Effect of Structural Modifications to Glyoxal-bis(thiosemicarbazonato

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The Effect of Structural Modifications to Glyoxalbis(thiosemicarbazanoto) Copper(II) Complexes on Cellular Copper Uptake, Coppermediated ATP7A Trafficking and P-Glycoprotein Mediated Efflux Karla M Acevedo, David J Hayne, Lachlan E McInnes, Asif Noor, Clare Duncan, Diane Moujalled, Irene Volitakis, Angela Rigopoulos, Kevin Jeffrey Barnham, Victor L. Villemagne, Anthony R White, and Paul S. Donnelly J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01158 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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Journal of Medicinal Chemistry

The Effect of Structural Modifications to Glyoxal-bis(thiosemicarbazonato)copper(II) Complexes on Cellular Copper Uptake, Copper-mediated ATP7A Trafficking and PGlycoprotein Mediated Efflux

Karla M. Acevedoa,b, David J. Hayneb,c, Lachlan E. McInnesb,c, Asif Noorb,c, Clare Duncana, Diane Moujalleda, Irene Volitakisd, Angela Rigopoulose, Kevin J. Barnhamc,d,e, Victor L. Villemagnef, Anthony R. White*a# and Paul S. Donnelly*b,c

a

Department of Pathology, bSchool of Chemistry, cBio21 Institute, dFlorey Institute of Neuroscience

and Mental Health and eDepartment of Pharmacology and Therapeutics, University of Melbourne, Melbourne, Victoria 3010, Australia University of Melbourne, Melbourne, Victoria, 3010, Australia, eOlivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia, fDepartment of Molecular Imaging & Therapy, Centre for PET, Austin Health, 145 Studley Road, Heidelberg, Victoria 3084, Australia. # Current address: Cell and Molecular Biology, Mental Health Program, QIMR Berghofer Medical Research Institute, Herston, Queensland, 4006, Australia.

Abstract Bis(thiosemicarbazonato)copper(II) complexes are of interest as potential therapeutics for cancer and neurodegenerative diseases as well as imaging agents for positron emission tomography (PET). The cellular uptake of six bis(thiosemcarbazonato)copper(II)complexes derived from glyoxal, with different functional groups, Cu(gtsx) where x = different functional groups, was investigated in SKOV-3, HEK293 and HEK293 P-gp cell lines. Treatment of the cells with the copper complexes increased intracellular copper and increased levels of p-ERK due to activation of the Ras-RafMEK-ERK pathway. Treatment of SKOV-3 cells with low concentrations (µM) of two of the copper complexes led to trafficking of the endogenous copper transporter ATP7A from the Golgi

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network to the cell membrane. Experiments in HEK293 and HEK293-P-gp cells suggest that Cu(gtsm) and Cu(gtse) are substrates for the P-gp efflux protein but the complex with a pyrrolidine functional group, Cu(gtspyr), is not. A PET experiment in mice showed that [64Cu]Cu(gtspyr) has reasonable brain uptake but high liver uptake.

Introduction The biological activity of copper(II) complexes of bis(thiosemicarbazone) ligands (Cu(btsc)) has led to them being investigated as potential anti-cancer agents, antimicrobials and neurotherapeutics. Treatment of a sarcoma 180 mouse model with the metal free bis(thiosemicarbazone), glyoxal-bis(N-4-methyl-3-thiosemicarbazone) H2gtsm (Figure 1), led to a reduction in tumor weight when compared to untreated controls.1 In this early investigation, the authors recognized ‘‘a mechanism of action involving inactivation or translocation of metal ions was possible”. The analogous bis(thiosemicarbazones) derived from 2,3-butanedione, featuring two methyl substituents on the backbone, such as H2atsm (Figure 1) were inactive in this model. Bis(thiosemicarbazones) derived from 1,2-diones, such as glyoxal, form charge neutral complexes with CuII with the ligand acting as a dianionic tetradentate N2S2 donor. The CuII complexes are stable (log KA ~ 18), relatively lipophilic and selected examples, such as Cu(gtsm) (Figure 1), have the ability to cross cell membranes and even the blood-brain barrier.2 The CuII complexes can be reduced to CuI complexes that are less stable than the CuII complexes. The CuII/I reduction potential is remarkably sensitive to the substituents on the backbone of the ligand as illustrated by the dramatically different reduction potentials for Cu(gtsm) (E0’ = –0.52 V vs SCE) and Cu(atsm) (E0’ = –0.65 V vs SCE) when measured in dimethylformamide.2-5 The inactivity of the ligands derived from 2,3-butanedione, H2atsm, in the early studies against Sarcoma 180 in Swiss mice was possibly due to the lower reduction potential of Cu(atsm) making it harder to release bioavailable copper.1


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It has been suggested that the reducing environment encountered inside cells is sufficient to reduce Cu(gtsm) to less stable CuI species that are susceptible to dissociation from the ligand making the copper ‘bioavailable’ to known copper binding proteins.5, 6 Copper is an essential trace nutrient necessary for many enzymes and cellular processes and its cellular biochemistry is carefully controlled by an array of transport and chaperone proteins.7,

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The Menkes copper-

translocating P-type ATPase (ATP7A) is a copper transport protein central to systemic copper absorption that uses the energy of ATP hydrolysis to transport copper from the cytosol into the lumen of the secretory pathway where copper is incorporated into various copper requiring enzymes.7 In polarized cells, the ATP7A protein resides in the trans-Golgi network and responds to increases in cellular copper concentrations by relocating to the plasma membrane.9 Treatment of the human neuroblastoma cell line BE(2)-M17 with the preformed copper complex, Cu(gtsm), resulted in cell cycle arrest that was not associated with the onset of apoptosis. Analysis of the treated cells by protein microarray techniques revealed that Cu(gtsm) rapidly and potently reduced cyclin D1 expression, while increasing Kip2 expression, decreased Cdk7 expression, activated CHK2 and resulted in a potent decrease of total and phosphorylated insulinlike growth factor receptor (IGF-IR).10 Treatment of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model with preformed Cu(gtsm) (2.5 mg/kg) significantly reduced prostate cancer burden (70%) and severity (grade) but caused mild kidney toxicity in the mice, associated primarily with interstitial nephritis and luminal distention.11 In a separate study, reatment of a HCT116 xenograft mouse model with another Cu(btsc) complex derived from glyoxal, glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazonato)copper(II), inhibited tumor growth by 95 ± 3.9% when compared to control mice.12 We have also investigated bis(thiosemicabazonato)copper(II) (Cu(btsc)) complexes as potential therapeutics for neurodegenerative diseases.13-16 Treatment of an amyloid mouse model (APP/PS1) of relevance to Alzheimer’s disease with Cu(gtsm) resulted in cognitive improvement associated with altered amyloid metabolism and microtubule tau protein phosphorylation mediated


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through modulation of kinase signaling including phosphatidylinositol-3-kinase (PI3K), glycogen synthase kinase 3β (GSK3β) and extracellular signal regulated kinase (ERK).6, 17 There are several positron-emitting isotopes of copper that are of interest in the development of copper based positron emission tomography (PET) imaging agents. Complexes of bis(thiosemicarbazones) formed with radioactive copper isotopes have been investigated as myocardial perfusion and hypoxia tracers.2, 18-22 Both [64Cu]Cu(gtsm) and [64Cu]Cu(atsm) cross the blood-brain barrier in mice and there is a significant increase in the uptake of [64Cu]Cu(gtsm) in a transgenic mouse model of amyloid pathology (APP/PS1) when compared to control animals (3.0 ± 0.25% ID/g and 1.58 ± 0.14% ID/g, respectively), possibly due to altered copper metabolism in the animal model of the disease. In addition, in the APP/PS1 model, [64Cu]Cu(gtsm) showed 40% higher brain retention than

64

Cu(atsm).23 A similar difference in brain uptake of copper following

administration of [64Cu]Cu(gtsm) was observed in an alternative transgenic amyloid model (TASTPM) when compared to wild-type control.24 The blood-brain barrier consists of a series of endothelial cells with tight junctions that control the passage of chemicals from the blood into the brain tissue. It is generally accepted that only certain small, MW < 600 Da, relatively lipophilic (LogP 0.9-3.0) molecules are capable of readily crossing the blood-brain barrier and it has been estimated that only 2% of central nervous system drug discovery compounds can cross the blood-brain barrier.25 The blood-brain barrier also contains several transporters that facilitate uptake or efflux and introduce yet further complications. Efflux transporters that are essential to the function of the blood-brain barrier include PGlycoprotein (P-gp) and Breast Cancer Resistant Protein (BCRP) and both can impede entry of imaging agents into the brain.26 P-gp is widely expressed and found in most barrier and excretory tissues including liver, kidney, intestine, testes and placenta. In the brain, P-gp is also found in astrocytes, microglia, neurons and the luminal side of brain capillary endothelial cells.27 In mice, Pgp is partially responsible for clearance of amyloid-β from the brain and patients with mild Alzheimer’s disease have compromised P-gp activity.28, 29


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Over expression of P-gp also plays a role in the multidrug resistance (MDR) of some human cancers and correlates with poor prognosis. There is an inverse relationship between P-gp expression and the efficacy of chemotherapy drugs, with high expression a predictor of poor prognosis in patients with advanced disease.30-33 Despite the importance of P-gp mediated efflux to chemotherapeutic drugs and imaging agents intending to target the brain, the precise molecular interactions that confer P-gp efflux remain poorly defined. There is a paucity of published examples where P-gp efflux had been purposefully circumvented. Functional groups that favour binding to Pgp are typically those adorned with hydrogen bond donors.34 The effect of P-gp expression on the cellular uptake and retention of the perfusion tracer, 64Cu(ptsm) and the hypoxia tracer 64Cu(atsm) was investigated in MES-SA (P-gp negative) and MES-SA/Dx5 (P-gp positive) cells under ambient oxygen conditions to investigate the influence of P-gp independent of hypoxia or perfusion. MESSA/Dx5 (P-gp positive) cells showed lower retention of both tracers in a time-dependent fashion and more rapid efflux.35 A structurally related N-heterocyclic thiosemicarbazone, di-2pyridylketone 4,4-dimethyl-3-thiosemicarbazone, is a substrate for P-gp.36 To our knowledge the interactions between P-gp and Cu(btsc) derived from glyoxal remain to be investigated. In this manuscript we substantiate the claim that Cu(btsc) complexes derived from glyoxal, Cu(gtsx) where x = different functional groups, ethyl (e), phenyl (p) and pyrolidine (pyr), cross cell membranes and release bioavailable copper inside cells by monitoring copper-induced trafficking of ATP7A between the trans-Golgi Network and the plasma membrane in SKOV3 cells. We also investigate the effects of P-gp expression on cellular uptake of Cu(gtsx) complexes in a HEK293 cell line that expresses the P-gp protein. A series of six complexes were synthesized, three compounds with different ‘N4’ substituents but which each retain a NH-R functional group (potential hydrogen bond donor) were compared to three N4-dialkyl derivatives that lack a potential H-bond donor in this position (Figure 1). It was hoped that we could use this information to help us predict derivatives with potential to be more effective in MDR tumors and derivatives with better


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blood-brain barrier penetration to provide superior PET imaging agents to enable study of copper metabolism in the brain.

Results The Cu(gtsx) complexes have quasi-reversible CuII/I reduction potentials The redox chemistry of each complex was investigated by cyclic voltammetry in dimethylformamide. Each of the six complexes displayed quasi-reversible reductions assigned to essentially metal based CuII/I processes. The CuII/I reduction potentials for the complexes with either one or two alkyl substituents on the N4-position were all similar ranging from Eo’ = 0.49 V for Cu(gtsm2) to Eo’ = 0.53 V for Cu(gtspyr) (vs SCE where ferrocene/ferricenium (FeII/III) Eo’ = 0.45 V in dimethylformamide) and consistent with previous reports (Figure 2).3-5 The aromatic functional group in Cu(gtsp) results in a significant shift in the CuII/CuI couple to Eo’ = 0.31 V (vs SCE).

Cell uptake and P-gp recognition of Cu(gtsx) complexes is dependent on their substituents The cellular uptake of Cu(gtsx) complexes was investigated in SKOV3 cells by measuring the concentration of Cu in cell pellets by ICP-MS following a 2 hour treatment with the complex (1 µM) (Figure 3). Measurements were based on the copper concentration (µmole/L) and the total protein in the cell pellet (Cu µmole/L/µg). Cell viability was not compromised following Cu(gtsx) treatment under these conditions as indicated by both 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays and lactate dehydrogenase (LDH) assays (see Supporting Information, Figure S1). Treatment with Cu(gtsm) led to the greatest increase in intracellular Cu when compared to Cu(gtsm2) [P

Effect of Structural Modifications to Glyoxal-bis(thiosemicarbazonato

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