, G. Y.; Guan, R. L.; Ji, L. N.; Chao, H. Synthesis, DNA interaction and anticancer activity of copper(II) complexes with 4'-phenyl-2,2':6',2''-terpyridine derivatives. J. Inorg. Biochem. 2014, 141, 17-27. (69) Lian, W. J.; Wang, X. T.; Xie, C. Z.; Tian, H.; Song, X. Q.; Pan, H. T.; Qiao, X.; Xu, J. Y. Mixed-ligand copper(ii) Schiff base complexes: the role of the co-ligand in DNA binding, DNA cleavage, protein binding and cytotoxicity. Dalton Trans. 2016, 45, 9073-9087. (70) Wei L.; Shao Y.; Zhou M.; Hu H. W.; Lu G. Y. Synthesis and enhanced DNA cleavage activities of bis-tacnorthoamide derivatives. Org. Biomol. Chem. 2012, 10, 8484-8492. (71) Zhou, X.-Q.; Li, Y.; Zhang, D.-Y.; Nie, Y.; Li, Z.-J.; Gu, W.; Liu, X.; Tian, J.-L.; Yan, S.-P. Copper complexes based on chiral Schiff-base ligands: DNA/BSA binding ability, DNA cleavage activity, cytotoxicity and mechanism of apoptosis. Eur. J. Med. Chem. 2016, 114, 244-256. (72) Zhou, C.-Q.; Lin, Y.-L.; Yang, J.-W.; Chen, J.-X.; Chen, W.-H. Synthesis, DNA-cleaving activities and cytotoxicities of C2-symmetrical dipyrrole-polyamide dimer-based Cu(II) complexes: A comparative study. Eur. J. Med. Chem. 2013, 66, 508-515. (73) Nguyen, T. X.; Abdelmalak, M.; Marchand, C.; Agama, K.; Pommier, Y.; Cushman, M. Synthesis and biological evaluation of nitrated 7-, 8-, 9-, and 10-hydroxyindenoisoquinolines as potential dual topoisomerase I (Top1)-tyrosyl-DNA phosphodiesterase I (TDP1) inhibitors. J. Med. Chem. 2015, 58, 3188-3208. (74) Yang, T.; He, W.; Cui, F.; Xia, J.; Zhou, R.; Wu, Z.; Zhao, Y.; Shi, M. 36



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MACC1 mediates acetylcholine-induced invasion and migration by human gastric cancer cells. Oncotarget 2016, 7, 18085-18094. (75) Coley, H. M.; Sarju, J.; Wagner, G. Synthesis and characterization of platinum(II) oxadiazoline complexes and their in vitro antitumor activity in platinum-sensitive and -resistant cancer cell lines. J. Med. Chem. 2008, 51, 135-141 (76) Ruden, M.; Puri, N. Novel anticancer therapeutics targeting telomerase. Cancer Treat. Rev. 2013, 39, 444-456. (77) Chakravarty, R.; Chakraborty, S.; Dash, A.

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Figure Legends Figure 1 The hypothesis of developing metal pro-drug based on the nature of cancer cell and IB sub-domain of the PA modified HSA. Figure 2 The structures of ligands 1a-6a and their Cu compounds 1b-6b. Figure 3 The experimental σA weighted 2Fo − Fc electron density map of 4b in IB subdomain (A) and PA7 in IIA sub-domain of HSA (B); (C) The overall structure of HSA complex. Figure 4 The binding cavity of 4b in IB subdomain (A) and PA7 in IIA sub-domain of HSA (B); the structural binding environment of 4b in IB subdomain (C) and PA7 in IIA sub-domain of HSA (D). Figure 5 (A) The uptake of 4b (0.6 µM) and HSA-PA-4b (0.6 µM) in A549 38



cells as a function of time at 37°C. (B) Uptake mechanisms of the HSA-PA-4b complex. (C) Western blot analysis of HSA in A549 cells treated with the HSA-PA-4b complex. β-Actin was used as the internal control. (D) Copper content released from HSA-4b or HSA-PA-4b in pH 4.7, pH 7.4 buffers and Dulbecco's Modified Eagle's medium (DMEM) with 10% FBS during 0−72 h, respectively. Statistical significance: (*) p < 0.05, (**) p < 0.005. Figure 6 (A) Absorption spectra of complex 4b (50 µM) upon the titration of CT-DNA (0−24 µM). (B) Fluorescence quenching curves of ethidium bromide (EB) bound to DNA: complex 4b. [DNA] = 8 µM, [EB] = 5 µM, and [complex] = 0−32 µM. (C) Relative viscosity increments of CT-DNA (200 µM) solution bound with 4b with increasing the [complex]/[DNA] ratio. (D) A molecular docked model for 4b with a DNA dodecamer duplex of sequence d(CGCGAATTCGCG)2. (E) Agarose gel electrophoresis patterns for the cleavage of pBR322 DNA by 4b at pH 7.0 at 37 °C for 5 h. Lane 1: DNA alone; Lanes 2−4: DNA with 4b at the concentrations of 20, 40, 80 µM, respectively. (F) Agarose GE patterns for the cleavage of pBR322 DNA by 4b (80 µM) at pH 7.0 and 37 °C for 5 h, in the presence of DMSO (1 M, Lane 3), MeOH (1 M, Lane 4), NaN3 (0.1 M, Lane 5), BDNPP (0.1 mM Lane 6), KI (0.1 M, Lane 7) and EDTA (0.1 M, Lane 8). Lane 1, DNA alone and Lane 2: DNA + 4b. Figure 7 (A) Agarose gel electrophoresis patterns showing the effect of different concentrations of 4b on the activity of Topo I. Lane 1, DNA control; lane 2, Topo I + DNA; Lanes 3 and 4: Topo I + DNA with 4b at the concentrations of 15 and 20 µM, respectively. (B) Molecular docked model of human DNA Topoisomerase1 with 4b. (C) In vitro proteasome-inhibitory

39


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activity of 4b. (D) The influence of 4b (0.6 µM) and the HSA-PA-4b complex (0.6 µM) on the telomerase activity of the A549 cells. Figure 8 (A) Western blot analysis of proteins related to the Bcl-2 family proteins response pathway. Proteins expressions in A549 cells treated with compound 4b and the HSA-PA-4b complex for 24 h; β-actin was used as the internal control. (B) Binding model of 4b with Bcl-xL. (C) Immunoblotting analysis of CDK2 and cyclin E1 in A549 cells treated with 4b and the HSA-PA-4b complex for 24 h; β-actin was used as the internal control. (D) The expression level of MACC1 in A549 cells induced by 4b and the HSA-PA-4b complex at the same concentration (0.6 µM) for 24 h, determined by Western blot analysis. (E) Relative expression profiles of telomeres/telomerase related genes in A549 cells after being treated with 4b and the HSA-PA-4b complex at the same concentration (0.6 µM) for 24 h (up: intersection, down: union). Figure 9 (A) Changes of tumor volume after intravenous injection (tail vein) of vehicle control, 4b and the HSA-PA-4b complex in A549 tumor-bearing nude mice. (B) Tumor weight (wet weight) separated from mice after different treatments. (C) Tumors are sectioned and stained with TUNEL (400×), red arrows show apoptotic cells. Results are mean ± SD (n = 6−7): (*) p < 0.05, (***) p < 0.001. Figure 10 (A) Body weight changes of A549 tumor-bearing mice after treatment with vehicle control, 4b and the HSA-PA-4b complex. (B) H&E staining analysis of organs (heart, liver, and kidney) sections treated with various treatments (400×). (C) Tissue copper of mice after treatment with 4b and the HSA-PA-4b complex. The background values of copper in these organs have been deducted.

40



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Table 1 Data collection statistics and crystallographic analysis of the HSA-PA-4b complex Data collection Space group

P1

Cell parameters, a, b, c (Å)

95.65, 96.64, 38.49

Cell parameters,

104.68, 89.97,101.43

Resolution range (Å)

32-2.6

Data redundancy

4.2

Completeness (%) a

97.9% (98.2%)

41


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I/σ

12.8 (4.1)

Rmerge (%) b

8.6% (29.8%)

Model refinement

a b

Rmodel (%) c

22.6%

Rfree (%) d

29.5%

R.m.s. deviation from ideal bond lengths

0.010 Å

R.m.s. deviation from ideal angles (°)

1.027

Values for the outermost resolution shell are given in parentheses. Rmerge=100×ΣhΣj| Ihj-Ih|/ΣhΣj Ihj, where Ih is the weighted mean intensity of the symmetry-related refractions Ihj.

c

Rmodel=100×Σhkl|Fobs -Fcalc|/ΣhklFobs, where Fobs and Fcalc are the observed and calculated structure factors, respectively.

d

Rfree is the Rmodel calculated using a randomly selected 5% sample of reflection data omitted from the refinement.

Table 2 IC50 values of compounds and HSA complexes toward various cell lines for 48 h. IC50 (µM) A549

T-24

Hela

NCI-H460

HL-7702

1a

>40

>40

>40

>40

>40

2a

>40

>40

>40

>40

>40

3a

>40

>40

>40

>40

>40

4a

>40

>40

>40

>40

>40

5a

>40

>40

>40

>40

>40

6a

>40

>40

>40

>40

>40

42



Page 44 of 54

1b

13.69 ± 0.97

10.76 ± 0.84

12.43 ± 0.78

10.35 ± 0.76

12.79 ± 0.90

2b

6.77 ± 0.42

5.34 ± 0.57

4.37 ± 0.46

4.25 ± 0.51

4.33 ± 0.44

3b

2.75 ± 0.30

2.89 ± 0.34

1.94 ± 0.28

3.71 ± 0.41

2.25 ± 0.24

4b

0.88 ± 0.08

1.41 ± 0.04

0.76 ± 0.08

1.54 ± 0.06

0.82 ± 0.07

5b

1.23 ± 0.30

1.65 ± 0.21

0.87 ± 0.12

2.34 ± 0.45

1.15 ± 0.19

6b

1.47 ± 0.06

1.78 ± 0.08

0.97 ± 0.05

5.17 ± 0.27

1.44 ± 0.33

HSA-PA-1b

11.73 ± 0.89

9.96 ± 0.79

10.12 ± 0.84

8.70 ± 0.74

12.54 ± 0.85

HSA-PA-2b

5.70 ± 0.24

5.09 ± 0.18

3.65 ± 0.43

3.25 ± 0.31

6.42 ± 0.27

HSA-PA-3b

2.25 ± 0.07

2.74 ± 0.06

1.56 ± 0.08

2.10 ± 0.04

3.69 ± 0.11

HSA-PA-4b

0.31 ± 0.05

1.22 ± 0.04

0.23 ± 0.09

0.39 ± 0.06

1.48 ± 0.12

HSA-PA-5b

0.78 ± 0.14

1.35 ± 0.09

0.55 ± 0.11

0.96 ± 0.07

2.89 ± 0.15

HSA-PA-6b

1.21 ± 0.07

1.66 ± 0.08

0.79 ± 0.04

1.54 ± 0.06

2.21 ± 0.10

Cisplatin

17.36 ± 0.75

22.47 ± 0.84

25.25 ± 0.66

13.24 ± 0.59

15.66 ± 0.81

HSA-PA

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

Cu

2+

43


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Figure 1 The hypothesis of developing metal pro-drug based on the nature of cancer cell and IB sub-domain of the PA modified HSA. 36x12mm (300 x 300 DPI)



Figure 2 The structures of ligands 1a-6a and their Cu compounds 1b-6b. 88x68mm (300 x 300 DPI)


Page 46 of 54

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Figure 3 The experimental σA weighted 2Fo − Fc electron density map of 4b in IB subdomain (A) and PA7 in IIA sub-domain of HSA (B); (C) The overall structure of HSA complex. 113x75mm (300 x 300 DPI)



Figure 4 The binding cavity of 4b in IB subdomain (A) and PA7 in IIA sub-domain of HSA (B); the structural binding environment of 4b in IB subdomain (C) and PA7 in IIA sub-domain of HSA (D). 113x87mm (300 x 300 DPI)


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Figure 5 (A) The uptake of 4b (0.6 µM) and HSA-PA-4b (0.6 µM) in A549 cells as a function of time at 37°C. (B) Uptake mechanisms of the HSA-PA-4b complex. (C) Western blot analysis of HSA in A549 cells treated with the HSA-PA-4b complex. β-Actin was used as the internal control. (D) Copper content released from HSA-4b or HSA-PA-4b in pH 4.7, pH 7.4 buffers and Dulbecco's Modified Eagle's medium (DMEM) with 10% FBS during 0−72 h, respectively. Statistical significance: (*) p < 0.05, (**) p < 0.005. 108x102mm (300 x 300 DPI)



Figure 6 (A) Absorption spectra of complex 4b (50 µM) upon the titration of CT-DNA (0−24 µM). (B) Fluorescence quenching curves of ethidium bromide (EB) bound to DNA: complex 4b. [DNA] = 8 µM, [EB] = 5 µM, and [complex] = 0−32 µM. (C) Relative viscosity increments of CT-DNA (200 µM) solution bound with 4b with increasing the [complex]/[DNA] ratio. (D) A molecular docked model for 4b with a DNA dodecamer duplex of sequence d(CGCGAATTCGCG)2. (E) Agarose gel electrophoresis patterns for the cleavage of pBR322 DNA by 4b at pH 7.0 at 37 °C for 5 h. Lane 1: DNA alone; Lanes 2−4: DNA with 4b at the concentrations of 20, 40, 80 µM, respectively. (F) Agarose GE patterns for the cleavage of pBR322 DNA by 4b (80 µM) at pH 7.0 and 37 °C for 5 h, in the presence of DMSO (1 M, Lane 3), MeOH (1 M, Lane 4), NaN3 (0.1 M, Lane 5), BDNPP (0.1 mM Lane 6), KI (0.1 M, Lane 7) and EDTA (0.1 M, Lane 8). Lane 1, DNA alone and Lane 2: DNA + 4b. 76x51mm (300 x 300 DPI)


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Figure 7 (A) Agarose gel electrophoresis patterns showing the effect of different concentrations of 4b on the activity of Topo I. Lane 1, DNA control; lane 2, Topo I + DNA; Lanes 3 and 4: Topo I + DNA with 4b at the concentrations of 15 and 20 µM, respectively. (B) Molecular docked model of human DNA Topoisomerase1 with 4b. (C) In vitro proteasome-inhibitory activity of 4b. (D) The influence of 4b (0.6 µM) and the HSA-PA4b complex (0.6 µM) on the telomerase activity of the A549 cells. 74x49mm (300 x 300 DPI)



Figure 8 (A) Western blot analysis of proteins related to the Bcl-2 family proteins response pathway. Proteins expressions in A549 cells treated with compound 4b and the HSA-PA-4b complex for 24 h; β-actin was used as the internal control. (B) Binding model of 4b with Bcl-xL. (C) Immunoblotting analysis of CDK2 and cyclin E1 in A549 cells treated with 4b and the HSA-PA-4b complex for 24 h; β-actin was used as the internal control. (D) The expression level of MACC1 in A549 cells induced by 4b and the HSA-PA-4b complex at the same concentration (0.6 µM) for 24 h, determined by Western blot analysis. (E) Relative expression profiles of telomeres/telomerase related genes in A549 cells after being treated with 4b and the HSA-PA-4b complex at the same concentration (0.6 µM) for 24 h (up: intersection, down: union). 113x128mm (300 x 300 DPI)


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Figure 9 (A) Changes of tumor volume after intravenous injection (tail vein) of vehicle control, 4b and the HSA-PA-4b complex in A549 tumor-bearing nude mice. (B) Tumor weight (wet weight) separated from mice after different treatments. (C) Tumors are sectioned and stained with TUNEL (400×), red arrows show apoptotic cells. Results are mean ± SD (n = 6−7): (*) p < 0.05, (***) p < 0.001. 70x44mm (300 x 300 DPI)



Figure 10 (A) Body weight changes of A549 tumor-bearing mice after treatment with vehicle control, 4b and the HSA-PA-4b complex. (B) H&E staining analysis of organs (heart, liver, and kidney) sections treated with various treatments (400×). (C) Tissue copper of mice after treatment with 4b and the HSA-PA-4b complex. The background values of copper in these organs have been deducted. 155x211mm (300 x 300 DPI)


Page 54 of 54

Developing an anticancer copper(II) multi-target pro-drug based on

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