Developing an Anticancer Copper(II) Pro-Drug Based on the His242

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Developing an anti-cancer copper(II) pro-drug based on the His242 residue of the human serum albumin carrier IIA sub-domain Jinxu Qi, Yao Zhang, Yi Gou, Zhenlei Zhang, Zuping Zhou, Xiaoyang Wu, Feng Yang, and Hong Liang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00938 • Publication Date (Web): 28 Mar 2016 Downloaded from http://pubs.acs.org on April 4, 2016

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Molecular Pharmaceutics

Developing an anti-cancer copper(II) pro-drug based on the His242 residue of the human serum albumin carrier IIA sub-domain Jinxu Qi1, Yao Zhang1, Yi Gou1, Zhenlei Zhang1, Zuping Zhou2, Xiaoyang Wu3, Feng Yang1*, Hong Liang1*

1

State Key Laboratory Cultivation Base for the Chemistry and Molecular

Engineering of Medicinal Resources, Ministry of Science and Technology of China, Guangxi Normal University, Guilin, Guangxi, China 2

Guangxi Universities Key Laboratory of Stem Cell and Pharmaceutical

Biotechnology, Guangxi Normal University, Guilin, Guangxi, China. 3

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

USA.

*To whom correspondence should be addressed: Feng Yang Email: [email protected] Address: 15 Yucai Road, Guilin, Guangxi, China. Zip code: 541004 Phone/Fax: 86−773−212−0958 Keywords: copper (II) compound; human serum albumin; pro-drug; tumor targeting; therapeutic effect.

1

ACS Paragon Plus Environment


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Abstract To increase delivery efficiency, anticancer activity and selectivity of anticancer metal agents in vivo, we proposed to develop the anticancer metal pro-drug based on His242 residue of the human serum albumin (HSA) carrier IIA sub-domain. To confirm our hypothesis, we prepared two Cu(II) compounds [Cu(P4mT)Cl and Cu(Bp44mT)Cl] by modifying Cu(II) compound ligand structure. Studies with two HSA complex structures revealed that Cu(P4mT)Cl bound to the HSA sub-domain IIA via hydrophobic interactions, but Cu(Bp44mT)Cl bound to the HSA sub-domain IIA via His242 replacement of a Cl atom of Cu(Bp44mT)Cl, and a coordination to Cu2+. Furthermore, Cu(II) compounds released from HSA could be regulated at different pHs. In vivo data revealed that the HSA-Cu(Bp44mT) complex increased copper’s selectivity and capacity of inhibiting tumor growth compared to Cu(Bp44mT)Cl alone.

2


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1 Introduction Although cisplatin is effective for treating a variety of cancers, cures with cisplatin and its derivatives are limited by side effects and inherited or acquired resistance.1-3 Therefore, less toxic and more effective metal-based anti-cancer compounds have been sought and developed,4-6 and drug delivery systems have been exploited to improve the selectivity of anti-cancer drugs and decrease their side effects.7 Many metal compounds have been evaluated in vitro and in vivo and some have reached clinical trials, but improving drug targeting and decreasing side effects is still challenging.8 Pro-drug and drug carrier strategies have been the most promising for overcoming these limitations.9-12 Human serum albumin (HSA), as the most abundant plasma protein, can bind to a diverse group of endogenous and exogenous compounds.13-17 It contains three structurally similar α-helical domains (I–III) that are comprised of sub-domains A and B, which contain six and four α-helices, respectively.18 HSA is a promising drug delivery system because it is non-toxic, non-antigenic, biocompatible and biodegradable and non-immunogenic.19-22 Two commonly applied HSA-based drug delivery strategies include chemical coupling of drugs to form albumin-drug conjugates and

encapsulation

of

drugs

in

albumin

nanoparticles

via

physical

interactions.23-27 However, these two methods can introduce exogenous chemicals or change albumin’s conformation. To overcome drawbacks associated with HSA-based drug delivery strategies, we attempted a novel 3



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approach for developing HSA-based drug delivery by complexing an anti-cancer drug with HSA.28-32 Preliminary data have shown that the HSA complex had greater anti-cancer activity and targeting ability than the drug alone.28-32 Furthermore, current studies confirmed by X-ray crystallography revealed that metal (Ru and Cu) compounds can bind to the IIA sub-domain of HSA via Lys199 and His242, replacing two ligands, and coordinating to the metal center.33, 34 Therefore, to synergize anti-cancer activity and targeting ability of metal agents in vivo, metal anti-cancer drugs that bind to the HSA IIA sub-domain may be promising.34-38 A potential problem with metal compound delivery to cancer cells via complexation with an HSA carrier in vivo is the premature released from HSA due to weak binding or a lack of release due to tight binding. The flexible property of the HSA IIA sub-domain allows drugs to bind based on their molecular structure.33, 39-44 Therefore, to enhance delivery efficiency, anticancer activity and selectivity of anticancer metal agents in vivo, we proposed a metal anti-cancer pro-drug with a leaving group that initially binds to the HSA IIA sub-domain, and then regulates His242 of HSA, displacing the metal pro-drug’s leaving group by structurally modifying the pro-drug’s ligand, coordinating to metal and forming a stable HSA complex (Figure 1). Subsequently, His242 of HSA is protonated in the cancer cell’s lysosomal acidic environment, which decreases its coordination ability with metal ion, allowing the metal agent to be released from the HSA carrier.34, 45 4


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Lung cancer is still too prevalent worldwide, causing many premature deaths and representing a significant healthcare cost burden.46 Furthermore, among next generation, metal-based anti-cancer compounds, Cu compounds may be promising as Cu is an essential element for human physiological functions due to its bioactivity and oxidative nature.47,48 Thus, to confirm our hypothesis about developing a metal anticancer pro-drug for better therapy of lung cancer based on cancer cell properties and the His242 residue of the HSA IIA sub-domain, the two Cu(II) compounds derived from a thiosemicarbazone Schiff-base and lung cancer cells (adenocarcinoma human alveolar basal epithelial cells, A549) were used to conduct the following studies: (1) prepared two Cu(II) compounds by modifying the structure of the Cu(II) compound thiosemicarbazone ligand [Cu(P4mT)Cl and Cu(Bp44mT)Cl] (Figure 2A), investigated their binding affinity and releasing behavior from HSA at pH 4.7 and pH 7.4; (2) provided the evidence about the on feasibility for developing a Cu(II) pro-drug based on His242 of the HSA carrier IIA subdomain and cancer cell properties; (3) determined whether an HSA carrier increases the selectivity and therapeutic efficacy of Cu(Bp44mT)Cl relative to Cu(Bp44mT)Cl alone in vivo. 2 Materials and Methods Fatty acid free HSA (catalogue number A3782) was purchased from Sigma Chemical Company and used without further purification. All other chemicals and solvents used were of high purity and available from commercial sources. Water used in the reactions was distilled prior to use. Elemental analyses (C, N, 5



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and H) were carried out on a Perkin-Elmer 2400 analyser. Infrared (IR) spectra were recorded using KBr pellets (4000–400 cm-1) on a Nexus 870 FT-IR spectrophotometer. 2.1 Preparation and characterization of two Cu compounds 2.1.1 Preparation of Cu(P4mT)Cl and Cu(Bp44mT)Cl The appropriate thiosemicarbazone (0.5mM) was dissolved in MeOH (20mL) and the appropriate MeOH solution (20mL) of CuCl2 (0.5mM) was added drop-wise with stirring, and the solution turned dark brown immediately. A few days later, single crystals of complexes suitable for X-ray diffraction were obtained from the solution. 2.1.2 Determination of structure of Cu(P4mT)Cl and Cu(Bp44mT)Cl X-ray crystallographic data of Cu(II) compounds were collected on a Bruker SMART Apex II CCD diffractometer using graphite-monochromated Mo-Kα (λ = 0.71073 Å) radiation. Empirical adsorption corrections were applied to all data using SADABS. The structures were solved by direct methods and refined against F2 by full-matrix least-squares methods using the SHELXTL version 5.1.49 All of the non-hydrogen atoms were refined anisotropically. All other hydrogen atoms were placed in geometrically ideal positions and constrained to ride on their parent atoms. The crystallographic data for Cu(P4mT)Cl and Cu(Bp44mT)Cl are summarized in Table S1. Selected bond lengths and angles are given in Table S2. Crystallographic data for the structural analyses have been deposited at the Cambridge Crystallographic Data Centre, reference numbers 6


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1015147

for

Cu(P4mT)Cl

and

1015145

for

Cu(Bp44mT)Cl.

The

crystallographic data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif. Cu(P4mT)Cl: The dark brown crystals were isolated, washed three times with distilled water and dried in a vacuum desiccator containing anhydrous CaCl2. (yield 81％). Anal. Calcd for C8H9ClCuN4S: C, 32.88%; H, 3.10%; N, 19.17%; S, 10.97%. Found:C, 33.12%; H, 2.59%; N, 19.86%; S, 10.91%. IR (main peaks) 3491 (s, amide), 2954 (m, aromatic hydrogen), 1502 (s), 1496 (s), 1389 (s, aromatic), 1301 (s, C=N), 1221 (s, thioamide), 1186s, 1011s, 918 (vs, C-H), 896 (vm, C=S), 689vs, 532vs. MS m/z (%) 255.98 (M − Cl, 100). Cu(Bp44mT)Cl: The dark brown crystals were isolated, washed three times with distilled water and dried in a vacuum desiccator containing anhydrous CaCl2. (yield 70％). Anal. Calcd for C15H15ClCuN4S: C, 46.87%; H, 4.46%; N, 14.58%; S, 8.34%. Found:C, 47.02%; H, 4.99%; N, 14.46%; S, 8.85%. IR (main peaks) 3212 (s, amide), 2921 (m, aromatic hydrogen), 1512 (s), 1483 (s), 1401 (s, aromatic), 1298 (s, C=N), 1256 (s, thioamide), 1172 (s), 903 (m, C-H), 845 (m, C=S), 801 (vs), 709 (vs), 628 (m). MS m/z (%) 346.05 (M − Cl, 100). 2.2 X-ray crystallography of HSA complexes Fatty acid (FA) free HSA was purified by removing HSA dimers and multimers as published.50 Palmitic acid (PA) was dissolved in alcohol, and diluted to 2.5 mM with 20 mM potassium phosphate (pH 7.5). HSA complexes were prepared by mixing 100 µL HSA (100 mg/mL), 380 µL PA (2.5 mM) and 7



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90 µL of each Cu(II) compound (5 mM) overnight, then mixtures were concentrated to 100 mg/ml with a Millipore spin filter (10,000 Da cutoff). Crystallization was carried out using sitting drop vapor diffusion at room temperature. An equal volume of the HSA complex was mixed with the reservoir solution, comprised of 28–32% (w/v) polyethylene glycol 3350, 50 mM potassium phosphate (pH 7.5), 5% glycerol, and 4% DMSO. Crystals were directly selected from the drop solution and then frozen in liquid nitrogen. X-ray diffraction data were collected under cryo-conditions (100 K) using the Shanghai Synchrotron Radiation Facility. The data were integrated and scaled with HKL2000. The two data sets obtained from the HSA complexes were processed in space group P1. The structure of HSA complexes were solved by molecular replacement with AMORE program using the HSA-MYR structure (PDB code 1BJ5) stripped of its ligands as the model. The model was initially refined using a rigid body protocol in CNS and then subjected to cycles of positional and B-factor refinement before the calculation of the initial Fo-Fc and 2Fo-Fc maps. These maps were used to guide the position of the fatty acid and ligands and to make manual adjustments to the protein prior to further cycles of refinement. Figures depicting the structure were prepared by PyMOL.51 Data collection details and unit cell parameters are given in Table 1. 2.3 Anti-cancer activity of HSA complex in vitro (MTT assay) A549 (adenocarcinoma human alveolar basal epithelial cells) and HL7702 (immortalized human hepatocyte cells) were grown as previously described at 8


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37 ℃ in a humidified atmosphere of 5% CO2/95% air in an incubator (Thermo-Fisher, city, state). We assayed toxicity of our compounds via MTT. Compounds were dissolved in DMSO as 10 mM stock solutions and diluted in PBS to a final DMSO concentration of 100

HL-7702

0.95 ± 0.09

0.15 ± 0.01

0.45 ± 0.01

15.66 ± 0.35

> 60

> 100

28


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Graphic Abstract 69x57mm (600 x 600 DPI)



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Figure 1 The hypothesis of developing metal pro-drug based on the His242 residue of HSA IIA subdomain. 37x16mm (600 x 600 DPI)


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Figure 2 (A) Chemical structure of Cu(P4mT)Cl and Cu(Bp44mT)Cl compounds; (B) Experimental sigmaA weighted 2Fo-Fc electron density map of Cu(P4mT)Cl and Cu(Bp44mT)Cl at IIA subdomain. 67x53mm (600 x 600 DPI)



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Figure 3 (A) The overall structure of HSA complex; (B) The surface of the binding pocket of Cu(II) compound with blue representing basic patches; (C) Structural binding environment of Cu(P4mT)Cl and Cu(Bp44mT)Cl to IIA subdomain of HSA. The amino acid chains that are close to the drug molecules are shown as sticks. Carbon, grey; nitrogen, blue; oxygen, red. 44x11mm (600 x 600 DPI)


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Figure 4 (A) Net tumor volume as a function of time of A549 xenografts after iv administration of vehicle control, HSA, Cu(Bp44mT) and HSA-Cu(Bp44mT). (B) Tumor weights (wet weight) from the study in (A) after sacrifice of the animals on day 26. Results are mean ± SD (n = 6−7 mice/condition). (C) Average body weights of nude mice for the study in (A) during treatment with the agents for 26 days. Results are mean ± SD (n = 6−7): (*) p < 0.05 (**) p < 0.01, (***) p < 0.001. 85x86mm (600 x 600 DPI)



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Figure 5 (A) Cu content in tumor of mice treated with Cu(Bp44mT) and HSA-Cu(Bp44mT) after 26 days. (B) The copper content released from HSA complex in pH 4.7 and 7.4 buffers, 0-48h, respectively. Results are mean ± SD (n = 6): (*) p < 0.05 (**) p < 0.01, (***) p < 0.001. 48x27mm (600 x 600 DPI)


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Developing an Anticancer Copper(II) Pro-Drug Based on the His242

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